The Interactive Fly

Zygotically transcribed genes


Endocytic pathway
Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila
Disruption of Drosophila melanogaster lipid metabolism genes causes tissue overgrowth associated with altered developmental signaling
Identification of Atg2 and ArfGAP1 as candidate genetic modifiers of the eye pigmentation phenotype of Adaptor Protein-3 (AP-3) mutants in Drosophila melanogaster
Efficient endocytic uptake and maturation in Drosophila oocytes requires Dynamitin/p50
Cooperative functions of the two F-BAR proteins Cip4 and Nostrin in regulating E-cadherin in epithelial morphogenesis
MiniCORVET is a Vps8-containing early endosomal tether in Drosophila
ARC syndrome-linked Vps33B protein is required for inflammatory endosomal maturation and signal termination
Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila
Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes
Scribbled optimizes BMP signaling through its receptor internalization to the Rab5 endosome and promote robust epithelial morphogenesis

Golgi complex
Distinct functional units of the Golgi complex in Drosophila cells
The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE
Golgi outposts shape dendrite morphology by functioning as sites of acentrosomal microtubule nucleation in neurons
GPI biosynthesis is essential for rhodopsin sorting at the trans-Golgi network in Drosophila photoreceptors
Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons
Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster
Golgi outpost synthesis impaired by toxic polyglutamine proteins contributes to dendritic pathology in neurons

BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior
AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila
Vesicle-mediated steroid hormone secretion in Drosophila melanogaster
The Arf family G protein Arl1 is required for secretory granule biogenesis in Drosophila
Synaptic control of secretory trafficking in dendrites
Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations
Golgi-resident Galphao promotes protrusive membrane dynamics

Asymmetric cell division
Lgl regulates Notch signaling via endocytosis, independently of the apical aPKC-Par6-Baz polarity complex
Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division

Synaptic function
A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis
Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release
Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture
Crimpy enables discrimination of presynaptic and postsynaptic pools of a BMP at the Drosophila neuromuscular junction
Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila
Immunolocalization of the vesicular acetylcholine transporter in larval and adult Drosophila neurons

Endosomal sorting complexes required for transport (ESCRT) machinery
De-regulation of JNK and JAK/STAT signaling in ESCRT-II mutant tissues cooperatively contributes to neoplastic tumorigenesis
ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo
The ESCRT machinery regulates the secretion and long-range activity of Hedgehog
The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors
Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB

Endosome-lysosomal pathway
Cell-free reconstitution of multivesicular body (MVB) cargo sorting
The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation
The lysosomal enzyme receptor protein (LERP) is not essential, but is implicated in lysosomal function in Drosophila melanogaster
FIG4 regulates lysosome membrane homeostasis independent of phosphatase function
The GATOR2 component Wdr24 regulates TORC1 activity and lysosome function
Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction
Coordination of autophagosome-lysosome fusion and transport by a Klp98A-Rab14 complex
Rab2 promotes autophagic and endocytic lysosomal degradation
A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila
Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress
Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis
Lysosomal degradation is required for sustained phagocytosis of bacteria by macrophages
Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster
Class III phosphatidylinositol-3-OH kinase controls epithelial integrity through endosomal LKB1 regulation
Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification

Cuticle Formation
The ABC transporter Snu and the extracellular protein Snsl cooperate in the formation of the lipid-based inward and outward barrier in the skin of Drosophila

Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila

The development of air-filled respiratory organs is crucial for survival at birth. A combination of live imaging and genetic analysis was used to dissect respiratory organ maturation in the embryonic Drosophila trachea. Tracheal tube maturation was found to entail three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, gas-filling-deficient mutants were identified showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 was shown to be required for protein secretion, luminal matrix assembly, and diametric tube expansion. sar1 encodes a small GTPase that regulates COPII vesicle budding from the endoplasmic reticulum (ER) to the Golgi apparatus. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation (Tsarouhas, 2007).

Branched tubular organs are essential for oxygen and nutrient transport. Such organs include the blood circulatory system, the lung and kidney in mammals, and the tracheal respiratory system in insects. The optimal flow of transported fluids depends on the uniform length and diameter of the constituting tubes in the network. Alterations in the distinct tube shapes and sizes cause pronounced defects in animal physiology and lead to serious pathological conditions. For example, tube overgrowth and cyst formation in the collecting duct are intimately linked to the pathology of Autosomal Dominant Polycystic Kidney Disease. Conversely, stenoses, the abnormal narrowing of blood vessels and other tubular organs, are associated with ischemias and organ obstructions (Tsarouhas, 2007 and references therein).

While the early steps of differentiation, lumen formation, and branch patterning begin to be elucidated in several tubular organs, only scarce glimpses into the cellular events of lumen expansion and tubular organ maturation are available. De novo lumen formation can be induced in three-dimensional cultures of MDCK cells. Recent studies in this system revealed that PTEN activation, apical cell membrane polarization, and Cdc42 activation are key events in lumen formation in vitro. In zebrafish embryos and cultured human endothelial cells, capillary vessels form through the coalescence and growth of intracellular pinocytic vesicles. These tubular vacuoles then fuse with the plasma membranes to form a continuous extracellular lumen. Salivary gland extension in Drosophila requires the transcriptional upregulation of the apical membrane determinant Crumbs (Crb), but the cellular mechanism leading to gland expansion remains unclear (Tsarouhas, 2007 and references therein).

The epithelial cells of the Drosophila tracheal network form tubes of different sizes and cellular architecture, and they provide a genetically amenable system for the investigation of branched tubular organ morphogenesis. Tracheal development begins during the second half of embryogenesis when 20 metameric placodes invaginate from the epidermis. Through a series of stereotypic branching and fusion events, the tracheal epithelial cells generate a tubular network extending branches to all embryonic tissues. In contrast to the wealth of knowledge about tracheal patterning and branching, the later events of morphogenesis and tube maturation into functional airways have yet to be elucidated. As the nascent, liquid-filled tracheal network develops, the epithelial cells deposit an apical chitinous matrix into the lumen. The assembly of this intraluminal polysaccharide cable coordinates uniform tube growth. Two luminal, putative chitin deacetylases, Vermiform (Verm) and Serpentine (Serp), are selectively required for termination of branch elongation. The analysis of verm and serp mutants indicates that modifications in the rigidity of the matrix are sensed by the surrounding epithelium to restrict tube length. What drives the diametric expansion of the emerging narrow branches to their final size? How are the matrix- and liquid-filled tracheal tubes cleared at the end of embryogenesis (Tsarouhas, 2007)?

This study used live imaging of secreted GFP-tagged proteins to identify the cellular mechanisms transforming the tracheal tubes into a functional respiratory organ. The precise sequence and cellular dynamics were characterized of a secretory and an endocytic pulse that precede the rapid liquid clearance and gas filling of the network. Analysis of mutants with defects in gas filling reveals three distinct but functionally connected steps of airway maturation. Sar1-mediated luminal deposition of secreted proteins is tightly coupled with the expansion of the intraluminal matrix and tube diameter. Subsequently, a Rab5-dependent endocytotic wave frees the lumen of solid material within 30 min. The precise coordination of secretory and endocytotic activities first direct tube diameter growth and then ensure lumen clearance to generate functional airways (Tsarouhas, 2007).

Two strong, hypomorphic sar1 alleles were identified in screens for mutants with tracheal tube defects. In wild-type embryos, the bulk of luminal markers 2A12, Verm, and Gasp has been deposited inside the DT lumen by stage 15. However, in zygotic sar1P1 mutants (hereafter referred to as sar1), luminal secretion of 2A12, Verm, and Gasp was incomplete. The tracheal cells outlined by GFP-CAAX partially retained those markers in the cytoplasm. sar1 zygotic mutant embryos normally deposited the early luminal marker Piopio by stage 13. Luminal chitin was also detected in sar1 mutants at stage 15. However, the luminal cable was narrow, more dense, and distorted compared to wild-type. To test if the sar1 secretory phenotype in the trachea is cell autonomous, Sar1 was reexpressed specifically in the trachea of sar1 mutants by using btl-GAL4. Such embryos showed largely restored secretion of 2A12, Verm, and Gasp. Thus, it is concluded that tracheal sar1 is required for the efficient secretion of luminal markers, which are predicted to associate with the growing intraluminal chitin matrix (Tsarouhas, 2007).

sar1 mRNA has been reported to be abundantly maternally contributed. At later stages, zygotic expression of sar1 mRNA is initiated in multiple epithelial tissues. To monitor Sar1 zygotic expression in the trachea, a Sar1-GFP protein trap line was used. Embryos carrying only paternally derived Sar1-GFP show a strong zygotic expression of GFP in the trachea. An anti-Sar1 antibody was used to analyze Sar1 expression in the trachea of wild-type, zygotic sar1P1, and sar1EP3575Δ28 null mutant embryos were generated. Both zygotic mutants showed a clear reduction, but not complete elimination, of Sar1 expression in the trachea. To test the effects of a more complete inactivation of Sar1, transgenic flies were generated expressing a dominant-negative sar1T38N form in the trachea. In btl > sar1T38N-expressing embryos, early defects were observed in tracheal branching and epithelial integrity as well as a complete block in Verm secretion. In contrast to btl > sar1T38N-expressing embryos, zygotic sar1P1 mutant embryos show normal early tracheogenesis with no defects in branching morphogenesis and epithelial integrity (Tsarouhas, 2007).

In summary, tracheal expression of Sar1 is markedly reduced in zygotic sar1 mutant embryos. While maternally supplied Sar1 is sufficient to support early tracheal development, zygotic Sar1 is required for efficient luminal secretion (Tsarouhas, 2007).

Given the conserved role of Sar1 in vesicle budding from the ER, its subcellular localization in the trachea was determined by using anti-Sar1 antibodies. Sar1 localizes predominantly to the ER (marked by the PDI-GFP trap). Continuous COPII-mediated transport from the ER is required to maintain the Golgi apparatus and ER structure. To test if zygotic loss of Sar1 compromises the integrity of the ER and Golgi in tracheal cells, sar1 mutant embryos were stained with antibodies against KDEL (marking the ER lumen) and gp120 (highlighting Golgi structures). In sar1 mutant embryos, a strongly disrupted ER structure and loss of Golgi staining was observed in dorsal trunk (DT) cells at stage 14. Additionally, TEM of stage-16 wild-type and sar1 mutant embryos showed a grossly bloated rough ER structure in DT tracheal cells. Consistent with its functions in yeast and vertebrates, Drosophila Sar1 localizes to the ER and is not only required for efficient luminal protein secretion, but also for the integrity of the early secretory apparatus (Tsarouhas, 2007).

To analyze tracheal maturation defects in sar1 mutant embryos, sar1 strains were generated and imaged that carry either btl > ANF-GFP btl-mRFP-moe or btl > Gasp-GFP. In sar1 mutants, luminal deposition of both ANF-GFP and Gasp-GFP is reduced. Like endogenous Gasp in the mutants, Gasp-GFP was clearly retained in the cytoplasm of sar1 embryos. ANF-GFP was also retained in the tracheal cells of sar1 mutants, but to a lesser extent. Strikingly, sar1 mutants failed to fully expand the luminal diameter of the DT outlined by the apical RFP-moe localization. This defect was quantified by measuring diametric growth rates in metamere 6 for wild-type and sar1 mutant embryos. While early lumen expansion commences in parallel in both genotypes, the later diametric growth of sar1 mutants falls significantly behind compared to wild-type embryos. The DT lumen in sar1 mutants reaches only an average of 70% of the wild-type diameter at early stage 16. Identical diametric growth defects were detected in fixed sar1 mutant embryos expressing btl > GFP-CAAX by analysis of confocal yz sections or TEM. Reexpression of sar1 in the trachea of sar1 mutant embryos not only rescued secretion, but also the lumen diameter phenotype at stage 16. In contrast to the diametric growth defects, DT tube elongation in sar1 embryos was indistinguishable from that in wild-type. This demonstrates distinct genetic requirements for tube diameter and length growth. It also reveals that the sar1 DT luminal volume reaches less than half of the wild-type volume. Prolonged live imaging showed that sar1 mutants are also completely deficient in luminal protein and liquid clearance. Up to 80% of the rescued embryos also completed luminal liquid clearance, suggesting that efficient tracheal secretion and the integrity of the secretory apparatus are prerequisites for later tube maturation steps. Taken together, the above-described results show that tracheal Sar1 is selectively required for tube diameter expansion. Additionally, subsequent luminal protein and liquid clearance fail to occur in sar1 mutants (Tsarouhas, 2007).

Do the tracheal defects of sar1 reflect a general requirement for the COPII complex in luminal secretion and diameter expansion? To test this, lethal P element insertion alleles were examined disrupting two additional COPII coat subunits, sec13 and sec23. Mutant sec13 and sec23 embryos were stained for luminal Gasp and for Crb and α-Spectrin to highlight tracheal cells. At stage 15, embryos of both mutants show a clear cellular retention of Gasp. Furthermore, stage-16 sec13 and sec23 embryos show significantly narrower DT tubes when compared to wild-type. The average diameter of the DT branches in metamere 6 was 4.8 μm and 4.4 μm in fixed sec13 and sec23 embryos, respectively, compared to 6.3 μm in wild-type. Therefore, sec13 and sec23 mutants phenocopy sar1. The phenotypic analysis of three independent mutations disrupting ER-to-Golgi transport thus provides a strong correlation between deficits in luminal protein secretion and tube diameter expansion (Tsarouhas, 2007).

The live-imaging approach defines the developmental dynamics of functional tracheal maturation. At the organ level, three sequential and rapid developmental transitions were identified: (1) the secretion burst, followed by massive luminal protein deposition and tube diameter expansion, (2) the clearance of solid luminal material, and (3) the replacement of luminal liquid by gas. Live imaging of each event additionally revealed insights into the startlingly dynamic activities of the tracheal cells. ANF-GFP-containing structures and apical GFP-FYVE-positive endosomes rapidly traffic in tracheal cells during the secretion burst and protein clearance. The direct live comparison between wild-type and mutant embryos further highlights the dynamic nature of epithelial activity during each pulse (Tsarouhas, 2007).

This study identified several mutations that selectively disrupt distinct cellular functions and concurrently interrupt the maturation process at specific steps. This clearly demonstrates the significance of phenotypic transitions in epithelial organ maturation and establishes that secretion is required for luminal diameter expansion and endocytosis for solid luminal material clearance (Tsarouhas, 2007).

The sudden initiation of an apical secretory burst tightly precedes diametric tube expansion. The completion of both events depends on components of the COPII complex, further suggesting that the massive luminal secretion is functionally linked to diametric growth. How does apical secretion provide a driving force in tube diameter expansion? In mammalian lung development, the distending internal pressure of the luminal liquid on the epithelium expands the lung volume and stimulates growth. Cl channels in the epithelium actively transport Cl ions into luminal liquid. The resulting osmotic differential then forces water to enter the lung lumen, driving its expansion. By analogy, the tracheal apical exocytic burst may insert protein regulators such as ion channels into the apical cell membrane or add additional membrane to the growing luminal surface. Since the ER is a crucial cellular compartment for intracellular traffic and lipogenesis, its disruption in sar1 mutants may disrupt the efficient transport of so far unknown specific regulators or essential apical membrane addition required for diametric expansion. Alternatively, secreted chitin-binding proteins (ChB) may direct an increase of intraluminal pressure and tube dilation. Overexpression of the chitin-binding proteins Serp-GFP or Gasp-GFP was insufficient to alter the diametric growth rate of the tubes, suggesting that lumen diameter expansion is insensitive to increased amounts of any of the known luminal proteins. In sar1 mutants, the secretion of at least two chitin-binding proteins, Gasp and Verm, is reduced. Chitin, however, is deposited in seemingly normal quantities, but assembles into an aberrantly narrow and dense chitinous cable. This phenotype suggests that the correct ratio between chitin and multiple interacting proteins may be required for the correct assembly of the luminal cable. Interestingly, sar1, sec13, and sec23 mutant embryos form a severely defective and weak epidermal cuticle. The luminal deposition of ChB proteins during the tracheal secretory burst may orchestrate the construction and swelling of a functional matrix, which, in turn, induces lumen diameter dilation. While this later hypothesis is favored, it cannot be excluded that other mechanisms, either separately or in combination with the dilating luminal cable, drive luminal expansion (Tsarouhas, 2007).

During tube expansion, massive amounts of luminal material, including the chitinous cable, fill the tracheal tubes. This study found that Dynamin, Clathrin, and the tracheal function of Rab5 are required to rapidly remove luminal contents, indicating that endocytosis is required for this process. Several lines of evidence argue that the tracheal epithelium activates Rab5-dependent endocytosis to directly internalize luminal material. First, the tracheal cells of rab5 mutants show defects in multiple endocytic compartments. These phenotypes of rab5 mutants become apparent during the developmental period matching the interval of luminal material clearance in wild-type embryos. Second, tracheal cells internalize two luminal markers, the endogenously encoded Gasp and the dextran reporter, exactly prior and during luminal protein clearance. The number of intracellular dextran puncta reaches its peak during the clearance process and ceases shortly thereafter. Lastly, intracellular puncta of both Gasp and dextran colocalize with defined endocytic markers inside tracheal cells. The colocalization of Gasp and dextran with GFP-Rab7 and of Gasp with GFP-LAMP1 suggests that the luminal material may be degraded inside tracheal cells. Taken together, these data show that the tracheal epithelium activates a massive wave of endocytosis to clear the tubes (Tsarouhas, 2007).

Endocytic routes are defined by the nature of the internalized cargoes and the engaged endocytic compartments. What may be the features of the endocytic mechanisms mediating the clearance of luminal material? The phenotype of chc mutants and the presence of intracellular Gasp in CCVs indicate that luminal clearance at least partly relies on Clathrin-mediated endocytosis (CME). In addition to CME, Dynamin and Rab5 have also been implicated in other routes of endocytosis, suggesting that multiple endocytic mechanisms may be operational in tracheal maturation. The nature of the endocytosed luminal material provides an additional perspective. While cognate uptake receptors may exist for specific cargos such as Gasp, Verm, and Serp, the heterologous ANF-GFP, degraded chitin, and the fluid-phase marker dextran may be cleared by either fluid-phase internalization or multifunctional scavenger receptors. Interestingly, Rab5 can regulate fluid-phase internalization in cultured cells by stimulating macro-pinocytosis and the activation of Rabankyrin-5. The defective tracheal internalization of dextran in rab5 mutants provides further loss-of-function evidence for Rab5 function in fluid-phase endocytosis in vivo. The above-described arguments lead to the speculation that additional Rab5-regulated endocytic mechanisms most likely cooperate with CME in the clearance of solid luminal material (Tsarouhas, 2007).

How is liquid cleared from the lumen? While very little is known about this fascinating process, some developmental and mechanistic arguments suggest that this last maturation step is mechanistically distinct. First, the interval of luminal liquid clearance is clearly distinct from the period of endocytic clearance of solids. Second, the dynamic internalization of dextran and the abundance of GFP-marked endocytic structures decline before liquid clearance. Finally, assessment of liquid clearance further suggests that it requires a distinct cellular mechanism (Tsarouhas, 2007).

Viewing the entire process of airway maturation in conjunction, some general conclusions may be drawn. First, the three epithelial pulses are highly defined by their sequence and exact timing, suggesting that they may be triggered by intrinsic or external cues. Second, the analysis of mutants that selectively reduce the amplitude of the secretory or endocyic pulses demonstrates the requirement for each epithelial transition in the completion of the entire maturation process. These pulses are induced in the background of basal secretory and endocytic activities that operate throughout development. Third, specific cellular activities exactly precede each morphological transition. Finally, the separate transitions are interdependent in a sequential manner. Efficient secretion is a prerequisite for the endocytic wave. Similarly, protein endocytosis is a condition for luminal liquid clearance. This suggests a hierarchical coupling of the initiation of each pulse to completion of the previous one in a strict developmental sequence (Tsarouhas, 2007).

This study provides a striking example of how pulses of epithelial activity drive distinct developmental events and mold the nascent tracheal lumen into an air delivery tube. These findings are likely to be relevant beyond the scope of tracheal development. The uniform growth of salivary gland tubes in flies and the excretory canal and amphid channel lumen in worms also require the assembly of a luminal matrix for uniform tube growth. Luminal material is also transiently present during early developmental stages in the distal nephric ducts of lamprey. Thus, the coordinated, timely deposition and removal of transient luminal matrices may represent a general mechanism in tubulogenesis (Tsarouhas, 2007).

Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila

UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. This study shows that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. These data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans (Nagy, 2016).

UVRAG encodes a homolog of yeast Vps38 in metazoans. UVRAG/Vps38 and Atg14 are mutually exclusive subunits of two different Vps34 lipid kinase complexes, both of which contain Vps34, Vps15 and Atg6/Beclin 1. It is well established that Vps38 is required for endosome maturation and vacuolar and lysosomal protein sorting, whereas Atg14 is specific for autophagy in yeast. However, the function of UVRAG is much more controversial in mammalian cells. Although UVRAG was originally found to have dual roles in autophagy through promotion of autophagosome formation and fusion with lysosomes in various cultured cell lines based on, predominantly, overexpression experiments, recent reports have described that autophagosomes are normally generated and fused with lysosomes in the absence of UVRAG in cultured mammalian (HeLa) cells and in the Drosophila fat body (Nagy, 2016 and references therein).

The discoveries of UVRAG mutations in colorectal cancer cells, and that its overexpression increases autophagy and suppresses the proliferation of certain cancer cell lines, altogether suggest that this gene functions as an autophagic tumor suppressor. Such a role for UVRAG is thought to be related to its binding to Beclin 1, a haploinsufficient tumor suppressor gene required for autophagy. UVRAG appears to play roles similar to yeast Vps38 in the Drosophila fat body, and developing eye and wing: its loss leads to the accumulation of multiple endocytic receptors and ligands in an endosomal compartment, impaired trafficking of Lamp1 and Cathepsin L to the lysosome, and defects in the biogenesis of lysosome-related pigment granules. However, whether this gene is also required for the maintenance of intestinal homeostasis in Drosophila was unclear because the loss of UVRAG did not lead to uncontrolled cell proliferation in the developing eye or wing according to these reports. The current results showing that Uvrag deficiency causes intestinal dysplasia suggest that this gene is also important for the proper functioning of the adult gut in Drosophila (Nagy, 2016).

A surprising aspect of this work is that UVRAG appears to function independently of autophagy in the intestine. There are other lines of evidence that also support that UVRAG has a more important role in endocytic maturation than in autophagy. First, it has been shown that truncating mutations in UVRAG that are associated with microsatellite-unstable colon cancer cell lines do not disrupt autophagy. Second, UVRAG depletion in HeLa cells does not prevent the formation or fusion of autophagosomes with lysosomes, but it does interfere with Egfr degradation. Third, a very recent paper has shown that overexpression of the colorectal-cancer-associated truncated form of UVRAG promotes tumorigenesis independently of autophagy status, that is, both in control and Atg5-knockout cells. That paper, again, relied on the overexpression of full-length or truncated forms of UVRAG, rather than the analysis of cancer-related mutations of the endogenous locus. Fourth, the endocytic function of UVRAG has been found to be required for developmental axon pruning that is independent of autophagy in Drosophila (Nagy, 2016 and references therein).

The results of this study indicate that UVRAG loss is accompanied with the sustained activation of JNK and STAT signaling in ISCs and EBs, and that these pathways are required for dysplasia in this setting. Sustained activation of these signaling routes is likely to be connected to the disruption of endocytic flux in the absence of UVRAG, because inhibiting endocytic uptake or degradation through dominant-negative dynamin expression or RNAi of Rab7, respectively, also leads to intestinal dysplasia. It is worth noting that the effects of inhibiting Shibire/dynamin function led to a much more severe hyperproliferation of ISCs and early death of animals. In line with this, the loss of early endocytic regulators, such as Rab5, in the developing eye causes overproliferation of cells and lethality during metamorphosis. Although eye development is not perturbed by the loss of the late endocytic regulators UVRAG or Rab7, these proteins are clearly important for controlling ISC proliferation and differentiation (Nagy, 2016).

A recent paper shows that hundreds of RNAi lines cause the expansion of the esg-GFP compartment in 1-week-old animals, which might be due to an unspecific ISC stress response in some cases. However, several lines of evidence support that impaired UVRAG-dependent endocytic degradation is specifically required to prevent intestinal dysplasia. First of all, activation of JNK stress signaling in esg-GFP-positive cells induces short-term ISC proliferatio, and almost all stem cells are lost through apoptosis by the 2- to 3-week age, the time when the Uvrag-mutant phenotype becomes obvious. In fact, UVRAG loss resembles an early-onset age-associated dysplasia that is normally observed in old (30-60 days) flies and involves the simultaneous activation of both JNK and STAT signaling. Second, UVRAG RNAi in ISCs and EBs leads to paracrine activation of the cytokine Unpaired3 in enterocytes, one of the hallmarks of niche appropriation by Notch-negative tumors. However, autocrine expression of the Unpaired proteins and JNK activation is observed in Uvrag-knockdown cells, unlike in Notch-negative tumors, and EBs with active Notch signaling accumulate in the absence of UVRAG, so the two phenotypes are clearly different. Third, it is the loss of autophagy that could be expected to mimic a stress response and perhaps induce stem cell tumors, but this does not seem to be the case – ISCs with Atg5 or Atg14 RNAi proliferate less in 3-week-old animals and an overall decrease of the esg-GFP compartment is seen, as opposed to the Uvrag-deletion phenotype (Nagy, 2016).

Taken together, this work indicates that endocytic maturation and degradation serves to prevent early-onset intestinal dysplasia in Drosophila, and its deregulation could be relevant for the development of colorectal cancer in humans (Nagy, 2016).

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. This study analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. These results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease (Fu, 2017).

Scribbled optimizes BMP signaling through its receptor internalization to the Rab5 endosome and promote robust epithelial morphogenesis

Epithelial cells are characterized by apical-basal polarity. Intrinsic factors underlying apical-basal polarity are crucial for tissue homeostasis and have often been identified to be tumor suppressors. Patterning and differentiation of epithelia are key processes of epithelial morphogenesis and are frequently regulated by highly conserved extrinsic factors. However, due to the complexity of morphogenesis, the mechanisms of precise interpretation of signal transduction as well as spatiotemporal control of extrinsic cues during dynamic morphogenesis remain poorly understood. Wing posterior crossvein (PCV) formation in Drosophila serves as a unique model to address how epithelial morphogenesis is regulated by secreted growth factors. Decapentaplegic (Dpp), a conserved bone morphogenetic protein (BMP)-type ligand, is directionally trafficked from longitudinal veins (LVs) into the PCV region for patterning and differentiation. These data reveal that the basolateral determinant Scribbled (Scrib) is required for PCV formation through optimizing BMP signaling. Scrib regulates BMP-type I receptor Thickveins (Tkv) localization at the basolateral region of PCV cells and subsequently facilitates Tkv internalization to Rab5 endosomes, where Tkv is active. BMP signaling also up-regulates scrib transcription in the pupal wing to form a positive feedback loop. These data reveal a unique mechanism in which intrinsic polarity genes and extrinsic cues are coupled to promote robust morphogenesis.

This study shows that the Scrib complex, a basolateral determinant, is a novel feedback component that optimizes BMP signaling in the PCV region of the Drosophila pupal wing (Gui, 2016).

During PCV development, limited amounts of Dpp ligands are provided by the Dpp trafficking mechanism. Furthermore, amounts of receptors appear to be limited since tkv transcription is down-regulated in the cells in which the BMP signal is positive, a mechanism that serves to facilitate ligand diffusion and sustain long-range signaling in the larval wing imaginal disc. To provide robust signal under conditions in which both ligands and receptors are limiting, additional molecular mechanisms are needed. Previous studies suggest that two molecules play such roles. Crossveinless-2 (Cv-2), which is highly expressed in the PCV region, serves to promote BMP signaling through facilitating receptor-ligand binding. Additionally, the RhoGAP protein Crossveinless-c (Cv-c) provides an optimal extracellular environment to maintain ligand trafficking from LVs into PCV through down-regulating integrin distribution at the basal side of epithelia. Importantly, both cv-2 and cv-c are transcriptionally regulated by BMP signaling to form a feedback or feed-forward loop for PCV formation (Gui, 2016).

Scrib, a third component, sustains BMP signaling in the PCV region in a different manner. First, to utilize Tkv efficiently, Scrib regulates Tkv localization at the basolateral region in the PCV cells, where ligand trafficking takes place. Regulation of receptor localization could be a means of spatiotemporal regulation of signaling molecules during the dynamic process of morphogenesis. Second, to optimize the signal transduction after receptor-ligand binding, Scrib facilitates Tkv localization in the Rab5 endosomes. Localization of internalized Tkv is abundant at Rab5 endosomes in the PCV region of wild-type, but not scrib RNAi cells. While the physical interaction between Scrib, Tkv and Rab5 in the pupal wing remains to be addressed, the data in S2 cells suggest that physical interactions between these proteins are key for preferential localization of Tkv at the Rab5 endosomes. Recently, Scrib has been implicated in regulating NMDA receptor localization through an internalization-recycling pathway to sustain neural activity. Therefore, Scrib may be involved in receptor trafficking in a context-specific manner, the molecular mechanisms of which, however, remain to be characterized. Third, BMP/Dpp signaling up-regulates scrib transcription in the pupal wing. Moreover, knockdown of scrib leads to loss of BMP signaling in PCV region but not loss of apical-basal polarity. These facts suggest that upregulation of Scrib is key for optimizing BMP signaling by forming a positive feedback loop (Gui, 2016).

Previous studies indicate that cell competition takes place between scrib clones and the surrounding wild-type tissues in the larval wing imaginal disc. Moreover, cell competition has been documented between loss-of-Dpp signal and the surrounding wild-type tissues. It is presumed that the mechanisms proposed in this study are independent of cell competition for the following reasons. First, scrib RNAi and AP-2μ RNAi data reveal that loss of BMP signal in the PCV region is produced without affecting cell polarity. Thus, cell competition is unlikely to occur in this context. Second, BMP signal is intact in scrib mutant clones of the wing imaginal disc, suggesting that cell competition caused by scrib clones is not a direct cause of loss of BMP signaling in scrib mutant cells (Gui, 2016).

Previous studies established that receptor trafficking plays crucial roles in signal transduction of conserved growth factors, including BMP signaling. Several co-factors have been identified as regulators of BMP receptor trafficking. Some of them down-regulate BMP signaling while others help maintain it. It is proposed that the Scrib-Rab5 system is a flexible module for receptor trafficking and can be utilized for optimizing a signal. During larval wing imaginal disc development, BMP ligands are trafficked through extracellular spaces to form a morphogen gradient. Although previous studies indicate that regulation of receptor trafficking impacts BMP signaling in wing imaginal discs, BMP signaling persists in scrib or dlg1 mutant cells in wing discs. Wing disc cells interpret signaling intensities of a morphogen gradient. In this developmental context, an optimizing mechanism might not be beneficial to the system. In contrast, cells in the PCV region use the system to ensure robust BMP signaling (Gui, 2016).

Taken together, these data reveal that a feedback loop through BMP and Scrib promotes Rab5 endosome-based BMP/Dpp signaling during PCV morphogenesis. Since the components (BMP signaling, the Scrib complex, and Rab5 endosomes) discussed in this work are highly conserved, similar mechanisms may be widely utilized throughout Animalia (Gui, 2016).

Disruption of Drosophila melanogaster lipid metabolism genes causes tissue overgrowth associated with altered developmental signaling

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, mutants were recovered that disrupt genes encoding serine palmitoyltransferase and Acetyl-CoA Carboxylase (ACC). Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EGFR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. This analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation (Sasamura, 2013).

The importance of lipid metabolism for the formation and maintenance of cell membranes is well established. Both serine palmitoyltransferase (SPT) and acetyl-CoA carboxylase (ACC) are critical enzymes that control different steps of lipid metabolism, and are highly conserved in diverse animal species. Genetic elimination of ACC1 or the SPT subunits Sptlc1 or Sptlc2 cause early embryonic lethality in mice, although the cellular basis for this lethality is unknown. In D. melanogaster, RNA-interfering disruption of ACC activity in the fat body results in reduced triglyceride storage and increased glycogen accumulation, and in oenocytes leads to loss of watertightness of the tracheal spiracles causing fluid entry into the respiratory system. This study demonstrates that D. melanogaster mutants lacking functional SPT or ACC exhibit endosomal trafficking defects, causing Notch, Wingless, EGFR, and Patched to accumulate abnormally in endosomes and lysosomes. These effects are accompanied by significant alterations in Notch and Wingless signaling, as revealed by changes in downstream target gene activation for both pathways. However, the mutants do not fully inactivate these developmental signaling pathways, and instead display phenotypes consistent with more complex, pleiotropic effects on Notch, Wingless, and potentially additional pathways in different tissues. These findings reinforce the importance of lipid metabolism for the maintenance of proper developmental signaling, a concept that has also emerged from studies demonstrating that: D. melanogaster mutants for phosphocholine cytidylyltransferase alter endosomal trafficking and signaling of Notch and EGFR; mutants for alpha-1,4-N-acetylgalactosaminyltransferase-1 affect endocytosis and activity of the Notch ligands Delta and Serrate; mutants for the ceramide synthase gene shlank disrupt Wingless endocytic trafficking and signaling, and mutants for the glycosphingolipid metabolism genes egghead and brainiac modify the extracellular gradient of the EGFR ligand Gurken (Sasamura, 2013).

Most strikingly, lace and ACC mutants also display prominent tissue overgrowth phenotypes. These tissue overgrowth effects are linked to changes in Notch and Wingless signaling outputs, and they involve gamma-secretase, Su(H), and Armadillo activities, suggesting that the overgrowth reflects an interplay of Wingless inactivation and Notch hyperactivation. Consistent with the findings, both Notch and Wingless regulate cell proliferation and imaginal disc size in D. melanogaster. Moreover, several observations indicate that Notch and Wingless are jointly regulated by endocytosis, with opposing effects on their respective downstream pathway activities, a dynamic process that might be especially sensitive to perturbations in membrane lipid constituents. Wingless itself exerts opposing effects on disc size that might depend on the particular developmental stage or disc territory. For example, hyperactivation of Wingless or inactivation of its negative regulators cause overproliferation, but Wingless activity can also constrain wing disc growth. Similar spatiotemporal effects might underlie the variability detected in studies with lace and ACC mutant clones, in which both tissue overgrowth and developmentally arrested discs were observed. Although no obvious changes were detected in downstream signaling for several other cell growth pathways that were examined, the trafficking abnormalities seen for other membrane proteins aside from Notch, Delta, and Wingless, as well as the incomplete suppression of the overgrowth phenotypes by blockage of Notch and Wingless signaling, suggest that other pathways might also be dysregulated in lace and ACC mutants, possibly contributing to the observed tissue overgrowth (Sasamura, 2013).

Wingless is modified by lipid addition, and lipoprotein vesicles have been suggested to control Wingless diffusion. In D. melanogaster embryos, endocytosis of Wingless limits its diffusion and ability to act as a long-range morphogen. Endocytosis can also affect Wingless signaling in receiving cells, where endocytosis both promotes signal downregulation and positively facilitates signaling. The apparently normal diffusion ranges for overaccumulated Wingless in lace and ACC mutant clones, yet reduced downstream target gene expression, is consistent with the idea that SPT and ACC act by promoting endocytic trafficking of Wingless in receiving cells rather than influencing the secretion and/or diffusion of Wingless from signal-sending cells (Sasamura, 2013).

The finding that lace and ACC mutant overgrowth phenotypes are also partially Notch-dependent is reminiscent of similar overproliferation phenotypes seen in certain D. melanogaster endocytic mutants, such as vps25, and tsg101. The overproliferation of disc tissue in these mutants is attributable to Notch hyperactivation, reflecting the fact that non-ligand-bound Notch receptors that are normally targeted for recycling or degradation are instead retained and signal from endosomes. Analogous effects are likely to contribute to the lace and ACC mutant overgrowth, where significant Notch overaccumulation was observed throughout the endosomal-lysosomal routing pathway. Some ectopic Notch signaling might emanate from the lysosomal compartment, which is enlarged and accumulates particularly high levels of Notch in lace and ACC mutant clones. Analysis of D. melanogaster HOPS and AP-3 mutants, which affect protein delivery to lysosomes, has identified a lysosomal pool of Notch that is able to signal in a ligand-independent, gamma-secretase-dependent manner (Sasamura, 2013).

How do SPT and ACC contribute to endosomal trafficking of Notch and other proteins? In the yeast SPT mutant lcb1, an early step of endocytosis is impaired due to defective actin attachment to endosomes, a phenotype that is suppressed by addition of sphingoid base. However, the trafficking abnormalities seen in lace and ACC mutants do not resemble those in the yeast lcb1 mutant, perhaps because endocytic vesicle fission is primarily dependent upon dynamin in D. melanogaster and mammals, instead of actin as in yeast. Nevertheless, the requirement for SPT and ACC in D. melanogaster endosomal compartments might reflect possible functions in endosome-cytoskeleton interactions. Another possibility is that the defective endosomal trafficking seen in lace and ACC mutants is caused by the inability to synthesize specific phospholipids needed for normal membrane homeostasis. Finally, lace and ACC might be important for the formation and/or function of lipid rafts, specialized membrane microdomains that have been implicated in both signaling and protein trafficking (Sasamura, 2013).

A remarkable feature of the lace and ACC mutant phenotypes that suggests an underlying defect in lipid biogenesis is the non-autonomous effect in mutant tissue clones, wherein nearby wildtype cells generate a secreted activity that diffuses several cell diameters into the mutant tissue and rescues the trafficking and signaling defects. One possibility is that these secreted activities are diffusible lipid biosynthetic products of SPT and ACC, which enter the mutant cells and serve as precursors for further biosynthetic steps that do not require SPT or ACC. An intriguing alternative is that the SPT and ACC enzymes are themselves secreted and taken up by the mutant cells. A precedent for this mechanism has recently been demonstrated for D. melanogaster ceramidase, a sphingolipid metabolic enzyme that is secreted extracellularly, delivered to photoreceptors, and internalized by endocytosis to regulate photoreceptor cell membrane turnover (Sasamura, 2013).

Recent work has highlighted the importance of lipid metabolism for oncogenic transformation, and ACC has been advanced as a promising target for cancer drug development. ACC is upregulated in some cancers, possibly as a result of high demands for lipid biosynthesis during rapid cell divisions. Sphingolipids and their derivatives are also thought to influence the balance of apoptosis and cell proliferation during tissue growth, and thus have also garnered attention as potential cancer therapy targets. The current findings regarding the requirements of SPT and ACC for proper trafficking and signaling of key developmental cell-surface signaling molecules, including Notch and Wingless, provide insights into how lipid metabolic enzymes might influence cell proliferation and tissue patterning in multicellular animals. Complex lipid biosynthesis is essential for the creation of the elaborate, interconnected, and highly specialized membrane compartments in which developmental pathways operate, and perturbations in lipid biosynthesis that are tolerated by the cell might nevertheless exert significant pleiotropic effects on developmental patterning, cell proliferation, and other cellular processes. Exploration of lipid metabolic enzymes as pharmacological targets must therefore take into account potentially unfavorable effects on critical signaling pathways controlling development and organogenesis (Sasamura, 2013).

Distinct functional units of the Golgi complex in Drosophila cells

A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. This study shows that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disc cells are functionally diverse. The UDP-sugar transporter Fringe-connection (Frc) is localized to a subset of the Golgi units distinct from those harboring Sulfateless (Sfl), which modifies glucosaminoglycans (GAGs), and from those harboring the protease Rhomboid (Rho), which processes the glycoprotein Spitz (Spi). Whereas the glycosylation and function of Notch are affected in imaginal discs of frc mutants, those of Spi and of GAG core proteins are not, even though Frc transports a broad range of glycosylation substrates, suggesting that Golgi units containing Frc and those containing Sfl or Rho are functionally separable. Distinct Golgi units containing Frc and Rho in embryos could also be separated biochemically by immunoisolation techniques. Tn-antigen glycan is localized only in a subset of the Golgi units distributed basally in a polarized cell. It is proposed that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification (Yano, 2005).

The pattern of glycosylation is extremely diverse, yet is highly specific to each protein. How can this specificity (and diversity) be achieved? There are >300 glycosylenzymes in humans and >100 in Drosophila, but is their enzymatic specificity sufficient to explain the precise modification of all substrates? One possible mechanism that might also contribute to the specific (and diverse) pattern of glycosylation would be the localization/compartmentalization of glycosylenzymes (Yano, 2005).

The Golgi complex, where protein glycosylation takes place, has been regarded as a single functional unit, consisting of cis-, medial-, and transcisternae in mammalian cells. However, the three-dimensional reconstruction of electron microscopic images of the mammalian Golgi structure has suggested the existence of more than one Golgi stack, with the individual stacks being connected into a ribbon by tubules bridging equivalent cisternae. Furthermore, during mitosis, the Golgi cisternae of mammalian cells become fragmented without their disassembly. In Drosophila, Golgi cisternae are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase, although there has been no evidence to date to indicate functional differences among the Golgi fragments (Yano, 2005).

The Golgi complex is a stack of cis-, medial-, and transcisternae in mammalian cells. In contrast, Golgi markers often do not overlap with each other in Saccharomyces cerevisiae, in which the Golgi cisternae are not stacked but disassembled. The Golgi cisternae of Drosophila are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase. To determine whether Drosophila imaginal disc cells have assembled or disassembled Golgi cisternae, the localizations were compared of the cis-cisternal marker dGM130, the transcisternal marker Syntaxin16 (Syx16), and the Golgi-tethered 120-kDa protein, which is commonly used to detect the Golgi complex in Drosophila. The 120-kDa protein was identified by immunoaffinity purification and protein sequencing as a Drosophila homolog of the vertebrate 160-kDa medial Golgi sialoglycoprotein (MG160), which resides uniformly in the medial-cisternae of the Golgi apparatus in vertebrate cells. An antibody specific for the 120-kDa protein also stained numerous Golgi fragments in imaginal disc cells. More than 80% of immunoreactivity for the 120-kDa protein was colocalized with both dGM130 and Syx16, suggesting that 120-kDa protein-positive fragments of the Golgi complex indeed comprise assembled cisternae; these fragments are referred to as 'Golgi units.' The distributions of the 120-kDa protein, dGM130, and peanut agglutinin (PNA), another transcisternal marker, also show that the markers are closely apposed but not identical, suggesting that the Golgi units are polarized. Interestingly, most of the PNA-positive transcisternae are oriented toward the basal side of the cell, within the Golgi complex, whereas most of the GM130-positive cis-cisternae are oriented toward the apical side of the cell. The cis-to-trans polarity of each Golgi unit thus appears to be correlated with the apico-basal polarity of the disc cells (Yano, 2005).

Drosophila mutant larvae defective in the UDP-sugar transporter Frc manifest a highly selective phenotype: the lack of Notch glycosylation in the presence of normal GAG synthesis (Goto, 2001). This limited phenotype is unexpected, given that Frc exhibits a broad specificity for UDP sugars used in the synthesis of various glycans including N-linked types, GAGs, and mucin types. However, given that the frcR29 allele studied previously (Goto, 2001) is hypomorphic, whether the selective glycosylation defect might be a consequence of partial loss of Frc activity was examined. With the use of imprecise excision, a new allele was generated, frcRY34, the presence of which results in the death of most larvae during the second-instar stage, much earlier than the death induced by frcR29. Real-time PCR analysis revealed that the amount of frc transcripts in the second-instar larvae of frcRY34 or frcR29 mutants was 4.2% and 24.4% of that in the wild type, respectively. About 1 kb of the gene, including the transcription initiation site, was deleted in the frcRY34 allele. Together, these observations suggest that frcRY34 is essentially a null allele (Yano, 2005).

Clonal cells of the frcRY34 mutant exhibited normal levels of GAGs, as detected by immunostaining with the 3G10 antibody, whereas the amount of GAGs was reduced in clones of tout-velu (ttv) mutant cells. Given that GAGs are required for signaling by Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp), the expression of corresponding target genes [patched (ptc) for Hh signaling and Dll for Wg and Dpp signaling] was examined in the wing discs of the frcRY34 mutant. Expression of ptc and that of Dll in the ventral compartment of the wing discs were unaffected in the mutant clones, suggestive of normal GAG function (Yano, 2005).

Given that Notch glycosylation by Fringe (FNG), a fucose-specific beta1,3-N-acetylglucosaminyltransferase, requires Frc activity, Notch glycosylation was examined in the frcRY34 mutant. The frcRY34 mutant clones in the dorsal compartment, but not those in the ventral compartment, of the wing discs induced wg expression at their borders, suggesting that Notch glycosylation is impaired in the frcRY34 mutant. The ectopic expression of Wg induced by the frcRY34 mutant clones is likely responsible for the observed induction of Dll expression in the dorsal compartment (Yano, 2005).

To determine why the loss of a UDP-sugar transporter with a broad specificity selectively affects Notch glycosylation, the subcellular localization of Frc was investigated. Frc tagged with the Myc epitope was expressed in imaginal discs under the control of the arm-Gal4 driver. The Gal4-induced expression of Frc-Myc rescues the frc mutant phenotype, suggesting that Frc-Myc is functional and properly localized. Immunostaining of imaginal discs of wild-type larvae expressing Frc-Myc with antibodies to Myc and to the 120-kDa protein revealed that Frc is localized to only a small subset of Golgi units. This differential immunostaining of different Golgi units is not likely to be due to differential penetration of the antibodies or cripticity of the epitopes. The penetration of antibodies would not vary within the cell, because the Golgi units were distributed evenly throughout the cell, not in a biased manner. Moreover, it is unlikely that degradation of the epitopes during the immunostaining experiments due to contaminating proteases might alter the cripticity of the epitopes in different Golgi units, since the percentage of different Golgi units among the anti-120-kDa-positive Golgi units was statistically constant in several independent experiments. Thus, it is hypothesized that the Golgi units might be functionally heterogeneous, and that those containing Frc might modify some proteins, including Notch, but not others (Yano, 2005).

To test this hypothesis, the localizations of various molecules involved in protein modification in the Golgi complex were compared with that of Frc. It was found that Sfl is also restricted to a subset of Golgi units, but that its distribution does not overlap with that of Frc. This differential localization of Sfl and Frc might thus explain the observation that frc mutant clones in wing discs do not show any defect in GAG synthesis by Sfl (Yano, 2005).

The Spi-processing enzyme Rho is also localized to a subset of Golgi units distinct from those containing Frc, in addition to its presence in other compartments. This result indicates the existence of at least two types of Golgi units, those containing Rho and those containing Frc. To determine whether these two types of Golgi units differ functionally, the glycosylation state and function of Spi was examined in frc mutants (Yano, 2005).

Given that the extent of Notch glycosylation, as detected by wheat germ agglutinin (WGA), is markedly reduced in frc mutants compared with that in the wild-type background, whether the WGA-reactive glycan of Spi is also affected by frc mutation was also examined. Myc epitope-tagged Spi was expressed in the wild type or the frcRY34 mutant. Spi-Myc was then precipitated from larval homogenates with antibodies to Myc and was examined for its glycosylation by SDS/PAGE and subsequent blot analysis with WGA. The reactivity of the Spi glycan with WGA was similar in the frc mutant and in the wild type. Whether the frcRY34 mutation affects the Spi glycan was examined by mobility shift analysis. The electrophoretic mobility of glycosylated Spi from the wild type is similar to that from the frc mutant. Deglycosylation of Spi by neuraminidase, peptide-N-glycosidase (PNGase) F, and O-glycanases also increases its mobility to the same extent in wild-type and frc mutant larvae, suggesting that the core protein is not affected by the frc mutation. Together, these results indicate that the function of Frc is not necessary for formation of the Spi glycan. It is also concluded that the function of the Rho-Spi pathway is not affected by frc mutation (Yano, 2005).

To confirm that the Golgi units containing Frc and those containing Rho are distinct, whether these Golgi units could be selectively isolated by using antibodies to Myc (for Myc-tagged Frc) or HA (for HA-tagged Rho) was examined. Because it was very difficult to collect enough of the imaginal discs, the starting material was switched to embryos, and whether Frc and Rho localize to distinct Golgi units was examined in embryos. Frc-Myc and Rho-HA were coexpressed in the embryos by the arm-Gal4 driver, and immunostaining with antibodies to Myc and to HA revealed that the Golgi units containing Frc-Myc (45.4% of total Golgi units) and those containing Rho-HA (43.0% of total Golgi units) are largely distinct: only 11.6% of total Golgi units were positive for both Frc-Myc and Rho-HA. Immunoisolation was attempted from embryonic lysates by using either antibody to Myc or HA, and how much Frc-Myc and Rho-HA were coisolated in each immunoisolate was examined. When Frc-Myc was immunoisolated with an antibody to Myc, the recovery of Frc-Myc was 5.7 times greater than that of Rho-HA. Moreover, when Rho-HA was immunoisolated with an antibody to HA, the recovery of Rho-HA was 18.3 times greater than that of Frc-Myc. The immunoblot analysis of these immunoisolates with the anti-120-kDa antibody confirmed that the Golgi units were concentrated in these immunoisolates. These results support the notion that Frc-Myc-containing fraction is distinct and could be separated from Rho-HA-containing fraction (Yano, 2005). Whether these distinct Golgi units contain different constituents was examined. Fringe (Fng) is one of the candidate molecules that may be colocalized with Frc. Therefore, expression of ectopically expressed Fng was examined in Rho- and Frc-containing immunoisolates. It was found that expression of Fng in Frc-containing immunoisolates was 26 times greater than in Rho-containing immunoisolates, supporting the idea that Fng is localized in the Frc-positive Golgi units rather than the Rho-positive Golgi units. Immunostaining analysis confirmed that FNG was colocalized mostly with Frc (88.1% of the FNG-positive Golgi units), but not with Rho (16.6% of the FNG-positive Golgi units), by immunostaining analysis (Yano, 2005).

The data suggest that different Golgi units perform different functions, a notion that is also supported by the observation that Tn antigen (O-linked N-acetylgalactosamine) was detected in only a subset of Golgi units in imaginal eye disc cells. In addition, most of these Tn antigen-positive Golgi units were found to be distributed in the basal region of the disc cells, suggesting that the differential distribution of Golgi units might contribute to the apicobasal polarity of glycan distribution (Yano, 2005).

In contrast to the larval stage, Frc is required for GAG synthesis at the early embryonic stage. To determine why the Frc requirement for GAG synthesis differs between the embryonic and larval stages, embryos were stained expressing Frc-Myc with antibodies to Sfl and to Myc. Sfl was found to be colocalized with Frc, likely explaining the importance of Frc for GAG synthesis at the embryonic stage. In addition, this embryonic requirement of Frc for GAG synthesis excludes the possibility that the selective defects in Notch and not in GAG synthesis observed in frc mutant larvae are caused by the selective Frc-dependent transport of a subset of UDP-sugars used only for glycosylation of Notch but not for GAGs synthesis (Yano, 2005).

These results provide evidence for the existence of functionally distinct Golgi units in Drosophila cells. Such functional heterogeneity of Golgi units is likely responsible for the diversity of protein glycosylation. At least two types of Golgi units containing either Frc or Sfl were shown to be present in larval disc cells. Two distinct sets of proteins, exemplified by Notch and GAG core proteins, might thus be selectively transported to Frc- or Sfl-containing Golgi units, respectively, where they undergo glycosylation by different sets of molecules (Yano, 2005).

The variety of Golgi units might be established by separate transport of secretory proteins and glycosylenzymes from the endoplasmic reticulum (ER) to the distinct Golgi units. In yeast, glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in vesicles distinct from those containing other secretory protein. Given that the GAG core protein Dally in Drosophila is anchored to the membrane by GPI, it is possible that Dally and Notch are loaded into distinct vesicles as they exit the ER (Yano, 2005).

Combinations of glycosylenzymes and transporters, such as Sfl and Frc, contained in Golgi units of Drosophila differ not only between embryos and larval disc cells but also among cell types. For example, it was found that Frc is localized to all Golgi units in salivary gland cells at the larval stage. It has also been shown that all of the Golgi complexes dispersed in oocytes may have the ability to process the Gurken precursor protein, which is usually cleaved in a subset of the Golgi complexes residing in the dorso-anterior region. The Golgi units may thus be altered in a manner dependent on development, cell type, and signaling processes (Yano, 2005).

The functional diversity of Golgi units also might contribute to the polarized distribution of glycans along the apicobasal axis of cells. It was found that Tn antigen is synthesized in the basal Golgi units of larval disc cells. Furthermore, certain types of glycans are distributed along the apicobasal axis of pupal ommatidia. These glycans might thus be synthesized differentially in the Golgi units that are asymmetrically distributed along the apicobasal axis and then be secreted at either the apical or basal cell surface (Yano, 2005).

Whereas Golgi units are dispersed throughout Drosophila cells, the Golgi complex in mammalian cells is thought to be a single entity that is located in the pericentriolar region through its association with the microtubule-organizing center in interphase and which is fragmented at the onset of mitosis. The Golgi fragments apparent in mammalian cells during mitosis are highly similar to the Golgi units of Drosophila cells in both electron and confocal microscopic images. The mammalian Golgi complex during interphase may therefore be comprised of functionally distinct units that are associated with the microtubule-organizing center and connected with each other (Yano, 2005).

The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE

During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. This study shows that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis. Pairing requires an intact actin cytoskeleton which in turn depends on Abi/Scar but not WASP. This actin-dependent pairing is not limited to flies but also occurs in mammalian cells. It is further shown that preventing the Golgi stack separation at G2 blocks entry into mitosis, suggesting that this paired organization is part of the mitotic checkpoint, similar to what has been proposed in mammalian cells (Kondylis, 2007).

During the cell cycle, the Golgi, like other organelles, has to duplicate in mass and/or number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. The process of Golgi duplication and inheritance in mammalian cells is still debated, as different modes of Golgi biogenesis have been proposed. One reason why this issue is not yet settled could be due to the elaborate organization of the Golgi stacks, which are interconnected to form a single-copy organelle capping the nucleus, thus impeding clear visualization of organelle duplication and segregation. Therefore, this study has exploited the relatively small number of Golgi stacks in Drosophila tissue-cultured S2 cells to revisit this issue (Kondylis, 2007).

In S2 cells, the Golgi stacks are found in close proximity to transitional endoplasmic reticulum (tER) sites, forming tER-Golgi units (Kondylis, 2003; Herpers, 2004). Their number nearly doubles at G2 phase. In an effort to identify factors mediating this process, focus was placed on cytoskeletal elements that have been involved in the organization of the mammalian Golgi apparatus. Microtubules are involved in mammalian Golgi ribbon maintenance, as their depolymerization leads to its reorganization into individual Golgi stacks in close proximity to ER exit sites (Kondylis, 2007 and references therein).

F-actin has also been implicated in the organization of the mammalian Golgi apparatus; its depolymerization leads to a compact appearance of this organelle without disruption of cisternal stacking. A key regulator of actin polymerization is the Arp2/3 complex. Its F-actin nucleation activity is triggered both by Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous (WAVE/Scar) proteins, which are in turn regulated by Rho small GTPases. WASP exists in an autoinhibited state that is released by the cooperative action of Cdc42, PI(4,5)P, and other SH3-containing proteins. In contrast, WAVE/Scar proteins, together with Sra-1, Kette (Nap1), Abi, and HSPC300, form a stable complex, which is itself regulated by Rac (Kondylis, 2007 and references therein).

Rho GTPases have recently been implicated in maintaining Golgi architecture. Cdc42 has been localized on the Golgi membrane and shown to recruit the Arp2/3 complex around this organelle via ARHGAP10. Furthermore, suppression of the brain-specific Rho-binding protein Citron-N in neurons was shown to lead to fragmentation of the Golgi apparatus, and Rho1 was proposed to exert its local effect on F-actin by regulating ROCK and profilin activity (Kondylis, 2007 and references therin).

This study shows that drug-induced F-actin depolymerization in S2 cells leads to doubling of the number of tER-Golgi units independent of anterograde transport. Using live cell imaging, electron microscopy, and three-dimensional (3D) electron tomography, this study shows that each Golgi is organized as a pair of stacks held together by an actin-based mechanism, both in Drosophila and in human cells. In S2 cells, this is mediated by Abi and Scar, suggesting a novel role for the Rac signaling cascade in Golgi architecture. Last, it was shown that the Golgi stacks undergo separation at G2 through modulation of Abi and Scar, and that blocking this separation prevents cells from entering mitosis, supporting the existence of a G2/M checkpoint related to Golgi structural organization (Kondylis, 2007).

The two Golgi stacks could be physically linked without displaying membrane continuity or being interconnected, for instance through intercisternal tubular connections, either permanent or transient. Tubules connecting cisternae of adjacent stacks are involved in the formation of the Golgi ribbon in mammalian cells and, recently, GM130 and GRASP65 have been proposed to be required for their integrity. However, the putative tubules connecting the two stacks in the pair would have different molecular requirements, at least in Drosophila, since depletion of dGM130 or dGRASP does not lead to their separation (Kondylis, 2003; Kondylis, 2005; Kondylis, 2007 and references therein).

F-actin could provide a physical link holding the paired Golgi stacks together, or it could help in the formation/maintenance of intercisternal tubules. In addition, short actin filaments have been proposed to link spectrin mosaics leading to the formation of a skeleton that surrounds the Golgi complex. One of its functions could be to hold the two Golgi stacks close enough to allow the formation and fusion of the tubules. It could also surround the tubules themselves, thus providing membrane stability. The localization of Abi and Scar at the periphery of the tER-Golgi units and between the two stacks in a pair is consistent with both proposed functions. These tomography studies so far have not revealed clear membrane continuities between Golgi cisternae, though examples have been found of a tubular network which is shared by the paired stacks (Kondylis, 2007).

tER sites behave similarly to the Golgi, as they also separate at G2 and upon F-actin depolymerization. Because little is known about the mechanism regulating the biogenesis of tER sites, it is difficult to envisage how the two parts could be held together. The spectrin-actin mesh described above could be common to Golgi and tER sites, and Golgi and tER site scission could be achieved in a synchronized fashion. Alternatively, either of these organelles could split first and lead to the scission of the other, perhaps by providing positional information. Recently, the centrosome component centrin 2 that is also localized to tER sites in Trypanosoma has been shown to give such positioning information. A more in-depth study combining immunogold labeling and 3D tomography would be required to elucidate such fine details of tER-Golgi structural organization (Kondylis, 2007).

Drosophila Rho1 is unlikely to have a role in holding the two Golgi stacks together. The overexpression of the Rho1 constitutively inactive mutant or treatment of S2 cells with ROCK or myosin light chain inhibitors (Y27632 and blebbistatin) did not affect the Golgi number. Cdc42 is also unlikely to participate as the depletion of its downstream effector WASP did not lead to Golgi separation, although the overexpression of the Cdc42T17N dominant negative did. However, this effect could be due to nonspecific sequestration of the guanine nucleotide exchange factor involved in maintaining the paired Golgi stacks and may be shared with other small GTPases (Kondylis, 2007).

Interestingly, the results are consistent with a role for Rac GTPases in Drosophila Golgi architecture. Expression of the constitutively inactive form of Rac1 led to a near-doubling in the Golgi number, and depletion of Scar/WAVE or Abi, which are regulated by Rac GTPases, led to a similar phenotype. The identical results obtained in Scar and Abi RNAi suggest that this well-established Scar/WAVE pentameric complex is involved in holding the paired Golgi stacks together by promoting F-actin polymerization. These data indicate that the Rac signaling pathway is involved. However, the Scar/Abi complex has recently been shown to also stimulate Arp2/3 and F-actin polymerization independently of Rac. This would need to be investigated further (Kondylis, 2007).

This study shows that the separation of the paired Golgi stacks occurs at G2, prior to mitosis. A similar phenomenon has already been reported during cell division in Toxoplasma gondii, where a single Golgi stack grows as a duplicated organelle and is separated as the cell divides. However, the mechanism underlying this separation is not known (Kondylis, 2007).

The Golgi doubling in number at G2 phase resembles many aspects of this observed upon F-actin depolymerization. In both cases, a similar increase in Golgi number and decrease in their size are observed. Furthermore, this study has shown that it is the modulation of the F-actin cytoskeleton and the activity of Abi/Scar at G2 that lead to Golgi stack separation. (1) It was found that both Scar and Abi localized to the Golgi, strongly arguing for having a role in actin remodeling around this organelle. (2) The Golgi stacks in G2 cells remain insensitive to F-actin depolymerization. (3) Cells depleted of Abi and Scar that exhibit separated Golgi stacks do not split them further at G2. (4) The overexpression of Abi prevents Golgi separation at G2. This strongly suggests that the F-actin/Abi/Scar-mediated link of the two stacks has been severed in a G2-specific manner, perhaps by kinases such as Polo (Kondylis, 2007).

Because tER sites and the Golgi apparatus ultimately disperse later in mitosis, both in mammalian and Drosophila S2 cells, the Golgi stack separation prior to dispersion might be part of the proposed Golgi G2/M checkpoint. Indeed, reagents that interfere with the GRASP65/55 phosphorylation by Polo and ERK/MEK, respectively, arrest or delay the cell cycle at the G2/M transition. This study shows that blocking Golgi separation at G2 by overexpressing Abi also prevents S2 cells from entering mitosis. This strengthens the relationship between Golgi organization and mitotic entry, although it cannot formally be excluded that the mitotic block observed is partly due to additional effects of Abi overexpression, for instance at the plasma membrane (Kondylis, 2007).

It is proposed that at G2, the paired stacks are separated along with the adjacent tER sites. As the cell enters mitosis, the Golgi membrane and the tER sites disperse, and are segregated into the two daughter cells, where the tER-Golgi units are rebuilt. The Golgi could be rebuilt as a very small paired stack in close association with Scar, Abi, and F-actin, or as a single stack that will duplicate by a mechanism that still needs to be unraveled. Since G1 cells are all sensitive to F-actin depolymerization, this suggests that the formation of the paired Golgi stack starts just after the exit from mitosis and persists until S phase, when the Golgi seems to grow significantly. A more detailed understanding will come from EM study of S and G2 cells (Kondylis, 2007).

One of the remaining questions regards the impact of the Abi/Scar role on Golgi organization during development. Using Scar/WAVE, Abi, Kette, and Sra-1 mutants, as well as transgenic flies carrying inducible RNAi constructs, it will be possible to assess whether any of the observed phenotypes (defects in oogenesis, cell and organ morphology, neuroanatomical malformations, and failure in cell migration) is in part due to defects in Golgi organization (Kondylis, 2007).

Golgi outposts shape dendrite morphology by functioning as sites of acentrosomal microtubule nucleation in neurons

Microtubule nucleation is essential for proper establishment and maintenance of axons and dendrites. Centrosomes, the primary site of nucleation in most cells, lose their function as microtubule organizing centers during neuronal development. How neurons generate acentrosomal microtubules remains unclear. Drosophila dendritic arborization (da) neurons lack centrosomes and therefore provide a model system to study acentrosomal microtubule nucleation. This study investigated the origin of microtubules within the elaborate dendritic arbor of class IV da neurons. Using a combination of in vivo and in vitro techniques, it was found that Golgi outposts can directly nucleate microtubules throughout the arbor. This acentrosomal nucleation requires gamma-tubulin and CP309, the Drosophila homolog of AKAP450, and contributes to the complex microtubule organization within the arbor and dendrite branch growth and stability. Together, these results identify a direct mechanism for acentrosomal microtubule nucleation within neurons and reveal a function for Golgi outposts in this process (Ori-McKenney, 2012).

Microtubules are organized into dynamic arrays that serve as tracks for directed vesicular transport and are essential for the proper establishment and maintenance of neuronal architecture. The organization and nucleation of microtubules must be highly regulated in order to generate and maintain such complex arrays. Nucleating complexes, in particular, are necessary because spontaneous nucleation of new tubulin polymers is kinetically limiting both in vivo and in vitro. Gamma(Γ)-tubulin is a core component of microtubule organization centers and has a well-established role in nucleating spindle and cytoplasmic microtubules. Previous studies have proposed that in mammalian neurons, microtubules are nucleated by γ-tubulin at the centrosome, released by microtubule severing proteins, and then transported into developing neurites by motor protein. Indeed, injection of antibodies against γ-tubulin or severing proteins inhibited axon outgrowth in neurons cultured for one day in vitro (DIV1) (Ori-McKenney, 2012).

However, proper neuron development and maintenance may not rely entirely on centrosomal sites of microtubule nucleation. Although the centrosome is the primary site of microtubule nucleation at DIV2, it loses its function as a microtubule-organizing center during neuronal development. In mature cultured mammalian neurons (DIV 11-12), γ-tubulin is depleted from the centrosome, and the majority of microtubules emanate from acentrosomal sites. In Drosophila dsas-4 mutants that lack centrioles, organization of eye-disc neurons and axon outgrowth are normal in third-instar larvae. Within the Drosophila peripheral nervous system (PNS), although dendritic arborization neurons contain centrioles, they do not form functional centrosomes, and laser ablation of the centrioles does not perturb microtubule growth or orientation (Nguyen, 2011). These results indicate that acentrosomal generation of microtubules contributes to axon development and neuronal polarity. How and where acentrosomal microtubule nucleation may contribute to the formation and maintenance of the more complex dendrites, and what factors are involved in this nucleation is unknown. Dendritic arborization (da) neurons provide an excellent system for investigating these questions. They are a subtype of multipolar neurons in the PNS of Drosophila melanogaster which produce complex dendritic arrays and do not contain centrosomes. Based on their patterns of dendrite projections, the da neurons have been grouped into four classes (I-IV) with branch complexity and arbor size increasing with class number. Class IV da neurons are ideal for studying acentrosomal microtubule nucleation because they have the most elaborate and dynamic dendritic arbor, raising intriguing questions about the modes of nucleation for its growth and maintenance (Ori-McKenney, 2012).

One potential site of acentrosomal microtubule nucleation within these neurons is the Golgi complex. A number of studies have shown that the Golgi complex can nucleate microtubules in fibroblasts. Although, in these cell types, the Golgi is tightly coupled to the centrosome, it does not require the centrosome for nucleation. It does, however, require γ-tubulin, the centrosomal protein AKAP450, and the microtubule binding proteins CLASPs. When the Golgi is fragmented upon treatment with nocodazole, the dispersed Golgi ministacks can still promote microtubule nucleation, indicating that these individual ministacks contain the necessary machinery for nucleation (Ori-McKenney, 2012 and references therein).

In both mammalian and Drosophila neurons, the Golgi complex exists as Golgi stacks located within the soma and Golgi outposts located within the dendrites. In cultured mammalian hippocampal neurons, these Golgi outposts are predominantly localized in a subset of the primary branches; however, in Drosophila class IV da neurons, the Golgi outposts appear throughout the dendritic arbor, including within the terminal branches (Ye, 2007). The Golgi outposts may provide membrane for a growing dendrite branch, as the dynamics of smaller Golgi outposts are highly correlated with dendrite branching and extension. However, the majority of larger Golgi outposts remains stationary at dendrite branchpoints and could have additional roles beyond membrane supply. It is unknown whether Drosophila Golgi outposts contain nucleation machinery similar to mammalian Golgi stacks. Such machinery could conceivably support microtubule nucleation within the complex and dynamic dendritic arbor. This study identifies a direct mechanism for acentrosomal microtubule nucleation within the dendritic arbor and reveal a role for Golgi outposts in this process. Golgi outposts contain both γ-tubulin and CP309, the Drosophila homolog of AKAP450, both of which are necessary for Golgi outpost-mediated microtubule nucleation. This type of acentrosomal nucleation contributes not only to the generation of microtubules at remote terminal branches, but also to the complex organization of microtubules within all branches of the dendritic arbor. Golgi outposts are therefore important centers of acentrosomal microtubule nucleation, which is necessary to establish and maintain the complexity of the class IV da neuronal arbor (Ori-McKenney, 2012).

This study has addressed how microtubules are organized and nucleated within the complex arbor of class IV da neurons and how essential these processes are for dendrite growth and stability. Microtubule organization within different subsets of branches in da neurons must require many levels of regulation. This study has identified the first direct mechanism for acentrosomal microtubule nucleation within these complex neurons and has uncovered a role for Golgi outposts in this process. The data are consistent with the observation that pericentriolar material is redistributed to the dendrites in mammalian neurons (Ferreira, 1993) and that γ-tubulin is depleted from the centrosome in mature mammalian neurons (Stiess, 2010). This suggests that the Golgi outposts may be one structure involved in the transport of centriole proteins such as γ-tubulin and CP309. This study found that microtubule nucleation from these Golgi outposts correlates with the extension and stability of terminal branches, which is consistent with the observation that EB3 comet entry into dendritic spines accompanies spine enlargement in mammalian neurons (Jaworski, 2009). It is striking that microtubule organization in shorter branches, but not primary branches, mimics the organization in mammalian dendrites, with a mixed microtubule polarity in the secondary branches and a uniform plus end distal polarity in the terminal branches. Kinesin-2 and certain +TIPS are necessary for uniform minus end distal microtubule polarity in the primary dendrites of da neurons. Golgi outpost mediated microtubule nucleation could also contribute to establishing or maintaining this polarity both in the terminal branches and in the primary branches. It will be of interest to identify other factors that may be involved in organizing microtubules in different subsets of branches in the future (Ori-McKenney, 2012).

In vivo and in vitro data support a role for Golgi outposts in nucleating microtubules at specific sites within terminal and primary branches. However, it is noted that not all EB1 comets originate from Golgi outposts, indicating other possible mechanisms of generating microtubules. One potentially important source of microtubules is the severing of existing microtubules by such enzymes as katanin and spastin, both of which are necessary for proper neuronal development. It is likely that both microtubule nucleation and microtubule severing contribute to the formation of new microtubules within the dendritic arbor; however, the current studies suggest that Golgi-mediated nucleation is especially important for the growth and maintenance of the terminal arbor. In γ-tubulin and CP309 mutant neurons, the primary branches contain a similar number of EB1 comets, but only a small fraction of the terminal branches still contain EB1 comets. This result indicates that severing activity or other sources of nucleation may suffice for microtubule generation within the primary branches, but γ-tubulin mediated nucleation is crucial in the terminal branches. As a result, the terminal branch arbor is dramatically reduced by mutations compromising the γ-tubulin nucleation activity at Golgi outposts (Ori-McKenney, 2012).

It is important to note that Golgi outposts are present in the dendrites, but not in the axons of da neurons; thus, this mode of nucleation is dendrite specific and likely contributes to the difference in microtubule arrays in axons and dendrites. While the axon is one long primary branch with uniform microtubule polarity, the dendrite arbor is an intricate array of branches where microtubule polarity depends on branch length. Therefore, this more elaborate branched structure may have evolved a variety of nucleation mechanisms, including Golgi outpost nucleation and microtubule severing. Intriguingly, in da neurons lacking cytoplasmic dynein function, the Golgi outposts are mislocalized to the axon, which appears branched and contains microtubules of mixed polarity (Zheng, 2008). It is speculated that in these mutants, Golgi-mediated microtubule nucleation within the axon is contributing to the mixed microtubule orientation and formation of ectopic dendrite-like branches (Ori-McKenney, 2012).

Only a subpopulation of Golgi outposts could support microtubule nucleation both in vivo and in vitro. The results show that Golgi outpost mediated microtubule nucleation is restricted to stationary outposts and dependent upon γ-tubulin and CP309, but why some outposts contain these proteins while others do not is unknown. γ-tubulin and CP309 could be recruited to the Golgi outposts in the cell body and transported on the structure into the dendrites, or they could be recruited locally from soluble pools throughout the dendritic arbor. Golgi outposts are small enough to be trafficked into terminal branches that are 150-300 nm in diameter, and therefore may provide an excellent vehicle for transporting nucleation machinery to these remote areas of the arbor. It will be interesting to determine how these nucleation factors are recruited to the Golgi outposts (Ori-McKenney, 2012).

It has been previously shown that GM130 can recruit AKAP450 to the Golgi complex, but whether the first coiled-coil domain of the Drosophila AKAP450 homolog, CP309, can also bind GM130 is unknown. Interestingly, this study has observed that predominantly stationary Golgi outposts correlated with EB1 comet formation, indicating that this specific subpopulation may contain γ-tubulin and CP309. What other factors may be necessary to properly position the Golgi outposts at sites such as branchpoints, and how this is achieved will be a fascinating direction for future studies (Ori-McKenney, 2012).

Whether the acentrosomal microtubule nucleation uncovered in this study also occurs in the dendrites of mammalian neurons is a question of great interest. Golgi outpost distribution in cultured hippocampal neurons is significantly different than that in da neurons, and hippocampal neurons do not form as elaborate arbors as da neurons. However, other types of mammalian neurons form much more complex dendritic arbors and may conceivably require acentrosomal nucleation for the growth and perpetuation of the dendrite branches (Ori-McKenney, 2012).

This study provides the first evidence that Golgi outposts can nucleate microtubules at acentrosomal sites in neurons, shedding new light on the longstanding question about the origin of the microtubule polymer in elongated neuronal processes. This source of nucleation contributes to the complex organization of microtubules within all branches of the neuron, but is specifically necessary for terminal branch development. It is thus conclude that acentrosomal microtubule nucleation is essential for dendritic branch growth and overall arbor maintenance of class IV da neurons, and that Golgi outposts are important nucleation centers within the dendritic arbor (Ori-McKenney, 2012).

GPI biosynthesis is essential for rhodopsin sorting at the trans-Golgi network in Drosophila photoreceptors

Sorting of integral membrane proteins plays crucial roles in establishing and maintaining the polarized structures of epithelial cells and neurons. However, little is known about the sorting mechanisms of newly synthesized membrane proteins at the trans-Golgi network (TGN). To identify which genes are essential for these sorting mechanisms, mutants were screened in which the transport of Rhodopsin 1 (Rh1), an apical integral membrane protein in Drosophila photoreceptors, was affected. Deficiencies in glycosylphosphatidylinositol (GPI) synthesis and attachment processes were found to cause loss of the apical transport of Rh1 from the TGN and mis-sorting to the endolysosomal system. Moreover, Na+K+-ATPase, a basolateral membrane protein, and Crumbs (Crb), a stalk membrane protein, were mistransported to the apical rhabdomeric microvilli in GPI-deficient photoreceptors. These results indicate that polarized sorting of integral membrane proteins at the TGN requires the synthesis and anchoring of GPI-anchored proteins. Little is known about the cellular biological consequences of GPI deficiency in animals in vivo. These results provide new insights into the importance of GPI synthesis and aid the understanding of pathologies involving GPI deficiency (Satoh, 2013).

In this study, 546 lethal lines were screened for potential defects in Rh1 by examining the localization of Arr2::GFP in FLP/FRT-mediated mosaic retinas using two-color fluorescence imaging. A mutation was found in the Drosophila homolog of human PIG-U (Drosophila PIG-U), which encodes a subunit of GPI transamidase. Mutations in other genes of the GPI synthesis pathway but not in the GPI modification pathway gave rise to the same phenotype. Furthermore, the GPI-linked protein, Chp accumulates in the ER whereas the stalk membrane Crumbs protein and basolaterally localized Na+K+-ATPase were mis-sorted to the rhabdomere. Rh1 was found to be degraded before entering the post-Golgi vesicles, but Crb and Na+K+-ATPase are misrouted into vesicles destined for the rhabdomere in PIG mutant cells (Satoh, 2013).

There are two previous reports concerning GPI requirements for the transport of transmembrane proteins. In zebrafish, GPI transamidase has been found to be essential for the surface expression of voltage-gated sodium channels. In yeast, GPI synthesis is required for the surface expression of Tat2p tryptophan permease, which is associated with detergent-resistant membrane (DRM) in wild-type cells. In GPI-deficient yeast, Tat2p and Fur4p fail to associate with DRM and are retained in the ER. Although DRM forms in the ER in yeast, in mammalian cells, it is likely that DRM formation occurs only after Golgi entry. The reason for this is thought to be that GPI lipid remodeling occurs in different places: the ER in yeast and the Golgi body in mammalian cells. In mammalian cells, lipid rafts are postulated to concentrate some fractions of apically destined proteins owing to their affinity for the TGN or recycling endosomes (Satoh, 2013).

Along with the raft model, there are two possible explanations for the sorting phenotype of PIG mutant fly photoreceptors: (1) the polarized sorting of Rh1 depends on its affinity for the raft/DRM and the raft/DRM is deficient in PIG mutants; (2) unidentified GPI-anchored protein(s) play crucial roles in the polarized sorting of Rh1 and Na+K+-ATPase, and the raft/DRM provides a platform for the GPI-anchored protein(s). The first model predicts raft/DRM deficiency in PIG mutants, Rh1 association with lipid rafts and a stronger phenotype caused by mutations in the genes involved in raft formation. By contrast, the second model predicts that GPI deficiency produces a stronger phenotype than that caused by raft deficiency (Satoh, 2013).

Analysis of lipid raft deficiency does not support the first model in which the loss of polarized sorting of Rh1/Na+K+-ATPase in PIG mutants is a consequence of raft deficiency; instead, the current results support the second model in which unidentified GPI-anchored protein(s) concentrate Rh1 and exclude Na+K+-ATPase and Crb from post-Golgi vesicles destined for the rhabdomeres. Thus, loss of the GPI-anchored sorting protein(s) might cause most Rh1 to be directed into the endocytotic pathway and degraded by lysosomes while simultaneously allowing Na+K+-ATPase and Crb to be loaded into post-Golgi vesicles destined for the rhabdomeres. Chp is the only GPI-anchored protein known to be expressed in fly photoreceptors in the late-pupal stages. However, chp2 mutants do not exhibit any mislocalization phenotype of Rh1 or Na+K+-ATPase. Identifying the GPI-anchored protein(s) responsible for the sorting in the TGN is an important step for understanding this mechanism of polarized transport (Satoh, 2013).

The biosynthetic pathway of GPI-anchored proteins has been well elucidated, but little was known to date about the phenotypic consequences of the loss of GPI synthesis in vivo. The present study demonstrates that GPI synthesis is essential for the sorting of non-GPI-anchored transmembrane proteins, including Rh1 and Na+K+-ATPase, without obvious defects in adherens junctions. Human PIGM or PIGV deficiency causes seizures or mental retardation. These neurological disorders might be also caused by the mis-sorting of some transmembrane proteins in addition to the defects in the formation of GPI-anchoring proteins. These findings aid the understanding of the pathology of diseases involving deficient GPI-anchoring protein synthesis (Satoh, 2013).

Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons

The golgi apparatus is optimized separately in different tissues for efficient protein trafficking, little is known of how cell signaling shapes this organelle. This study finds that the Abl tyrosine kinase signaling pathway controls the architecture of the golgi complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena), selectively labels the cis-golgi in developing PRs. Overexpression or loss-of-function of Ena increases the number of cis and trans-golgi cisternae per cell, and Ena overexpression also redistributes golgi to the most basal portion of the cell soma. Loss of Abl, or of its upstream regulator, the adaptor protein Disabled, lead to the same alterations of golgi as does overexpression of Ena. The increase in golgi number in Abl mutants arises in part from increased frequency of golgi fission events and a decrease in fusions, as revealed by live imaging. Finally, it was demonstrated that the effects of Abl signaling on golgi are mediated via regulation of the actin cytoskeleton. Together, these data reveal a direct link between cell signaling and golgi architecture. Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion (Kannan, 2014).

The Abl tyrosine kinase signaling pathway controls golgi morphology and localization in Drosophila photoreceptors through its regulation of the actin cytoskeleton. Ena, the main effector of Abl in morphogenesis, is associated with the cis-golgi compartment, and it regulates golgi localization and dynamics under the control of Abl and its interacting adaptor protein, Dab. Reducing the levels of Abl or Dab, or overexpressing Ena, led to similar defects in golgi fragmentation state and subcellular distribution. During golgi biogenesis, Abl increases the frequency of fusion of golgi cisternae, and decreases fission events. Abl evidently controls golgi organization through its regulation of actin structure, as the effect of Abl signaling on golgi could be blocked by modulating actin structure genetically or pharmacologically. Collectively, these data reveal an unexpected link between a fundamental tyrosine kinase signaling pathway in neuronal cells and the structure of the golgi compartment (Kannan, 2014).

The data reported here suggest that the Abl signaling pathway controls golgi morphology and localization through its control of actin structure. This is consistent with previous reports that altering the levels of actin modulators perturbs the structure and function of the golgi apparatus. A variety of proteins that modulate actin dynamics have been localized to golgi. Ultra-structural studies established the association of actin filaments with golgi membranes and the association of β and γ actin with the golgi. In cultured cell models, including neurons, actin depolymerization leads to golgi compactness, fragmentation and altered subcellular distribution. It is noted, moreover, that the reported golgi-associated signaling proteins include several that have been linked to Abl signaling, including the Abl target Abi, the Abi binding partner WAVE, and various effectors of Rac GTPase including ADF/cofilin, WASH and Arp2/3. Thus, for example, Abi and WAVE have been implicated in actin dependent golgi stack reorganization and in scission of the golgi at cell division to allow faithful inheritance of golgi complex to daughter cells in Drosophila S2 cell cycles (Kondylis, 2007). These data reinforce the importance of actin-regulating signaling pathways for controlling golgi biogenesis (Kannan, 2014).

Two lines of evidence suggest that the increase observed in golgi number in Abl pathway mutants is due primarily to net fragmentation of pre-existing golgi cisternae and not to de novo synthesis of golgi. First, live imaging of golgi dynamics in neurons of the Drosophila eye disc reveals that the steady-state number of golgi cisternae reflects an ongoing balance of fusion and fission events, much as observed previously in yeast. Quantification of these events in wildtype vs Abl mutant tissue demonstrated directly that loss of Abl significantly increased the frequency of fission events, and reduced the frequency of fusions. Second, the absolute volume of cis-golgi in Abl mutant photoreceptors was not substantially greater than that in wildtype, as judged by direct measurement of the volume of GM130- immunoreactive material in deconvoluted image stacks of photoreceptor clusters. While a small apparent increase was observed in golgi volume in the mutants (~55%, based on pixel counts), it is noted that golgi cisternae are small on the length scale of the point spread function of visible light, such that the fluorescent signal from a single cisterna extends into the surrounding cytoplasm. The increase in apparent golgi volume is therefore within the range expected due simply to fluorescence 'spillover' from the three-fold greater number of separate golgi cisternae in the mutants (Kannan, 2014).

It is striking that both increase and decrease of Ena led to net fragmentation of golgi. Why might this be? It is known that both fission and fusion of membranes requires actin dynamics: at scission, polymerization provides force for separating membranes, while in fusion, actin polymerization is essential for bringing membranes together and for supplying membrane vesicles, among other things. As a result, altering actin dynamics is apt to change the probabilities of multiple aspects of both fission and fusion events, making it impossible to predict a priori how the balance will be altered by a given manipulation, just as either increase or decrease of Ena can inhibit cell or axon motility, depending on the details of the experiment, due to the non-linear nature of actin dynamics. Indeed, this study also observed net golgi fragmentation when actin was stabilized with jasplakinolide, just as was done from depolymerization with cytochalasin or latrunculin. More direct experiments will be necessary to fully understand this dynamic, however. deficits selectively disrupt dendritic morphogenesis but not axogenesis, and perhaps consistent with this, Abl/Ena function is essential for dendrite arborization in these cells but has not been reported to affect their axon patterning. Finally, in some contexts, neuronal development requires local translation of guidance molecules in the growth cone rather than translation in the cell soma. It is likely that the need for actin dynamics to target different subcellular compartments in different cell types will be reflected in different patterns of Abl/Ena protein localization (Kannan, 2014).

This study reports the role of Abl/Ena-dependent regulation of actin structure on overall golgi structure and localization but there may be more subtle effects on golgi function as well. For example, recent evidence supports a role for actin-dependent regulation of the specificity of protein sorting in the golgi complex. Preferential sorting of cargos is achieved by nucleation of distinct actin filaments at the golgi complex. In Hela cells, for example, Arp2/3 mediated nucleation of actin branches at cis-golgi regulates retrograde trafficking of the acid hydroxylase receptor CI-MPR, while Formin family mediated nucleation of linear actin filaments at golgi regulates selective trafficking of the lysosomal enzyme cathepsin D. Similarly, the actin-severing protein ADF/cofilin, the mammalian ortholog of Drosophila twinstar, sculpts an actin-based sorting domain at the trans-golgi network for selective cargo sorting. It will be important to investigate whether the effects of Abl/Ena on golgi morphology have functional consequences on bulk secretion or protein sorting (Kannan, 2014).

Protein trafficking and membrane addition in neurons need to be coordinated with the growth requirements of the axonal and dendritic plasma membranes, but the mechanisms that do so have been obscure. Abl pathway proteins associate with many of the ubiquitous guidance receptors that direct axon growth and guidance throughout phylogeny, including Netrin, Roundabout, the receptor tyrosine phosphatase DLAR, Notch and others. The data therefore suggest a potential link between the regulatory machinery that senses guidance information and the secretory machinery that helps execute those patterning choices. Indeed, preliminary experiments suggest that some of the axonal defects of Abl pathway mutants may arise from alterations in golgi function. Beyond this, Abl signaling is essential in neuronal migration, epithelial polarity and integrity, cell adhesion, hematopoiesis and oncogenesis, among other processes The data reported in this study now compel a reexamination of the many functions of Abl to ascertain whether some of these effects arise, at least in part, from regulation of secretory function (Kannan, 2014).

Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster

Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. This study provides evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. The gene omelette (omt) was found encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster. Rab1 protein colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. It was further demonstrated that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. It is proposed that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis (Sechi, 2017).

Golgi outpost synthesis impaired by toxic polyglutamine proteins contributes to dendritic pathology in neurons

Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. This study shows that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, these results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway (Chung, 2017).

Neurons have tremendously higher amounts of plasma membrane (PM) than other cell types, due to their highly elongated morphology. Recently, dendritic satellite organelles have been reported to be involved in the maintenance of integrity and dynamics of the PM in distal dendrites out of reach from somatic perinuclear organelles. The dendritic organelles have functional overlaps with the canonical perinuclear organelles. However, the restricted space appears to require the miniaturization of the satellite organelle systems, such as the endoplasmic reticulum (ER) and Golgi outposts (GOPs), compared to those localized around the nucleus, suggesting that the dendritic organelles may show different functional capacity from their perinuclear counterparts. Additionally, the distal areas of the dendrites may have quite different cellular environments, such as variations in the concentration of ions and proteins, compared to the perinuclear region. These differences may account for the vulnerability of dendrites over other neuronal domains to neuropathological insults, including protein toxicity conferred by the accumulation of mutant or misfolded proteins in neurons (Chung, 2017).

In Drosophila dendritic arborization (da) sensory neurons, GOPs were found to be located in dendrites, but not in axons, suggesting that GOPs may act as local stations to supply membranes to the PM in nearby dendrites (Ye, 2007). The synthesis of GOPs was reported to be regulated by secretory pathway-related genes, such as Sec31 and the transcription factor Cut (Iyer, 2013). Additionally, GOPs were shown to be transported toward or away from the soma by machinery consisting of a lava lamp protein (LVA), a golgin coiled-coil adaptor protein, and a motor dynein-dynactin protein complex, and their transport was found to be closely associated with the dynamics of dendrite growth. Moreover, GOPs have been implicated in the growth and maintenance of the dendrite arbor. For example, laser ablation of local GOPs decreased branch dynamics in da neurons (Ye, 2007), and the disruption of GOP trafficking blocked dendrite growth in developing hippocampal neurons, leading to the decreased length of dendrites (Chung, 2017).

Despite such functional roles of the GOPs in the growth and maintenance of the dendrite arbor under non-pathological conditions, it still remains unclear whether the GOPs can also contribute to dendrite aberration observed under pathological conditions induced by protein toxicity. Previously, nuclear proteotoxicity has been reported to cause severe dendrite pathology, but the mechanisms underlying dendrite pathology caused by protein toxicity remain largely elusive. Therefore this study investigated a mechanistic link of the GOPs to dendrite pathology caused by toxic nuclear polyQ proteins using Drosophila da sensory neurons that have been extensively utilized by various researchers as an in vivo cellular model for the study of the mammalian neuronal dendrite system with high fidelity dendrite pathology (Chung, 2017).

Toxic nuclear polyQ proteins cause terminal dendrite defects in C4 da neurons. This study examined morphological characteristics of dendrites of class IV (C4) da neurons expressing pathogenic Machado-Joseph's Disease (MJD) protein, MJDtr-78Q (78Q), known to induce nuclear protein toxicity. For this analysis, dendrite images of C4 da neurons expressing 78Q (78Q OE) and wild-type C4 da neuron controls were compared. A mechanistic model is proposed that links toxic nuclear polyQ proteins to the impaired elongation of terminal dendrites in 78Q OE through the CBP-CrebA-COPII-GOP pathway. Toxic nuclear polyQ proteins decreased the number of GOPs in the dendrites of C4 da neurons. mRNA sequencing revealed that the secretory pathway, including the COPII pathway, was a major pathway that was downregulated by toxic polyQ proteins. The disruption of the COPII pathway decreased GOP synthesis. TF enrichment analysis identified CrebA as a key regulator of the genes involved in the COPII pathway. CrebA overexpression restored the downregulation of the COPII pathway and the loss of GOPs caused by toxic polyQ proteins. It was further found that CBP regulated CrebA expression cooperatively with Cut and that toxic polyQ proteins interfered with CBP upstream of CrebA in the regulation of GOP synthesis. Finally, the co-overexpression of CrebA and Rac1 synergistically restored polyQ-induced defects in dendrite branching and elongation (Chung, 2017).

According to a mechanistic model, however, how the overexpression of Cut, unlike CBP, can potentially contribute to the restoration of GOPs is unclear, although it cooperatively regulates CrebA expression with CBP and interacts with CBP at the protein level. This can be explained by alterations in the stoichiometry between CBP and Cut for their complex formation under polyQ-expressed conditions. The results showed that polyQ proteins appeared to sequester CBP, but not Cut. The sequestration of CBP by polyQ proteins leads to a decreased amount of CBP available for the CBP-Cut complex formation. Accordingly, CBP overexpression can increase the amount of non-sequestered CBP under polyQ-expressed conditions, thereby restoring the CBP-Cut complex formation. By contrast, Cut overexpression does not contribute to an increase in complex formation because it has no effect on the depletion of CBP by polyQ proteins. Moreover, Rac1 was included as a component in the mechanistic model. Rac1 was connected to the polyQ-CBP-CrebA pathway based on the following results. First whether polyQ expression affected the expression of Rac1 was tested and a significant decrease in Rac1 expression was found in polyQ-expressing fly heads compared to w1118. Then TFs were sought that could be affected by polyQ expression and could regulate Rac1 expression. Previously, Cut was shown to regulate Rac1 expression. Thus, to examine the effect of polyQ proteins on Cut, whether polyQ proteins altered the intracellular distribution of Cut proteins was checked in polyQ-expressing da neurons, and no noticeable alteration was found in its distribution. As previously mentioned, Cut interacted with CBP in fly heads. Furthermore, Rac1 was shown to be epigenetically suppressed by H3K27me3, which can be antagonized by CBP-dependent H3K27ac. These data together provide a mechanistic connection of the CBP-Cut-Rac1 pathway to the polyQ-CBP-CrebA pathway through CBP (Chung, 2017).

This study proposed a polyQ-CBP-CrebA pathway for polyQ-induced terminal dendrite defects. However, an alternative possibility that dendrite perturbation may precede through CBP-CrebA-independent pathways, which then can result in altered gene expression, cannot be excluded. Consistent with such a possibility, nuclear polyQ proteins were shown to sequester nuclear pore complex proteins, potentially impairing the transport of proteins and RNA, independently of CBP or CrebA (Grima, 2017). Moreover, nuclear polyQ proteotoxicity induces the mis-localization of several TFs (e.g., TBP, Sp1, and p53) other than CBP, leading to the altered expression of their target genes. The perturbed functions of nuclear pore complexes and TFs may result in dendrite defects via CBP-CrebA-independent mechanisms and consequently alterations in gene expression. However, the restoration of GOP synthesis and PM protein supply via the overexpression of CBP or CrebA and the restoration of dendritic arborization defects via the co-overexpression of CrebA and Rac1 in polyQ-expressing neurons demonstrates that the CBP-CrebA pathway should be one of the important pathways that functionally regulates the polyQ-induced dendrite defects. Nevertheless, the relative importance of the CBP-CrebA pathway to the pathways that include other TFs affected by polyQ proteins can be questioned. However, this study found that the overexpression of the three key TFs (HLH106, gt, and Dref) did not restore the number of GOPs in polyQ-expressing neurons. Moreover, the effect of Sp1, which is known to be downregulated by polyQ proteins, on GOP synthesis was examined, and Sp1 knockdown did not significantly decrease the number of GOPs compared to the controls. Furthermore, it was also found that the overexpression of Cut and Scr, known upstream TFs of CrebA, failed to restore the loss of GOPs in polyQ-expressing neurons comparable to CrebA overexpression. Therefore, the data suggest that the mechanism by which polyQ toxicity induces dendrite pathology can be attributed to the polyQ-CBP-CrebA pathway proposed in this study (Chung, 2017).

The loss of GOPs was one of the most apparent dendrite defects caused by polyQ proteins. Thus, among many cellular processes downregulated by the polyQ proteins, this study focused on three cellular processes, (1) vesicle-mediated transport (VT) and (2) membrane organization (MO) and lipid biosynthetic process (LP), that were considered to be related to GOPs. However, these processes can also be related to other secretory-pathway-related processes in addition to GOP synthesis. For example, neuropeptide maturation and trafficking were previously shown to be modulated by the secretory pathway, suggesting the potential association of neuropeptide-containing vesicle trafficking with the three processes. Thus the effect of polyQ proteins on neuropeptide-containing vesicle trafficking was examined by measuring the amount of neuropeptide-containing vesicles in w1118 and polyQ-expressing neurons using a reporter. PolyQ expression was found to decrease the amount of ANF-EMD vesicles in dendrites and soma compared to controls, suggesting that polyQ perturbs the function of the secretory pathway related to neuropeptide-containing vesicle trafficking in addition to GOP synthesis. Additionally, the three processes can be associated with the synthesis of somatic Golgi. However, the data showed a larger loss of ManII-eGFP-positive puncta in the dendrites of C4 da neurons compared to the soma, suggesting that the three processes can be considered to be related more to GOP synthesis than to somatic Golgi synthesis. Thus, although the polyQ-induced downregulation of genes associated with the abovementioned three processes may impact the secretory pathway in general, the results appear to indicate that the synthesis of GOPs responds more to the downregulation of those genes compared to the synthesis of somatic Golgi (Chung, 2017).

In mRNA-sequencing analysis, fly heads were used to identify genes affected by toxic polyQ proteins. Considering that fly heads contain no C4 da neurons, alterations in gene expression obtained from mRNA sequencing in polyQ-expressing fly heads, compared to w1118 fly heads, should be defined by non-C4 da cells. Due to a small number (~50) of C4 da neurons per larva, however, it is challenging to isolate a sufficient number (>105 cells) of C4 da neurons for mRNA sequencing. Instead of C4 da neurons, fly heads were used for mRNA sequencing to obtain a clue for alteration of gene expression by polyQ proteins. Moreover, mRNA sequencing identified 5,385 differentially expressed genes between polyQ-expressed and control samples, of which 5,325 (98.9%) were downregulated. Such a large number and high percentage of downregulated genes suggests that there might be widespread cell death in polyQ-expressing fly brains. Thus, the extent of cell loss was checked in the polyQ-expressing adult fly brains on which mRNA sequencing was performed by measuring the number of cells that displayed cleaved caspase-3 through immunohistochemistry analysis. PolyQ-expressing brains showed minimal cell loss compared to controls in the condition where mRNA sequencing was performed. Additionally, no observable decrease was found in rRNA in polyQ-expressed adult fly heads compared to w1118 heads. These data suggest that the downregulation of the large number of genes is not likely to be caused by cell death. One alternative explanation can be the previously reported dysregulation of the following histone modifications caused by polyQ proteins: impaired acetylation of H3K27 and dysregulated tri-methylation of H3K9 and H3K27. These dysregulated histone modifications may result in the downregulation of 98.9% of DEGs through a global epigenetic silencing (Chung, 2017).

The normal length of the polyQ tract in humans has been reported to be less than 36 for the MJD protein. In flies, numerous studies have used the truncated MJD protein with only 27 polyQ repeats (MJDtr-27Q) as a control. However, in this study, w1118 rather than 27Q OE was used as the control for 78Q OE in most of the experiments because 27Q OE exhibited strong dendrite pruning defects compared to 78Q OE. A previous study also showed that Httex1p-Q93 caused strong pruning defects in C4 da neurons, whereas Httex1p-Q20 caused no such defects. Taken together, these data suggest that the pruning defects are likely not due to the context of the host proteins but to polyQ toxicity. However, there appears to be a threshold length (between 20Q and 27Q) of the polyQ tract to confer such polyQ toxicity. Nevertheless, further detailed studies should be performed to understand the mechanism underlying the protein toxicity of MJDtr-27Q to induce pruning defects (Chung, 2017).

Finally, this study identified three different nuclear polyQ toxicity models that exhibited decreased GOP synthesis and PM protein supply, indicating a potentially shared mode of nuclear proteotoxicity. Of note, the accumulation of misfolded proteins in the nucleus has been observed in other neuronal diseases or during aging. Thus, the proposed mechanistic model underlying terminal dendrite defects caused by the accumulation of nuclear toxic polyQ proteins can provide significant insights into other neuronal maladies linked to nuclear proteotoxicity and aging that can be further examined in detailed functional studies (Chung, 2017).

BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior

Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. This paper shows that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution (Corrigan, 2014).

Seminal fluid synthesized by male reproductive glands has a powerful influence on fertility, affecting multiple sperm activities and altering female behavior, in some cases directly conflicting with female reproductive interests. Several previous studies have revealed an important function for seminal peptides in Drosophila in these processes. However, this study presents the first in vivo evidence that exosomes also play a key role and identify a completely novel role for BMP signaling in regulating this process (Corrigan, 2014).

Exosome biogenesis, secretion, and uptake have been previously studied in Drosophila. However, the small size of exosomes, MVBs, and fly tissues makes these processes difficult to analyze in vivo. The AG contains only nanoliter volumes of secretions, making it impractical to use standard exosome analysis techniques, such as ultracentrifugation and Nanosight Tracking Analysis. Like other studies in flies, this study used genetic and imaging approaches to test the identity of SC-specific CD63-positive puncta. In addition, Western blot analysis of transferred seminal fluid and live imaging of giant MVBs in SCs were used to test the hypothesis that SCs produce exosomes (Corrigan, 2014).

The human CD63-GFP tetraspanin marker was used in this analysis. However, GFP-positive puncta were also observed in large secretory compartments of SCs expressing cytosolic GFP, and exosome-sized vesicles in MVBs and the AG lumen were observed in EM analysis of wild-type glands, confirming their presence in nontransgenic flies. Because exosomes can be loaded with many cellular components, the findings provide a potential explanation for the observation that AGs of several insects, including Drosophila, secrete intracellular proteins (Corrigan, 2014).

Other evidence strongly supports the idea that CD63-positive puncta secreted from SCs are exosomes and not vesicles shed from the plasma membrane. This includes the observation that CD63-positive puncta are found inside both acidic Rab7-positive MVB-like compartments as well as nonacidic Rab11-positive vacuoles and require the ESCRT and ESCRT-associated proteins Hrs and ALiX, as well as several Rabs linked to mammalian exosome secretion, to be formed and secreted. Secreted puncta counts have been used previously in flies to study genetic control of exosome secretion. A criticism of this approach is that reduced puncta numbers may merely reflect aggregation. However, the transfer of CD63-GFP to females was drastically reduced in mutant backgrounds, arguing against a simple aggregation model. Furthermore, because genetic manipulation of ESCRT function does not alter other secretory processes in SCs, this strongly implicates the endocytic pathway in secretion of tagged CD63 (Corrigan, 2014).

Studies of exosomes in Drosophila as well as mammals already suggest that multiple exosome subtypes exist and may be regulated differently, e.g., different roles for ALiX, Hrs, and Evi. If different exosome subtypes are made in SCs, these cells should offer an ideal system to study their differential regulation (Corrigan, 2014).

The remarkably large size of endosomal compartments in SCs provides new opportunities to study exosome biogenesis in vivo. To date, many studies of the intracellular exosome biogenesis machinery and endolysosomal trafficking in higher eukaryotes have relied on expressing an activated form of Rab5 or addition of the ionophore monensin in cell culture to artificially enlarge the endolysosomal compartments, disrupting normal trafficking events. Hence, this new SC in vivo model should allow reinvestigation of previously reported regulators of exosome biogenesis and identify functional differences in trafficking phenotypes, as has been seen for Hrs and ALiX (Corrigan, 2014).

This study has already revealed a surprisingly dynamic interaction between the secretory and endolysosomal systems in SCs. Communication between these compartments using vesicular transport and tubulation processes has been reported in other cell types in flies and mammals, but this study suggests that direct fusion can also be involved. Indeed, the data are also consistent with mMVBLs forming after fusion between SVs and iLEs, suggesting that fusion events may play a critical role in establishing distinct compartments within SCs. In light of this dynamic flux between compartments, it remains unclear whether CD63-GFP-labeled exosomes might be released by the classical route involving mMVBL fusion to the plasma membrane or via an intermediate secretory compartment (Corrigan, 2014).

Although most analysis of the fly AG has highlighted roles for MC products, such as SP, in reprogramming female postmating responses, several recent studies have also suggested a central but poorly defined function for SCs. A transcriptional program regulated by the Hox gene Abd-B controls vacuole formation in SCs (Gligorov, 2013). These findings now indicate that at least one of the effects mediated by SCs, altered receptivity to remating, requires exosome secretion (Corrigan, 2014).

It is difficult to accurately estimate the frequency of SC exosome-sperm fusion events in each female fly because they can probably only be visualized transiently, and many may involve fusion to the very long sperm tail. Sperm play an essential role as mediators of SP-dependent postmating effects in females, so it is plausible that exosome fusion to sperm may modulate specific SP functions. Another appealing hypothesis is that SC exosomes also interact with the female reproductive tract to influence female behavior. However, whatever the target tissues, the data clearly demonstrate a role for SC exosomes in female reprogramming. Furthermore, like human prostasomes, SC exosomes fuse with sperm, highlighting possible conserved roles for exosomes in male reproductive biology. In prostate cancer, prostasomes are inappropriately secreted into the bloodstream, so that other cells in the body may be subjected to these powerful reprogramming functions, potentially supporting tumor-stroma interactions and metastasis (Corrigan, 2014).

Reducing BMP signaling in SCs inhibits exosome secretion and leads to the formation of a novel mMVBL compartment that is filled with fluorescent CD63-GFP. A simple interpretation of this result is that MVBL compartments in these cells do not mature properly, blocking exosome secretion. Consistent with this, increasing BMP signaling in these cells produces a highly enlarged acidic compartment (Corrigan, 2014).

Previous studies have shown that blocking endosomal maturation by knockdown of the early ESCRT component Hrs increases the size of immature endosomal class E compartments lacking ILVs and also results in increased BMP signaling. The data demonstrate that elevated BMP signaling increases mMVBL size, suggesting that there is a complex bidirectional interaction between mMVBL maturation and size and the level of BMP signaling in SCs (Corrigan, 2014).

The findings are consistent with a model in which BMP signaling also controls SC growth by driving endolysosomal trafficking and maturation events. Late endosomes and lysosomes have previously been shown to house major nutrient sensors and cell growth machinery, including the mTORC1 complex, which is activated by intraluminal amino acids. Interestingly, the growth rate of knockdown cells with reduced ESCRT function appears to correlate with mMVBL size rather than exosome secretion rate. Whether growth in these cells is mTORC1 dependent needs to be tested (Corrigan, 2014).

Whatever the explanation for the growth defects in SCs, these data very clearly implicate BMP signaling in the regulation of endolysosomal trafficking and exosome secretion. It will now be important to test whether BMP signaling plays a similar role in mammalian glands that secrete exosomes, such as prostate and breast, and determine whether this role is affected in diseases such as cancer (Corrigan, 2014).

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in Drosophila were used to investigate the molecular pathway for biogenesis of the mucin-containing 'glue granules' that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules (Burgess, 2011).

Constitutive secretion of proteins and lipids from the trans-Golgi network (TGN) toward the cell surface is believed to operate in all cells. Constitutive secretion is characterized by the rapid deployment of newly synthesized cargo toward its final cellular destination. Specialized secretory cells such as endocrine, neuroendocrine, and exocrine cells contain an additional pathway termed the regulated secretory pathway. One hallmark of this pathway is the storage of regulated secretory proteins at high concentration in dense-core secretory granules that can be released in response to an external signal. How secreted proteins enter the regulated secretory pathway is a source of debate and may prove to be cargo and cell-type specific. In the case of endocrine and neuroendocrine cells, sorting of secreted cargo is believed to be content driven, with selective aggregation of regulated secretory proteins at the TGN playing a major role in secretory granule biogenesis (Burgess, 2011).

Little is known about the coat proteins that might be required on the cytoplasmic face to promote budding of lumenal regulated secretory cargo from the TGN. Initial studies in AtT20 pituitary cells noted that condensing secretory products accumulate in dilated regions of the TGN that are coated with clathrin. Similarly, in β-cells treated with monensin to perturb intracellular trafficking, proinsulin accumulates in a clathrin-coated compartment related to the TGN. These observations raise the possibility that the formation of regulated secretory granules might require clathrin at the TGN (Burgess, 2011).

Coat proteins selectively incorporate cargo into vesicles and provide a scaffold for vesicle formation. Clathrin and its associated heterotetrameric adaptor proteins (APs) make up a major class of vesicular coats. APs bind to sorting motifs found in the cytoplasmic tails of membrane cargo and function as links between vesicular cargo and the clathrin lattice, although some AP-3 and AP-4 coats lack clathrin. The four different AP complexes (AP-1-4) have distinct sites of action in the cell. Of these, the AP-1 complex has perhaps the most diverse roles, acting at the TGN to promote constitutive secretion (Chi, 2008), at the TGN and endosomes to sort mannose 6-phosphate receptors, and at immature secretory granules of specialized secretory cells to retrieve missorted proteins. Indeed, a coat composed of clathrin and AP-1 is required for maturation and condensation of regulated secretory granules. In contrast to granule maturation, the roles of AP-1 and clathrin in initial stages of secretory granule formation are less well established. AP-1 and clathrin were shown to be required for formation of Weibel-Palade bodies (Lui-Roberts, 2005), secretory organelles that store the hemostatic protein von Willebrand factor. However, a dominant-negative clathrin construct did not interfere with insulin granule production in neuroendocrine cells, suggesting these granules form through a clathrin-independent mechanism. Thus it is not clear how general a role AP-1 and clathrin play in granule biogenesis (Burgess, 2011).

The larval salivary gland in Drosophila provides an excellent system for molecular genetic analysis of factors required for formation of regulated secretory granules. During the last half of third-instar larval development, prior to pupariation, salivary gland cells initiate production of mucin-type secretory granules termed 'glue' granules. These granules contain highly glycosylated mucin-type glue proteins that are required to adhere the pupal case to a solid substrate during metamorphosis. Of the six known glue proteins (also called salivary gland secretion or Sgs proteins), Sgs1, Sgs3, and Sgs4 contain extended amino acid repeats that are likely sites of oligosaccharide linkage. These proteins, which are synthesized in response to a low-titer pulse of the steroid hormone ecdysone at the mid-third-instar larval stage, are stored until an additional high-titer pulse of ecdysone promotes their release at the onset of pupariation (Burgess, 2011).

Secreted mucin-type glycoproteins are ubiquitous in metazoans and serve important roles in animal physiology. This study analyzed the mechanism of mucin-type glue granule biogenesis in third-instar larval salivary gland cells. It was shown that AP-1 and clathrin localize to the TGN prior to glue production, colocalize with newly synthesized glue proteins during early stages of granule formation, and are found at later stages on maturing glue granules. Genetic disruption or knockdown of AP-1 subunits strongly reduces clathrin localization to the TGN. Moreover, AP-1 and clathrin are required for glue granule formation; loss of AP-1 causes glue cargo to accumulate at the TGN and in small, highly aberrant granules. These results reveal a requirement for AP-1 and clathrin in the formation of mucin-type secretory granules (Burgess, 2011).

To identify coats that might function in granule biogenesis, the subcellular distribution of clathrin heavy chain was examined, as well as subunits of the clathrin adaptor protein complexes AP-1 and AP-3, which reside on intracellular organelles (note that Drosophila lacks AP-4). First clathrin, AP-1, and AP-3 were examined in salivary gland cells at stage 0, just prior to glue production. At this stage, Golgi bodies are easily visualized using antibodies directed against the golgin Lava lamp (Lva), which localizes to the cis-Golgi. Note that the cis-Golgi has a cup-shaped appearance. A monomeric red fluorescent protein fusion to clathrin heavy chain (RFP-Chc) predominantly localized to large puncta adjacent to the concave face of the cis-Golgi, consistent with a previous report showing localization of endogenous Chc to intracellular puncta in these cells (Wingen, 2009). Endogenous AP-1γ showed a similar distribution. A projection constructed from serial confocal sections revealed numerous Golgi units scattered throughout the cytoplasm. There was a one-to-one correspondence between AP-1γ- and Lva-positive structures, with the cis-Golgi cups surrounding AP-1γ in a manner consistent with AP-1 localizing to the TGN. Indeed AP-1γ and RFP-Chc colocalized with the trans-Golgi protein EpsinR (also called Liquid facets-Related or LqfR). In contrast, AP-1 showed only minimal overlap with the recycling endosome regulator Rab11. AP-1γ and RFP-Chc colocalized at the TGN, although AP-1γ distribution appeared slightly more diffuse in salivary gland cells expressing RFP-Chc than in nonexpressing cells. Localization of AP-1 to the TGN is adaptor-protein specific, because a functional monomeric cherry fluorescent protein (mCherry) fusion to AP-3δ (called Garnet in Drosophila) showed no overlap with a Venus fluorescent protein (VFP) fusion to AP-1μ (called AP-47 in Drosophila), but rather colocalized with the late endosome marker Rab7. Given the high degree of colocalization of clathrin and AP-1, it was asked whether AP-1 might be required to recruit clathrin to the TGN (Burgess, 2011).

To test whether AP-1 recruits clathrin to the TGN, use was made of a μ1-adaptin null allele, AP-47SHE-11. To bypass late embryonic lethality caused by this allele, mosaic clones were generated in the salivary gland using FLP-FRT-based recombination. Briefly, the wild-type chromosome carries a copy of green fluorescent protein (GFP) such that homozygous mutant cells are marked by the absence of GFP expression and heterozygous and wild-type cells are marked by one or two copies of GFP, respectively. AP-47SHE-11 clones were generated during embryogenesis and analyzed in third-instar larval salivary glands at stage 0, just prior to glue production. To determine whether other AP-1 subunits can localize to the TGN in the absence of AP-47, the distribution of AP-1γ was examined, and its punctate localization was found to be entirely lost in AP-47SHE-11 mutant cells. Hence AP-47 is required for efficient recruitment or stability of AP-1γ, similar to what was previously observed in μ1-adaptin-deficient mouse embryonic fibroblasts. Not all trafficking markers were affected by the loss of AP-47, as the early endosome marker Rab5 was unperturbed (Burgess, 2011).

Strikingly, in AP-47SHE-11 mutant cells, RFP-Chc localization to the Golgi was dramatically reduced. The effect on RFP-Chc distribution was also observed in salivary gland cells in which expression of a double-stranded RNA was used to knock down expression of AP-1γ by RNA interference (RNAi). Most cells depleted of AP-1γ exhibited strong delocalization of RFP-Chc, with only a few cells retaining weak RFP-Chc localization at the TGN. Hence the TGN is the major site of clathrin localization in these cells, and AP-1 plays a pivotal role in clathrin recruitment. Importantly, Golgi integrity per se (as assessed by distribution of Lva) was not affected by disruption of AP-1 (Burgess, 2011).

This study has provided compelling evidence of a previously unknown function for clathrin and AP-1 in the formation of mucin-type secretory granules. Clathrin and AP-1 were shown to localize to the TGN prior to synthesis of secretory cargo, colocalize with newly synthesized secretory cargo, and are required for secretory granule formation. Hence AP-1 and clathrin play a crucial role in early stages of secretory granule formation in salivary gland cells. Consistent with this idea, clathrin becomes delocalized upon AP-1 depletion, indicating that other adaptors cannot recruit clathrin in the absence of AP-1 at this stage of salivary gland development (Burgess, 2011).

The results suggest that formation of mucin-containing glue granules and Weibel-Palade bodies might be similar. Weibel-Palade bodies have an unusual cigar-shaped appearance and it was proposed that AP-1 and clathrin might participate in their formation at the TGN by allowing lumenal cargo to properly fold and aggregate or by preventing premature scission. Indeed, depletion of AP-1 in endothelial cells results in the formation of small, round von Willebrand factor-containing organelles lacking other Weibel-Palade body markers. The data demonstrate that the requirement for clathrin and AP-1 is not restricted to one specific type of granule. Depletion of clathrin or AP-1 in Drosophila salivary glands resulted in the accumulation of glue protein both at the TGN and in small organelles of aberrant morphology. This finding extends the role of AP-1 and clathrin to the formation of granules containing mucoprotein cargo and suggests a broader requirement for this coat complex in granule production (Burgess, 2011).

How might AP-1 participate in glue granule formation? One possibility is that AP-1 and clathrin are directly involved in packaging glue granule cargo at the TGN. In mammalian cells, several transmembrane proteins are targeted to regulated secretory granules, including peptidyl-α-amidating monooxygenase, muclin, and phogrin. Indeed, phogrin has been shown to bind to AP-1 and AP-2 through well-conserved tyrosine and dileucine sorting motifs present in its cytosolic tail. How AP-1, a cytosolic coat protein, might interact with lumenal glue proteins in salivary cells remains to be determined. Because none of the known granule proteins contains a predicted transmembrane domain, a yet-unidentified transmembrane receptor might mediate this interaction (Burgess, 2011).

A distinct possibility is that AP-1 might be required to maintain a steady-state distribution of proteins that shuttle between the TGN and endosomes such that they are available at the TGN during granule formation. For instance, the protein convertase furin recycles between the TGN and endosomes and is required to process numerous secreted proteins such as von Willebrand factor. Importantly, furin is no longer concentrated at the TGN in μ1A-deficient fibroblasts. Thus failure to recycle transmembrane enzymes that play a crucial role in processing secreted cargo could also contribute to defective granule formation (Burgess, 2011).

Reduced levels of AP-1 resulted in intermediate-sized granules, suggesting AP-1 might have an additional role during glue granule maturation. The development of Drosophila glue granules is characterized by an overall increase in size and decrease in number, consistent with homotypic fusion of smaller granules over time (Farkas, 1999). Whether small and large granules are equally capable of fusing and whether fusion events are temporally regulated is not known. AP-1 might regulate granule maturation by sorting or retrieving membrane proteins required for homotypic fusion and eventual exocytosis. Additionally, AP-1 might function directly on maturing granules to remove missorted proteins, such as lysosomal hydrolases, similar to what has been reported for other types of secretory granules. In support of this view, live imaging revealed a dynamic association of AP-1 with immature granules. Further studies are needed to resolve whether AP-1 functions in the addition and/or removal of proteins from maturing glue granules (Burgess, 2011).

On the basis of the small size of mutant cells, AP-1 likely participates in additional trafficking pathways. In mammalian cells, AP-1A is ubiquitously expressed and required for trafficking between TGN and endosomes, whereas AP-1B is present only in polarized epithelial cells and is required for basolateral sorting from recycling endosomes. The sole AP-1 complex in Drosophila might mediate both functions in a single cell type. Interestingly, depletion of AP-1γ in salivary glands after granule formation caused the basolateral protein Discs large to redistribute to the apical surface, suggesting that AP-1 is required for basolateral targeting of proteins in this tissue. However, an independent analysis of AP-1μ null cells in the dorsal thorax epithelium failed to reveal a similar polarity defect (Benhra, 2011). This discrepancy might be due to cell type-specific requirements for AP-1 or to differences in RNAi versus mutant clones (Burgess, 2011).

The observation that the abundance of Sgs3-DsRed protein and several Sgs mRNAs is reduced upon AP-1 knockdown suggests the existence of a negative-feedback loofp, whereby a block in anterograde secretory trafficking results in down-regulation of secretory genes. A block in secretion at the TGN could potentially induce the unfolded protein response, analogous to what happens upon depletion of the Arf1 GEF GBF1. However, GBF1 functions early in the secretory pathway, and knockdown of two Arf-GEFs that act on the TGN did not elicit a similar response. Alternatively, a block in anterograde trafficking might repress transcriptional activation of secretory genes by Drosophila CrebA and Forkhead (Fkh) by some as-yet-unknown mechanism (Burgess, 2011).

In addition to the AP-1 complex, the Drosophila genome encodes two other Golgi-localized clathrin adaptor proteins, EpsinR/LqfR and Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding (GGA) protein (Drosophila has only one GGA). LqfR partially colocalizes with AP-1 at the TGN in salivary gland cells and lqfR mutants exhibit small salivary glands, suggesting defects in granule biogenesis. It will be interesting to determine whether LqfR and GGA participate in glue granule biogenesis, especially since these clathrin adaptors might facilitate sorting of other types of cargo. For example, EpsinR has been shown to bind SNARE proteins and could function to provide vesicle identity to nascent glue-containing granules. SNAP-24 was previously identified as a glue granule-specific SNARE, although whether this SNARE mediates homotypic fusion of granules or functions during exocytosis of granules at the plasma membrane is unclear. Given the apparent similarities between glue granule and Weibel-Palade body biogenesis, as well as the high degree of conservation of TGN sorting machinery in Drosophila, the current findings suggest that Drosophila salivary glands are of great utility to further elucidate the mechanisms of biogenesis of regulated secretory granules (Burgess, 2011).

Vesicle-mediated steroid hormone secretion in Drosophila melanogaster

Steroid hormones are a large family of cholesterol derivatives regulating development and physiology in both the animal and plant kingdoms, but little is known concerning mechanisms of their secretion from steroidogenic tissues. This study presents evidence that in Drosophila, endocrine release of the steroid hormone ecdysone is mediated through a regulated vesicular trafficking mechanism. Inhibition of calcium signaling in the steroidogenic prothoracic gland (PG) results in the accumulation of unreleased ecdysone, and the knockdown of calcium-mediated vesicle exocytosis components in the gland caused developmental defects due to deficiency of ecdysone. Accumulation of synaptotagmin-labeled vesicles in the gland is observed when calcium signaling is disrupted, and these vesicles contain an ABC transporter that functions as an ecdysone pump to fill vesicles. It is proposed that trafficking of steroid hormones out of endocrine cells is not always through a simple diffusion mechanism as presently thought, but instead can involve a regulated vesicle-mediated release process (Yamanaka, 2015)

This study provides several lines of evidence demonstrating that the insect steroid hormone E is secreted from the PG not by simple diffusion, but rather through a calcium signaling-regulated vesicle fusion event. Three major points come from these findings: (1) Atet, an ABCG transporter, can facilitate E passage through membranes in an ATP-dependent manner, (2) GPCR-regulated calcium signaling in the PG promotes E release, and (3) the significance of steroid hormone release by vesicle exocytosis and its implication for other steroid hormone/cholesterol trafficking processes (Yamanaka, 2015)

Atet was originally cloned in Drosophila as an ABC transporter-encoding gene with unknown function. It was found to be highly expressed in embryonic trachea, leading to its name ABC transporter expressed in trachea or Atet. In an in situ hybridization experiment, however, this study found little expression of Atet in embryonic trachea, but instead saw specific high level expression in the PG, consistent with its expression pattern in the third instar larva. Since Atet has an atypical membrane topology and can transport E across membranes in vitro, renaming this gene Atypical topology ecdysone transporter is proposed, thereby retaining the Atet gene designation (Yamanaka, 2015)

Atet belongs to the ABCG subfamily of ABC transporters, members of which in mammals have been shown to transport cholesterol as well as other steroids, such as estrogens and their metabolites, in many biological systems. The atypical membrane topology, with the N-terminal ABC domain on the non-cytoplasmic side of the membrane, has not been reported for any ABC transporter to date. However, this topology may have a strong advantage in facilitating tight control on E release by preventing Atet from functioning on the plasma membrane, due to the lack of ATP in extracellular space. This configuration therefore prevents E transport directly through the plasma membrane and confines it to a vesicle-mediated fusion process, although it requires a separate molecular mechanism to transport ATP into the secretory vesicles. This mechanism remains unclear at this point, but it may involve a specific transporter like the recently described VNUT/SLC17A9. In this context, it is interesting to note that the human Atet orthologs ABCG1 and ABCG4 are also strongly predicted by membrane topology algorithms to position their N-terminal ABC domain on the non-cytoplasmic side. These transporters mediate cellular cholesterol efflux and have recently been shown to work not on the plasma membrane but in intracellular endosomes. Clearly, additional studies on the membrane topology of ABCG transporters are warranted (Yamanaka, 2015)

The results of the RNAi screening demonstrate that CG30054, a Gαq subunit, and Plc21C, a PLCβ class enzyme, are both required for proper PG function. These findings strongly implicate the existence of an unknown GPCR and cognate ligand as mediators of the calcium signaling event that is suggested to stimulates E release from the PG. On the other hand, it is known that the PTTH receptor is Torso, a receptor tyrosine kinase and its primary role is to promote E production by inducing E biosynthetic enzyme gene transcription. These observations suggest that, at least in Drosophila, E production and release are likely regulated separately. This machinery might help the GPCR ligand to generate large pulses of steroid in a timely fashion. The identification of the GPCR as well as its ligand is necessary to further pursue this possibility (Yamanaka, 2015)

The mechanism of steroid hormone transit through lipid membranes has not been well studied and in many physiology textbooks the issue is not even discussed. When this topic is mentioned, the explanation most often given is that they can freely diffuse through lipid membranes. Despite this prevailing assumption, there are only a few reports where such transbilayer transfer of steroids by free diffusion has been analyzed. In one theoretical study, it was shown in silico that a free energy of solvation-based mechanism can produce rapid flux of estradiol, testosterone, and progesterone through a simple membrane in concordance with measured rates. However, it is well known that steroid hormone transport across membranes can indeed be an active process in some situations: there are a number of reports on transporter involvement in either uptake or elimination of steroid hormones in eukaryotes ranging from yeast to human. These reports are suggestive enough to rationalize a potential mechanism that incorporates steroid hormones into secretory vesicles, which enables regulated secretion of steroid hormones from steroidogenic tissues (Yamanaka, 2015)

Historically, the possibility of vesicle-mediated steroid hormone release has been examined using ultrastructural and biochemical approaches in multiple biological systems, including the corpus luteum in sheep. The proposed vesicle-mediated progesterone release from the sheep corpus luteum, however, was later challenged, since the peptide oxytocin was shown to be present in dense granules by immuno-EM methods and release of oxytocin and progesterone responded differently to various secretagogues. Since that time, studies investigating the possibility of vesicle-mediated steroid release in any biological system have rarely been reported. One relevant and intriguing set of studies, however, involved ultrastructural localization of E in the PG of the waxworm Galleria mellonella using immuno-EM methods. These studies suggested that E in the PG is concentrated into what appear to be secretory granules that fuse with the plasma membrane, but once again no follow up studies have been reported in the literature (Yamanaka, 2015)

In considering the various models for steroid passage through membranes, it is important to note that steroids such as progesterone, testosterone, and estradiol are significantly more hydrophobic than E. Therefore, the free energy of solvation into a lipid bilayer of E is likely to be much more positive than for sex steroids; this may preclude the use of a simple diffusion mechanism for E. In this respect, E is more similar to bile acids, which are also highly hydrophilic and need active transporters to traverse lipid bilayers. Thus, depending on their specific physiochemical properties, different steroids might use either simple passive diffusion through the plasma membrane, active transporters or some combination of these mechanisms (Yamanaka, 2015)

In summary, this work provides strong evidence that E is released from the PG by calcium-stimulated, vesicle-mediated exocytosis. Therefore, it is suggested that the prevailing 'free diffusion' model of steroid hormone secretion needs to be reconsidered. It also follows that if E uses an active export process, then the import of many hormones, in particular 20E, is also likely controlled by transporters. Given the diversity of physiological processes regulated by steroid hormones, additional characterization of the mechanisms responsible for their import and export from various cell types and tissues will have significant impact on both basic and clinical aspects of steroid hormone physiology (Yamanaka, 2015)

The Arf family G protein Arl1 is required for secretory granule biogenesis in Drosophila

The small G protein Arf like 1 (Arl1) is found at the Golgi apparatus, and in the GTP-bound form it recruits to the Golgi several effectors including GRIP-domain containing coiled-coil proteins, and the Arf1 exchange factors Big1/2. To investigate the role of Arl1, this study has characterised a loss of function mutant of the Drosophila Arl1 orthologue. The gene is essential, and examination of clones of cells lacking Arl1 shows that it is required for recruitment of three of the four GRIP domain golgins to the Golgi, with dGCC185 being less dependent on Arl1. At a functional level, Arl1 is essential for formation of secretory granules in the larval salivary gland. When Arl1 is missing, the Golgi are still present but there is a dispersal of AP-1, a clathrin adaptor that requires Arf1 for its membrane recruitment and which is known to be required for secretory granule biogenesis. Arl1 does not appear to be required for AP-1 recruitment in all tissues, suggesting that it is critically required to enhance Arf1 activation at the trans-Golgi in particular tissues (Torres, 2014).

Synaptic control of secretory trafficking in dendrites

Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER) from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. This study, carried out in mammalian neuronal cell cultures, monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor Kif17 (see Drosophila KIF17) by Ca2+/calmodulin-dependent protein kinases (CaMK) (see for example Drosophila CaMKII). Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport (Hunus, 2014. PubMed ID: 24931613).

Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. TG-A, but not TG-B, was found to be modified concomitantly by N-myristoylation and S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca2+ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. TG-A was shown to be secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes (Shibata, 2017).

Golgi-resident Galphao promotes protrusive membrane dynamics

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. This study found that the Galphao-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Galphao induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Galphao, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Galphao interactors, this study pinpoints several basic cellular activities, including vesicular trafficking, as being regulated by Galphao. It was further found small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Galphao. This KDELR --> Galphao --> Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues (Solis, 2017).

Lgl regulates Notch signaling via endocytosis, independently of the apical aPKC-Par6-Baz polarity complex

The Drosophila melanogaster junctional neoplastic tumor suppressor, Lethal-2-giant larvae (Lgl), is a regulator of apicobasal cell polarity and tissue growth. Previous studies have shown in the developing Drosophila eye epithelium that, without affecting cell polarity, depletion of Lgl results in ectopic cell proliferation and blockage of developmental cell death due to deregulation of the Hippo signaling pathway. This study shows that Notch signaling is increased in lgl-depleted eye tissue, independently of Lgl's function in apicobasal cell polarity. The upregulation of Notch signaling is ligand dependent and correlates with accumulation of cleaved Notch. Concomitant with higher cleaved Notch levels in lgl- tissue, early endosomes (Avalanche [Avl+]), recycling endosomes (Rab11+), early multivesicular bodies (Hrs+), and acidified vesicles, but not late endosomal markers (Car+ and Rab7+), accumulate. Colocalization studies revealed that Lgl associates with early to late endosomes and lysosomes. Upregulation of Notch signaling in lgl- tissue requires dynamin- and Rab5-mediated endocytosis and vesicle acidification but is independent of Hrs/Stam or Rab11 activity. Furthermore, Lgl regulates Notch signaling independently of the aPKC-Par6-Baz apical polarity complex. Altogether, these data show that Lgl regulates endocytosis to restrict vesicle acidification and prevent ectopic ligand-dependent Notch signaling. This Lgl function is independent of the aPKC-Par6-Baz polarity complex and uncovers a novel attenuation mechanism of ligand-activated Notch signaling during Drosophila eye development (Parsons, 2014).

This study demonstrates a novel function for the cell polarity regulator lgl in regulation of endocytosis and Notch signaling. In the developing Drosophila eye epithelium that (1) Notch targets, E(spl)m8, CycA, and Rst, are upregulated in lgl- tissue; (2) Notch upregulation contributes to the lgl- mosaic adult eye phenotype; (3) Notch upregulation in lgl- clones is ligand dependent and requires endocytosis; (4) Lgl colocalizes with intracellular Notch and endocytic markers; (5) lgl- tissue accumulates Avl+ EEs, Hrs+MVBs, Rab11+ REs, endocytic compartments, and acidified vesicles, but not LE markers, Rab7, and Car; (6) Notch upregulation in lgl- clones is independent of the ESCRT-0 complex (Hrs/Stam) or Rab11, but it requires Rab5 function and acidification of endosomes; and (7) Notch upregulation in lgl- clones is independent of aPKC-Baz-Par6. Altogether, these data reveal a novel role for Lgl in attenuating ligand-activated Notch signaling via restricting the acidification of endocytic compartments, independently of its role in regulating the aPKC-Baz-Par6 cell polarity complex (Parsons, 2014).

The data have revealed that increased vesicle acidification in lgl- tissue is responsible for elevated Notch signaling. Because S3 cleavage (by γ-secretase) of Notch extracellular domain (Next) to form the intracellular domain (Nicd) depends on acidification of endosome, this suggests that increased vesicle acidification in lgl- tissue leads to aberrant γ-secretase activity and cleavage of Next to Nicd, resulting in upregulation of Notch signaling (Parsons, 2014).

The precise endocytic compartment in which the Notch receptor undergoes γ-secretase-mediated S3 proteolytic processing is controversial. In Drosophila epithelial tissues, V-ATPase function is implicated in Notch activation in the EEs or the MVBs; however, whether this is ligand dependent or independent is unclear. In contrast, ligand-independent generation of Nicd in the LE and/or lysosome compartment has been described. Because increased Notch signaling in lgl- tissue depends on Rab5 EE activity, but Rab7 and Car LE compartments were not perturbed in lgl- tissue, a model is favored in which Lgl restricts vesicle acidification and activation of Notch signaling in the EE and/or early MVB compartments (Parsons, 2014).

It is speculated that Lgl regulates Notch signaling by two possible mechanisms: (1) via direct regulation of vesicle acidification or (2) via regulation of endosomal maturation, which indirectly affects vesicle acidification. In the first model, Lgl might inhibit V-ATPase activity by regulating levels and/or subunit composition or the association and/or dissociation of the V-ATPase complex. In the second model, Lgl might regulate endosomal maturation, and alterations in this process subsequently lead to accumulation of acidic vesicles and ectopic Notch activation. The data showing that lgl- tissue accumulates EEs (Avl+), REs (Rab11+), and early MVBs (Hrs+), but not LEs (Rab7+ or Car+), suggest that Lgl regulates a specific step in endosome maturation after early MVB formation. Further studies are required to determine whether Lgl controls vesicle acidification by affecting the V-ATPase or endosome maturation to regulate Notch signaling (Parsons, 2014).

Previous work has revealed that alterations in components of endocytic compartments, such as Rab5 overexpression or mutation of tsg101/ept or vps25, disrupt epithelial cell polarity and upregulate Notch signaling. However, in these cases, it is unclear whether perturbation of endocytosis alters Notch signaling via cell polarity disruption or whether changes in endocytic compartments directly impact on Notch. This study shows that without cell polarity loss, lgl- tissue displays altered endocytic compartments and upregulates Notch signaling, indicating that changes in endocytosis alone are sufficient to upregulate Notch pathway activity. Moreover, this study shows that reducing aPKC-Baz-Par6 complex activity does not rescue Notch pathway upregulation in lgl- tissue, revealing that Lgl's roles in regulating cell polarity and endocytosis are separable. Interestingly, the Crb polarity protein also has separable roles in the regulation of cell polarity and Notch signaling and endocytosis, via different mechanisms to Lgl (Parsons, 2014).

The discovery that Lgl depletion increases Notch activation without cell polarity loss has implications for tumorigenesis. In the developing eye epithelium, increased Notch signaling results in upregulation of the cell cycle regulator, CycA, and the cell survival regulator, Rst, which in lgl- tissue, is expected to contribute to increased cell proliferation and survival, concomitant with impaired Hippo signaling. Because elevated Notch signaling is associated with various human cancers, the finding that Lgl regulates Notch signaling warrants investigation of whether elevated Notch signaling in human cancer is associated with Lgl depletion. Notably, Lgl1 knockout in the mouse brain induces hyperproliferation and decreased differentiation, associated with increased Notch signaling, and mutation of zebrafish Lgl disrupts retinal neurogenesis, dependent on increased Notch signaling. Moreover, the finding that Lgl plays a novel role in regulating endosomal acidification and the striking suppressive effect of chloroquine on the adult lgl- mosaic phenotype reveal the importance of acidification in tumor growth, perhaps by also modulating Hippo signaling. The data, together with evidence that many cancers show higher acidity due to increased V-ATPase activity, which contributes to tumorigenesis, posit the question of whether Lgl dysfunction might contribute to acidification defects in human cancer (Parsons, 2014).

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate. Previous studies have shown that Notch/Delta molecules internalized in the mother cell traffic through Sara endosomes and are directed to the pIIa daughter. This study shows that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. The data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis (Loubery, 2014).

The Notch signaling pathway plays multiple roles in organisms ranging from flies and worms to mammals. A powerful model system to elucidate the cell biology of Notch signaling is the Drosophila sensory organs. Each sensory organ precursor (SOP) cell divides asymmetrically to produce a pIIa cell and a pIIb daughter cell, which perform directed Notch signaling: pIIb signals to pIIa. Four independent endocytic mechanisms control asymmetric signaling in the SOP. These include asymmetric endocytic events mediated by the E3 ubiquitin ligase Neuralized, recycling endosomes, and the endocytic adaptors α- and γ-adaptin together with Numb (Loubery, 2014).

During SOP cytokinesis, a fourth mechanism involves a population of endosomes marked by the adaptor protein Sara. Sara endosomes contain as cargo a pool of endocytosed Notch and Delta molecules. Notch and Delta reach the Sara endosome 20 min after their endocytosis in the SOP; this pool is dispatched into pIIa during cytokinesis. In contrast, the pools of Notch in endosomal populations upstream (Rab5 early endosomes) or downstream (Rab7 late endosomes) of Sara endosomes are segregated symmetrically. The specific pool of Notch in Sara endosomes is relevant for signaling: it is cleaved in a ligand- and gamma-secretase-dependent manner to release the transcriptionally active Notch intracellular domain (NICD) in pIIa (Loubery, 2014).

A key question is what machineries control the asymmetric targeting of these endosomes. Is the cargo (the ligand Delta or its receptor Notch) playing a role on the specific targeting of these endosomes? To unravel the machinery regulating the behavior of Sara endosomes during SOP mitosis, candidate factors from previously reported proteomics approaches or genetic screens were tested for Notch signaling. Thus, Uninflatable was identified as a factor involved in the asymmetric dynamics of Sara endosomes (Loubery, 2014).

MARCM homozygous mutant clones were generated for a null allele of Uninflatable (Uif2B7) an the trafficking of Delta, Notch, and the Notch effector Sanpodo through Sara endosomes was monitored. To look at the motility of the endogenous population of Sara endosomes, the cohort of internalized Delta molecules 20 min after its endocytosis was followed in the SOP by means of a pulse-chase antibody uptake assay. Delta, Notch, and Sanpodo traffic normally through Sara endosomes in the absence of Uif, and these endosomes are targeted to the cleavage plane (the central spindle) in cytokinesis (Loubery, 2014).

In Uif mutants or RNAi knockdown conditions, iDl20'/Sara endosomes fail to be asymmetrically dispatched to pIIa after their targeting to the central spindle. These results indicate that Uif is not required to bring Notch to the Sara endosomes or to target the endosomes to the central spindle. However, once in the spindle, Uif is essential for the specific dispatch of Sara endosomes from the spindle into the pIIa cell (Loubery, 2014).

This function of Uninflatable is specific to the asymmetric segregation of Sara endosomes. To gain mechanistic insights into the mechanism of action of Uif, this study has analyzed the density of microtubules in the central spindle and has shown that Uninflatable does not regulate the organization of the microtubular cytoskeleton. In contrast, it was found that Uif controls the residence time of Sara endosomes on the central spindle: in control SOPs, Sara endosomes depart from the central spindle with a decay time of 103 ± 21 s, whereas upon Uif downregulation this decay time goes up to 175 ± 42 s. These data indicate that Uif is not involved in the organization of the spindle, but rather in the motility properties of the endosomes, particularly their last step of departing from the central spindle and end up in pIIa (Loubery, 2014).

Consistent with the role of Uif in the asymmetric targeting of Sara endosomes, Uif contributes to Notch-dependent cell fate assignation in the SOP lineage. To address this, the composition of SOP lineages was examined in homozygous Uif2B7 MARCM clones or upon Uif RNAi. In wild-type animals, the SOP lineage consists of four different cells: two external cells (the shaft and the socket) originating from pIIa and two internal cells (the sheath and the neuron) from pIIb, which can be identified by immunostaining. In Uif mutant clones, instead of a sheath and a neuron per SOP lineage, two sheath cells can be frequently observed in the notum, indicating a symmetric division in the pIIb lineage. Similarly, upon Uif downregulation in the postorbital SOPs, duplications of sockets were observed, which is diagnostic of symmetric divisions in the pIIa lineage. These data uncover a role for Uninflatable in Notch-dependent asymmetric cell fate assignation that is mediated by the asymmetric dispatch of the Sara endosomes (Loubery, 2014).

The Uif phenotype during asymmetric endosomal targeting and cell fate assignation prompted us to look whether Uif is a cargo of Sara endosomes. To detect the endogenous protein, anti-Uif antibodies were generated. To look at Uif trafficking in vivo, transgenic flies were generated expressing a Uif-GFP protein, which can provide activity to rescue the lethality of a Uif lethal mutation at least partly (Loubery, 2014).

Uif-GFP is strongly colocalized with both Sara-GFP and iDelta20'. Since a cargo of Sara endosomes is Notch itself (73% ± 2.7% of the vesicular population of Notch molecules is in Sara endosomes), the presence of Notch cargo was examined in Uif vesicles: 44% ± 4.7% of Uif-positive vesicular structures contain Notch. Therefore, a population of Uninflatable and Notch traffics through Sara endosomes during SOP asymmetric mitosis (Loubery, 2014).

The fact that Uninflatable controls the asymmetric dispatch of the Sara endosomes, which contain internalized Notch and Uninflatable, prompted a look at a possible molecular interaction between Uninflatable and Notch. Uif- and Notch-expressing plasmids were cotransfected in S2 cells and immunoprecipitation experiments were performed by using anti-Uif-coupled beads, followed by immunoblotting with a clean anti-Notch antibody that was purified from a hybridoma cell line (DSHB #C17.9C6). Uif was shown to immunoprecipitate Notch. This coimmunoprecipitation can be reproduced from lysates of S2 cells expressing Notch and Uif tagged with the PC peptide tag and anti-PC-coupled beads; as a control, other transmembrane proteins such as Tkv-GFP are not coimmunoprecipitated with Uif-PC. Together, these results indicate a specific molecular interaction between Notch and Uif (Loubery, 2014).

Uninflatable is a transmembrane protein that, like Notch, contains an array of epidermal growth factor (EGF) repeats. It has been shown that Notch is engaged in protein-protein interactions through its EGF repeats with other factors containing EGF repeats. These include its ligand Delta, but also a number of noncanonical Notch ligands, secreted or membrane proteins lacking the DSL domain characteristic of canonical Notch ligands (Dlk-1, Dlk-2, DNER, Trombospondin, LRP1, EGFL7, and Weary). Consistently, it has recently been reported that a synergistic genetic interaction between Uif and Notch depends on Notch EGF repeats. Therefore, studies were performed to discover which EGF repeats of Uif could be involved in the molecular interaction with Notch. A coimmunoprecipitation experiment was performed in S2 cells coexpressing Notch and an N-terminal, truncated form of Uif tagged with PC (UifΔCter-PC) that lacks the four EGF domains flanking the transmembrane domain but still contains the other 17 EGF repeats and other extracellular domains. While full-length Uif-PC coimmunoprecipitates Notch, UifΔCter-PC does not. This indicates that the interaction between Uif and Notch may be mediated by the four EGF domains of Uif flanking its transmembrane domain (Loubery, 2014).

Although Uif binds and colocalizes with Notch, it does not play a role in core Notch signaling: embryos deprived of maternal and zygotic Uif in germline clones do not show a Notch signaling phenotype, whereas they display loss of inflation of the trachea as previously reported. Consistently, loss of Uif in wing mosaics does not cause a defect of Notch-dependent expression of Wingless at the wing margin. This indicates that Uninflatable interaction with Notch is not essential during core Notch signaling, but rather during the asymmetric dispatch of Notch-containing Sara endosomes during asymmetric cell division. This prompted the possibility that Notch itself is required for the asymmetric motility of the endosomes (Loubery, 2014).

To study whether Notch plays a role during the asymmetric dispatch of Sara endosomes, the trafficking was studied of a Notch-GFP fusion expressed at endogenous levels. The idea was to confirm previous observations using a Notch antibody uptake assay to follow Notch expressed at endogenous levels. To achieve this, a reporter transgenic fly strain was set up in which Notch-GFP fusion is driven by the Notch endogenous promoter and is expressed at endogenous levels. In this fusion, GFP is inserted in the middle of the Notch-intra domain. Since in protein fusions GFP is frequently cleaved out, whether the fusion protein is intact was examined. This would be particularly important in this case, since a cleavage event would lead to a truncated Notch-intra peptide (Loubery, 2014).

In these transgenic Notch-GFP flies, GFP is very efficiently cleaved out (74% of total GFP is cleaved, leading to truncated Notch-intra peptides that can only partially support Notch function and thereby cause a highly penetrant mutant phenotype. This precludes the usage of this reagent as a bona fide marker for Notch. In particular, the cytosolic GFP signal cannot be used as a readout of signaling as previously reported: a nuclear accumulation of the GFP signal in these flies does not solely reflect the accumulation of Notch-intra-GFP, but rather the overall accumulation of different GFP-containing fragments (Loubery, 2014).

Whether, in these conditions, the pool of membrane associated GFP-Notch traffics through Sara endosomes and is asymmetrically dispatched to the pIIa cell was studied. Only 11% ± 1.3% of the total GFP signal in these flies is membrane associated (plasma membrane and intracellular vesicular structures). The rest, representing the vast majority (89%), corresponds to cytosolic and nuclear cleaved GFP (Loubery, 2014).

In Notch-GFP flies, 3.1% of the total GFP signal is associated with intracellular vesicular structures. These correspond to various intracellular vesicular compartments, including Notch in the secretory pathway, as well as in early endosomes, Sara endosomes, recycling endosomes, and late endosomes. To measure the size of the specific pool of Notch in Sara endosomes, a Notch antibody internalization assay was performed, and internalized Notch was chased 20 min after its endocytosis (iNotch20'). As previously established, 73% ± 2.7% of Notch-GFP vesicles are positive for iNotch20'. Of this iNotch20'-positive pool, 79% would be targeted to pIIa . This is consistent with only 65% ± 3.1% of the total pool of Notch-GFP being dispatched to pIIa (Loubery, 2014).

Whether Notch itself plays a role on the asymmetric targeting of Sara endosomes was addressed. Notch was depleated in the SOP by expressing a previously validated Notch dsRNA, and the behavior of Sara endosomes was examined. Upon Notch knockdown in the SOP, iDl20'/Sara endosomes are still targeted to the central spindle, but the subsequent directed dispatch to pIIa is defective. This indicates that Notch itself contributes to the endosomal recruitment of the machinery that endows the Sara endosomes with their asymmetric behavior (Loubery, 2014).

It has been shown that the targeting of Notch to Sara endosomes does not depend on Uninflatable; it was then determined whether the recruitment of Uninflatable on Sara endosomes depended on Notch. Interestingly, it was found that, conversely, the targeting of Uif to Sara endosomes is not controlled by Notch. This implies that these two molecules use different machineries to get to the endosome, where they can interact and are both required for the asymmetric motility of the endosome (Loubery, 2014).

Since the Notch receptor itself is required for the asymmetric targeting of Sara endosomes, it was asked whether Notch signaling plays a role in the process. Notch signaling was blocked by inactivating the ligand Delta through overexpression of Tom in the SOP cell; Tom overexpression leads to inactivation of the Ubiquitin ligase Neuralized and thereby blocks endocytosis-dependent activation of Delta. In the absence of Notch signaling, targeting of Sara endosomes to the central spindle and their asymmetric dispatch to the pIIa cell remains intact. This indicates that although the Notch receptor is essential for the asymmetric targeting of Sara endosomes, Notch signaling is not (Loubery, 2014).

This report has started to unravel the machinery that mediates asymmetric endosome motility during asymmetric cell division. Both Notch and Uninflatable were shown to play a key role in the last step of the asymmetric motility of endosomes: the final, specific stride of the Sara endosomes from the central spindle into the anterior pIIa cell. This is based on the following four key sets of observations (Loubery, 2014).

First, it was confirmed that a functional Notch-GFP fusion expressed at endogenous level does traffic through Sara endosomes, which are indeed dispatched asymmetrically during SOP mitosis. Second, Notch binds Uninflatable, and both colocalize in Sara endosomes. Third, neither Notch nor Uninflatable is essential for the targeting of Notch/Delta/Uif to the Sara endosomes or the targeting of those endosomes to the central spindle, but they are essential for the final dispatch from the central spindle into the pIIa cell. Although Notch is necessary for this process, Notch signaling is not. Fourth, Uninflatable is not an integral component of the Notch signaling pathway, but it plays a role during asymmetric Notch signaling in the SOP, and therefore mutant Uif conditions lead to a lineage identity phenotype. It remains to be elucidated what machineries downstream of Notch/Uninflatable implement the control of the final step toward pIIa and what is asymmetrical in the cytoskeleton so that this final step occurs toward pIIa and not pIIb (Loubery, 2014).

Identification of Atg2 and ArfGAP1 as candidate genetic modifiers of the eye pigmentation phenotype of Adaptor Protein-3 (AP-3) mutants in Drosophila melanogaster

The Adaptor Protein (AP)-3 complex is a molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. A forward screening was performed for genetic modifiers of the eye pigmentation AP-3 (carmine) gene in Drosophila. One modifier was the Atg2 gene, encoding a conserved protein involved in autophagy. Loss of one copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, Biogenesis of lysosome-related organelles complex 1, subunit 1 (Blos1) and lightoid (Rab32), and even increased the eye pigment content of wild-type flies. A second modifier was the ArfGAP1 gene, encoding a conserved GTPase-activating protein. Loss of a single copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants. Strikingly, loss of the second copy of the gene elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 or lightoid. These results provide evidence for functional links connecting the machinery for biogenesis of LROs with autophagy and small GTPase regulation (Rodriguez-Fernandez, 2015).

Efficient endocytic uptake and maturation in Drosophila oocytes requires Dynamitin/p50

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. This study examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. It is therefore proposed that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor co-localized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. These results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation (Liu, 2015).

Cooperative functions of the two F-BAR proteins Cip4 and Nostrin in regulating E-cadherin in epithelial morphogenesis

F-BAR proteins are prime candidates to regulate membrane curvature and dynamics during different developmental processes. This study analyzed nostrin (nost), a novel Drosophila F-BAR protein related to Cip4. Genetic analyses revealed a strong synergism between nost and cip4 functions. While single mutant flies are viable and fertile, combined loss of nost and cip4 results in reduced viability and fertility. Double mutant escaper flies show enhanced wing polarization defects and females exhibit strong egg chamber encapsulation defects. Live-imaging analysis suggests that the observed phenotypes are caused by an impaired E-cadherin membrane turnover. Simultaneous knock-down of Cip4 and Nostrin strongly increases the formation of tubular E-cadherin vesicles at adherens junctions. Cip4 and Nostrin localize at distinct membrane subdomains. Both proteins prefer similar membrane curvatures but seem to form different membrane coats and do not heterooligomerize. These data suggest an important synergistic function of both F-BAR proteins in membrane dynamics. A cooperative recruitment model is proposed in which first Cip4 promotes membrane invagination and early actin-based endosomal motility while Nostrin makes contact with microtubules through the kinesin Khc-73 for trafficking of recycling endosomes (Zobel, 2015).

Members of the Fes-CIP4 homology Bin-amphiphysin-Rvs161/167 (F-BAR) protein family form crescent-shaped dimers that are able to shape membranes into vesicles and tubules. F-BAR proteins have been grouped into six subfamilies, the Cdc42-interacting protein 4 (Cip4) subfamily, the Fes subfamily of non-receptor tyrosine kinases, the protein kinase C and casein kinase substrate in neurons protein (pacsin) subfamily, the Slit-Robo RhoGTPase-activating proteins (SrGAPs), the FCH-domain-only (FCHO) and the proline-serine-threonine phosphatase-interacting protein (PSTPIP) subfamilies. The phylogenetic subgrouping is mainly based on structural similarities of the N-terminal F-BAR module, and on the composition and architecture of C-terminal domains. Distinct differences of the intrinsic F-BAR domain curvature observed among the different F-BAR-domain proteins are thought to reflect characteristic preferences in sensing and/or inducing membrane invaginations of different curved geometry. Consistent with this idea, members of FCHO subfamily bind to very low membrane curvatures and are found to be essential for the initial step of membrane invagination in endocytosis. Other F-BAR proteins, such as members of the Cip4 subfamily, which includes the Cdc42-interacting protein 4 (Cip4), the transducer of Cdc42-dependent actin assembly (Toca-1) and the formin-binding protein 17 (FBP17), have a preference for higher membrane curvatures present in later steps during vesicle formation (Zobel, 2015 and references therein).

Unlike those of the FCHO subfamily, Cip4 subfamily proteins contain a C-terminal SH3 domain that binds dynamin and factors that promote actin filament formation. All three members of the Cip4 subfamily are able to activate N-WASP by promoting Arp2/3-mediated actin nucleation in vitro. Cip4 and Toca-1 also associate with Cdc42 through a central coiled-coil region. The current view is that Cip4-related proteins may stabilize plasma membrane invaginations and, subsequently, recruit dynamin and WASP proteins to the neck of endocytic pits that mediate the constriction and scission of vesicles. Recruitment of WASP proteins to newly formed vesicles also promotes the formation of actin comet tails that provide the driving force for endocytic vesicle movement. However, understanding of how F-BAR proteins function in vivo in a physiological context is still incomplete because loss-of-function studies in higher organisms are limited. Mice that lack Cip4 are viable and show only a weak endocytosis defect of the insulin-responsive glucose transporter Glut4. Mutant animals also display a reduced platelet production and defective integrin-dependent T-cell adhesion. Both defects are probably caused by decreased WASP-dependent actin polymerization, rather than impaired endocytosis (Chen, 2013). Given the mild phenotypes, the two other Cip4-like subfamily members Toca-1 and FBP17 might have redundant functions and could compensate for the loss of Cip4 function (Zobel, 2015).

Initial RNA interference (RNAi) studies in Drosophila melanogaster revealed that Cip4 regulates dynamin-dependent endocytosis of E-cadherin at adherens junctions. As in mammals, function of cip4 is not essential for fly development. cip4 mutants show duplicated wing hairs because of an impaired endocytosis. Further analyses revealed that Cip4 acts downstream of Cdc42 to activate the WASP-WAVE-Arp2/3 pathway in the notum and the wing epithelium. In addition, a postsynaptic, endocytosis-independent function of Cip4 has been identified at the neuromuscular junction. This function also depends on an interaction with the Cdc42-WASP-Arp2/3 pathway but does not require a functional F-BAR domain (Zobel, 2015 and references therein).

The Drosophila genome contains an additional, not yet characterized gene encoding a Cip4-like F-BAR protein with highest similarities to human Nostrin (CG42388). Human Nostrin was originally identified as an interaction partner of the endothelial nitric oxide synthase (eNos). Cell culture studies further suggest that Nostrin regulates N-WASP and/or dynamin-dependent trafficking and the activity of endothelial nitric oxide synthase (eNos). However, an in vivo role of Nostrin in the regulation of eNos activity or endocytosis has not yet been found. A recent loss-of-function study in zebrafish and mice revealed a role of Nostrin in endothelial cell morphology during vascular development. Antisense morpholino oligonucleotide (MO)-mediated knockdown of Nostrin in developing zebrafish affects the migration of endothelial tip cells of intersegmental blood vessels. Remarkably, nostrin-knockout mice are viable and show only mild retinal angiogenesis defects. This suggests that other F-BAR proteins compensate for nostrin function in mutant mice (Zobel, 2015 and references therein).

As Drosophila contains only a single gene copy of each F-BAR subfamily, studies in flies are well-suited to address putative functional redundancies within and between F-BAR domain subfamilies. This study presents a functional analysis of CG42388, which encodes the Drosophila Nostrin protein, and its physiological relationship to Cip4. Flies that lack Nostrin are viable and fertile. However, loss of both nostrin and cip4 results in reduced viability and fertility. Double mutant flies show a strong multiple wing hair phenotype and females are semi-sterile. Egg chambers of double mutant flies show strong encapsulation defects that are likely to be caused by an impaired membrane turnover of E-cadherin. Cip4 and Nostrin, preferentially, bind similar membrane curvatures but localize at distinct subdomains of membrane structures in cells. These data suggest an important, non-redundant function of Cip4 and Nostrin in the regulation of membrane dynamics in epithelial morphogenesis (Zobel, 2015).

F-BAR proteins play an important role in the regulation of membrane curvatures in a sequential manner during endocytosis. Despite their pivotal functions in linking actin and membrane dynamics, recent single-knockout studies revealed that many F-BAR proteins are not essential for development and, thus, might have redundant or cooperative functions in vivo. In fission yeast, the Cip4-like F-BAR proteins Cdc15 and Imp2 are examples for such synergistic function of two F-BAR proteins. Cells deficient for either Cdc15 or Imp2 show mild defects in cytokinesis but are still able to divide. However, deletion of both C-terminal SH3 domains of the proteins completely restricts the division of the cells (Zobel, 2015 and references therein).

This study provides first evidence for cooperative function of the two F-BAR proteins Cip4 and Nostrin in the multicellular context of Drosophila development. Like Cip4, members of the Nostrin subfamily show a remarkably high evolutionary conservation and single orthologs can be found from porifera to humans. All the more surprising is the fact that nostrin loss-of-function mutant flies have no obvious phenotype. Only after removal of both cip4 and nostrin, was a strong enhancement found of the phenotypic traits already observed in cip4 single mutants. Double mutants show a substantial reduction in the number of offspring. Both mutant females and males display reduced fertility, indicating a common function of both F-BAR proteins in early morphogenesis. Female sterility of double mutant flies is caused by strong defects in egg chamber morphogenesis. The formation of compound egg chambers results from a defective encapsulation in the germarium, a phenotype that has not yet been described for many mutants. Most mutations that have been reported of so far, either affect gene functions directly through the regulation of cell division or control of follicle cell differentiation through Notch/Delta signaling. However, multicyst egg chambers in nost;;cip4 double mutants display neither defects in cell division nor in the differentiation of follicle cells (Zobel, 2015).

Interestingly, loss of Maelstrom (Mael), a high-mobility group box protein that regulates microtubule organization leads to egg chambers with cell division defects but also results in an encapsulation defect with misplaced oocytes that is similar to the one observed in nost;;cip4 double mutants. Mael forms a complex with the components of the microtubule-organizing center (MTOC) -- including centrosomin and γ-tubulin, which seems to be required not only for early oocyte determination but also egg chamber packing and oocyte positioning in the germarium. Interestingly, in mael mutant multicyst egg chambers E-cadherin is not enriched on the oocyte cortex and not apically concentrated in the follicle epithelium as in wild type. nost;;cip4 double mutant egg chambers show a similar E-cadherin mislocalization, suggesting that the microtuble cytoskeleton plays an important role in E-cadherin localization. Consistently, Nostrin mainly localizes to Rab11-positive vesicles that move along microtubules. Thus, Nostrin might act on Rab11-dependent E-cadherin trafficking along microtubules. A strong requirement for Rab11 in E-cadherin trafficking in germline stem cells (GSC) and in the maintenance of GSC identity has recently been identified. Mosaic egg chambers are severely disorganized, comprising mispositioned oocytes. Most importantly, compound egg chambers can be found that contain two or more germline cysts surrounded by a single continuous follicle epithelium, as was observed in this study for nost;;cip4 mutants. However, rab11-null follicle stem cells (FSC) give rise to the normal number of cells that enter polar, stalk and epithelial cell differentiation pathways. Like Rab11, Nostrin and Cip4 functions do not seem to be required in follicle cell differentiation (Zobel, 2015).

Given the dramatic switch in Nostrin expression in the germline cysts between region 2a and region 2b, when protein levels drop from highest to very low or no expression, an important function is proposed of Nostrin in germ cells rather than in somatic follicle cells. The germline cyst undergoes a dramatic change in shape within this region, as is reflected by a transformation from a spherical to a lens-shaped structure. This morphological transition might imply important changes in the adhesiveness mediated by homophilic E-cadherin cell-cell contacts between germ cells within the cyst. Thus, it is proposed that Nostrin and Cip4 are involved in the regulation of this transition by controlling E-cadherin endocytosis and vesicle recycling in germline cells. A failure of nost;;cip4 mutant cysts in adopting a lens-shaped morphology might interfere with their encapsulation by follicle cells, which results in the formation of compound egg chambers (Zobel, 2015).

Given the substantial mislocalization of E-cadherin that becomes obvious in double mutant egg chambers, an additional role of both F-BAR proteins in the maintenance of E-cadherin cell-cell contacts between germline and somatic follicle cells is suggested (Zobel, 2015).

Cip4 and Nostrin partially mark the same membrane structures but they localize to distinct subregions in vivo. Consistently, in vitro liposome studies showed that both proteins prefer defined membrane curvatures of similar diameter, i.e. both might associate with similarly shaped membrane compartments. However, the different appearance of Cip4- and Nostrin-decorated vesiculo-tubular structures might also reflect differences in lattice formation of these two F-BAR domain proteins. It is hypothesized that these regular arrays of electron-dense structures at liposomes represent regular Cip4 lattices formed upon self-association by head-to-tail and lateral interactions as previously supposed by real-space reconstruction. Because such patterns were not observed at tubular structures following incubation in the presence of Nostrin, it is suggested that Nostrin does not polymerize into rigid helical coats that are thought to be the structural basis for membrane invagination. Consistently, unlike Cip4, overexpression of Nostrin in S2R+ cells did not induce membrane tubulation. How do Cip4 and Nostrin cooperate in membrane remodeling and vesicle trafficking? In cells, Cip4 mainly localizes to Rab5-positive early endosomes, whereas Nostrin marks both Rab5- and Rab11-positive vesicles. Thus, a cooperative recruitment model is proposed, in which first Cip4 promotes membrane invagination, vesicle scission and motility of Rab5-positive membrane compartments by recruiting dynamin and the WASP-WAVE-Arp2/3 pathway. Nostrin will then be recruited to Cip4-positive membrane structures because Nostrin prefers the curvature induced by the highly organized Cip4 coats. Yet, at these membrane compartments, both proteins still occur in a spatially segregated manner, as visualized in EM analyses of Cip4- and Nostrin-coated membrane tubules. This segregation might reflect that Nostrin is not interacting with Cip4, and that Cip4 has the ability to bind PE - which Nostrin does not have. Furthermore Nostrin protein arrays seem less organized, as reflected by the broader range of curvatures induced in vitro and by the lack of regular structures of Nostrin-coated membranes. Strong formation of rigid lattices might explain why Cip4 tubulates membranes effectively in vitro and in vivo, and why anti-Cip4 labeling usually outlines tubular structures. In contrast, Nostrin is confined to Cip4-free segments of these structures and predominantly appears at the end of such Cip4-coated tubules because the end does not accommodate a Cip4 coat optimized for cylindrical surfaces (Zobel, 2015).

Interestingly, Kif13A (a kinesin motor that directly binds Rab11) is most enriched at the tips of membrane tubules. Moreover, Kif13A localizes to distinct Rab11-positive subdomains within sorting endosomes and is thought to initiate the formation of recycling endosomal tubules along microtubules through its motor activity. Interestingly, Nostrin directly interacts with the Drosophila Kif13A homologue Khc73 and colocalizes with Khc73-marked endosomes that move along microtubules. A close link between Nostrins and kinesin motors seem to be evolutionarily conserved and the interaction is likely mediated by a conserved bipartite tryptophan-based kinesin-1 binding motif. Thus, in the current model of cooperative recruitment, Cip4 stabilizes endosomal tubules, whereas Nostrin defines subdomains of recycling intermediates of endosomal tubules and makes contact with microtubules through the kinesin Khc-73 for long-range trafficking of recycling endosomes (Zobel, 2015).

A similar scenario might also take place during cell polarization of the wing epithelium. Here, Cip4 and Nostrin act together to control the polarized outgrowth of a single actin-rich protrusion called prehair, a process that also requires tight coupling of membrane trafficking and the cytoskeleton. The restriction of wing hair formation at the most distal apical vertex of each wing cell depends on the Frizzled-PCP signaling pathway. A key step in the cell polarization is the asymmetric localization of core PCP proteins at adjacent cell membranes within the plane of the epithelium. Thus, one of the central questions in understanding PCP signaling is how this asymmetric localization is achieved. Based on live-imaging studies, selective endocytosis and directional transport of Frizzled along polarized non-centrosomal microtubules have been proposed as possible mechanisms for asymmetric polarization. Previous studies that had used microtubule antagonists already revealed an important role of the microtubule cytoskeleton in order to localize prehair initiation to the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. Multiple pre-hair formation is also caused by overexpression of Frizzled, presumably through an ectopic activation of the pre-hair nucleation machinery. However, the multiple wing hair phenotype in nost;;cip4 double mutant wings does not seem to affect the asymmetric distribution of Frizzled. Interestingly, a similar Frizzled-independent multiple wing hair phenotype has recently been observed in mutants that affect casein kinase 1γ (CK1γ, also known as CSNK1G). Loss of CK1γ disrupts the apical localization of Rab11 at the base of prehairs, suggesting that Ck1γ regulates Rab11-mediated polarized vesicle trafficking that is required for prehair nucleation. Consistently, expression of either a dominant-negative or a dominant-active Rab11 variant strongly induces the formation of multiple wing hairs. Overexpression of Cip4 or Nostrin alone also phenocopies the multiple wing hair defect of nost;;cip4 double mutants. Like CK1γ and Rab11, Cip4 and Nostrin accumulate at the base of forming prehairs. Because Nostrin colocalizes with Rab11-positive vesicles that move along microtubules, it is proposed that Nostrin is involved in Rab11-mediated polarized vesicle trafficking in the developing wing (Zobel, 2015).

Polarized Rab11-dependent vesicle trafficking of E-cadherin is also needed for the hexagonal packing of wing cells. During this process, irregularly shaped cells adopt a hexagonal geometry by coordinated endocytosis and Rab11-dependent recycling of junctional E-cadherin. Hexagonal packing starts shortly after pupal molt and ends just before wing hair formation but, remarkably, also depends on components of the PCP pathway. The underlying molecular mechanism that links hexagonal packing and hair formation in the wing is unknown. However, both processes depend on vesicle trafficking because suppression of Rab11, Rab23 or the simultaneous knockdown of cip4 and nostrin results not only in multiple wing hairs but also affects the regular hexagonal array of wing epithelial cells. A similar E-cadherin-dependent process of cell packing and remodeling can also be observed in the dorsal thorax, an epithelium that originally derived from the fused proximal parts of two wing imaginal discs. A role in E-cadherin membrane turnover has already been reported for Cip4 in the developing thorax epithelium of Drosophila. In cells that express cip4 dsRNA, E-cadherin-GFP accumulates in apical punctate structures and elongated malformed tubules form at the cell cortex. These long and defective endocytic structures do not tolerate fixation and could only be observed in live-imaging experiments. This study observed an even stronger defect on E-cadherin membrane dynamics upon simultaneous downregulation of Cip4 and Nostrin. The number of elongated malformed tubules that form at the cell cortex is clearly increased. Moreover, knockdown of both cip4 and nostrin cause obvious defects in the formation of E-cadherin junctions, a phenotype that was never observed when suppressing either cip4 or nostrin. These strong junctional defects might be responsible for the lethality of late pupae following RNAi transgene expression by the aptereous-Gal4 driver. Thus, it is concluded that both F-BAR proteins play an important cooperative rather than a redundant function in E-cadherin trafficking and junction maintenance (Zobel, 2015).

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. This study shows that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. Vps8 forms a complex and acts together with Vps16A, Deep Orange/Vps18 and Carnation/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Light/Vps41. This study thus identifies a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila (Lorincz, 2016).

ARC syndrome-linked Vps33B protein is required for inflammatory endosomal maturation and signal termination

Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) sense microbial ligands and initiate signaling to induce inflammatory responses. Although the quality of inflammatory responses is influenced by internalization of TLRs, the role of endosomal maturation in clearing receptors and terminating inflammatory responses is not well understood. This study reports that Drosophila and mammalian Vps33B proteins play critical roles in the maturation of phagosomes and endosomes following microbial recognition. Vps33B was necessary for clearance of endosomes containing internalized PRRs, failure of which resulted in enhanced signaling and expression of inflammatory mediators. Lack of Vps33B had no effect on trafficking of endosomes containing non-microbial cargo. These findings indicate that Vps33B function is critical for determining the fate of signaling endosomes formed following PRR activation. Exaggerated inflammatory responses dictated by persistence of receptors in aberrant endosomal compartments could therefore contribute to symptoms of Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, a disease linked to loss of Vps33B (Akbar, 2016).

A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis

Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. This study identified and characterized a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. It is proposed that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis (Yao, 2009).

Ca2+ influx triggers both SV exo- and endocytosis. Since SV retrieval requires much lower Ca2+ levels than those that elicit release, it was proposed that the Ca2+ levels needed to initiate endocytosis derived from diffusion of VGCC-dependent Ca2+ influxes from active zones. However, several reports have documented a requirement for extracellular Ca2+ during endocytosis. Furthermore, it has been proposed that a specific Ca2+ channel is required for SV endocytosis in Drosophila. The present study identified and characterized a SV- and presynaptic membrane-associated protein with three or four transmembrane domains that is evolutionarily conserved but has not been previously characterized in any species. Animals that lack Flower display the typical features of endocytic mutants. These include supernumerary boutons, a low number of SVs in boutons at rest, a severe depletion of SVs upon stimulation (except at active zones), enlarged SVs, a decrease in FM1-43 uptake, a rundown in neurotransmitter release upon repetitive stimulation, and an accumulation of endocytic intermediates (Yao, 2009).

flower mutants exhibit impaired Ca2+ handling, even when the boutons are at rest. This argues that Flower plays a role in Ca2+ homeostasis at rest as well as during endocytosis. Since Flower is associated with SVs and the presynaptic membrane, a reduction in Flower levels may cause lower resting Ca2+ levels because Ca2+ efflux from SVs or Ca2+ influx from the extracellular compartment is impaired. However, a role for Flower in SVs is unlikely. First, experimental evidence suggests that SVs have no or a very limited role in the sequestration of Ca2+ at NMJs in Drosophila. Second, although single SV fusions with the plasma membrane in flower mutants elicit larger amplitudes than in wild-type animals, our data suggest that this is due to the fact that the SVs in flower mutants are larger than wild-type SVs. Indeed, there is a near perfect correlation between the SV size and the size of the mEJPs, as observed in some other endocytic mutants. This suggests that the Flower protein present in the presynaptic membrane affects the resting Ca2+ levels, but does not exclude a role for the protein in SVs (Yao, 2009).

The second TM domain of Flower contains a 9 aa motif similar to the selectivity filters identified in Ca2+-gating TRPV channels. TRPV5 and 6 channels are homo- or multimeric channels that have been shown to form a pore lined by four negatively charged amino acids, similar to VGCCs. This study shows that Flower can form tetramers and higher order multimers and that its heterologous expression in salivary gland cells promotes Ca2+ influx. In addition, substitution of a conserved, negatively charged glutamate (E) residue in the second TM domain with a neutral amino acid (Q) abolishes this Ca2+ influx. Furthermore, the purified Flower protein in proteoliposomes can form a Ca2+-permeable cationic channel. A simple model is proposed to account for the data. SV exocytosis is triggered by VGCCs located at active zones. Subsequently, SVs, and hence Flower proteins are integrated in the presynaptic membrane, where they mostly localize to periactive zones, known sites for endocytosis). A homomultimeric Flower complex then promotes Ca2+ influx which triggers clathrin-mediated endocytosis. This is also supported by the observation that higher extracellular Ca2+ alleviates the endocytic defect. It is proposed that endocytosis removes most, but not all, of the Flower protein, thereby removing a key trigger for endocytosis. Thus, Flower may perform a simple autoregulatory role for itself during endocytosis. This model also addresses how exo-endocytosis coupling may be mediated at presynaptic terminals. The remainder of the Flower protein that is not endocytosed may help to regulate basal Ca2+ levels (Yao, 2009).

Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture

Neurotransmission requires anterograde axonal transport of dense core vesicles (DCVs) containing neuropeptides and active zone components from the soma to nerve terminals. However, it is puzzling how one-way traffic could uniformly supply sequential release sites called en passant boutons. In this study Drosophila neuropeptide-containing DCVs are tracked in vivo for minutes with a new method called simultaneous photobleaching and imaging (SPAIM). Surprisingly, anterograde DCVs typically bypass proximal boutons to accumulate initially in the most distal bouton. Then, excess distal DCVs undergo dynactin-dependent retrograde transport back through proximal boutons into the axon. Just before re-entering the soma, DCVs again reverse for another round of anterograde axonal transport. While circulating over long distances, both anterograde and retrograde DCVs are captured sporadically in en passant boutons. Therefore, vesicle circulation, which includes long-range retrograde transport and inefficient bidirectional capture, overcomes the limitations of one-way anterograde transport to uniformly supply release sites with DCVs (Wong, 2012).

To determine how the uniform neuropeptide stores in Drosophila motoneuron type Ib boutons are supplied, the Geneswitch (GS) system was used to induce expression of Emerald GFP-tagged Atrial Natriuretic Factor (Anf-GFP), a reporter of native neuropeptide packaging and release in Drosophila larvae. Independent of Anf-GFP labeling, boutons were detected with a TRITC-conjugated anti-horseradish peroxidase antibody (TRITC-HRP) and numbered from distal to proximal. Surprisingly, neuropeptide accumulated initially in the most distal bouton (#1) and only later was distributed uniformly among en passant boutons #1-4. Similar results were obtained with heat shock induction of GFP-tagged Drosophila proinsulin-like peptide 2 (Dilp2-GFP). Therefore, the initial distal accumulation cannot be attributed to a particular neuropeptide or induction mechanism. Finally, when neuropeptide release was inhibited by removing extracellular Ca2+, fluorescence recovery after photobleaching (FRAP) of constitutively expressed Anf-GFP (i.e., driven by elav-GAL4) also revealed preferential neuropeptide accumulation in the most distal bouton. This finding independently verifies the polarized neuropeptide accumulation detected with pulse labeling and furthermore proves that this pattern cannot be explained by differential release by en passant boutons (Wong, 2012).

One-way anterograde transport of DCVs was believed to fully explain neuropeptide delivery to nerve terminals. However, SPAIM in different neuronal compartments (i.e., en passant boutons, axonal branches, the proximal axon, and the soma) showed that neuropeptide accumulation in nerve terminal release sites is mediated by sporadic anterograde and retrograde capture of DCVs circulating between the proximal axon and distal boutons. These findings cannot be attributed to phototoxicity as they are supported by SPAIM-independent induction and mutant experiments, the match between flux measured without photobleaching, the consistency of DCV behavior after multiple bouts of photobleaching, and the detection of release from single DCVs, which demonstrates viability and function (Wong, 2012).

It was surprising that DCVs travel such far distances and that their presynaptic capture is inefficient, but this elegantly overcomes the limitations of relying solely on one-way transport to produce the uniform neuropeptide accumulation that characterizes en passant boutons. Indeed, the conundrum of how to ensure that all potential release sites can function effectively regardless of distance from the soma or presence on different branches, which is critical for neurons, is resolved by vesicle circulation. Vesicle circulation also accounts for the long known, but mysterious, abundance of retrograde DCVs in axons and ensures that DCVs do not readily return to the soma to be degraded. Furthermore, vesicle circulation provides a novel explanation for the finding that perturbing retrograde DCV transport in neurons affects anterograde transport: interrupting retrograde transport prevents circulating retrograde DCVs from contributing to anterograde flux and so would reduce total anterograde transport. Finally, because the same kinesin motor transports neuropeptides and many other presynaptic proteins, vesicle circulation could be a general strategy for maintaining the synaptic function of en passant boutons. In this light, it is interesting that retrograde transport is compromised in neurodegenerative diseases. A previously unconsidered contributing factor to the early onset loss of synaptic function could be that diseases that affect dynactin-dependent retrograde transport perturb vesicle circulation that normally maintains the terminal. Therefore, vesicle circulation may be significant under physiological and pathological conditions (Wong, 2012).

SPAIM detects DCVs in a native synapse for many minutes. In contrast, it was found that photoactivation-based approaches using PAGFP, tdEosFP and mOrange did not work well with Drosophila DCVs. This may be due to suboptimal targeting, difficulty in inducing photoactivation in the mildly acidic and oxidizing lumen of Drosophila DCVs, and the lower brightness and poorer folding of many photoactivatable proteins compared to emerald GFP. Given the availability of bright, well characterized GFP constructs, SPAIM represents a tenable method for tracking DCVs in native Drosophila neurons. In addition to detecting vesicle circulation, SPAIM could be used to study how presynaptic DCV mobilization is induced by activity in a native synapse and how DCV traffic changes with synaptic development and plasticity. Furthermore, SPAIM can detect release from single DCVs induced by nerve stimulation, which opens the possibility of studying peptidergic transmission at the level of individual vesicles in a native synapse. Therefore, SPAIM could answer many questions concerning neuronal DCVs (Wong, 2012).

In principle, SPAIM could be applied to any fluorescent organelle. However, a dual scanner confocal microscope, which was used in this study, might not be ideal for detecting very dim signals. When optical sectioning is not required, this limitation could be overcome by using a single scanner system for initially photobleaching a region of interest and then for continually photobleaching newcomers while simultaneously imaging individual organelles with a sensitive camera. In fact, performing SPAIM experiments by coupling a camera with a common single scanner confocal microscope may be an attractive option to overcome both the insensitivity and the expense of dual scanner systems. With the appropriate setup, SPAIM could be used to study traffic in terminals, dendrites, cilia, and filopodia (Wong, 2012)

Crimpy enables discrimination of presynaptic and postsynaptic pools of a BMP at the Drosophila neuromuscular junction

Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. This study presents evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, it is proposed that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal (James, 2014).

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4 GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. This study reports that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network (Dey, 2017).

Immunolocalization of the vesicular acetylcholine transporter in larval and adult Drosophila neurons

Vesicular acetylcholine transporter (VAChT) function is essential for organismal survival, mediating the packaging of acetylcholine (ACh) for exocytotic release. However, its expression pattern in the Drosophila brain has not been fully elucidated. To investigate the localization of VAChT, an antibody against the C terminal region of the protein was developed; this antibody recognizes a 65KDa protein corresponding to VAChT on an immunoblot in both Drosophila head homogenates and in Schneider 2 cells. Further, the expression is reported of VAChT in the antennal lobe and ventral nerve cord of Drosophila larva, and the expression was confirmed of the protein in mushroom bodies and optic lobes of adult Drosophila. Importantly, it was shown that VAChT co-localizes with a synaptic vesicle marker in vivo, confirming previous reports of the localization of VAChT to synaptic terminals. Together, these findings help establish the vesicular localization of VAChT in cholinergic neurons in Drosophila and presents an important molecular tool with which to dissect the function of the transporter in vivo (Boppana, 2017).

De-regulation of JNK and JAK/STAT signaling in ESCRT-II mutant tissues cooperatively contributes to neoplastic tumorigenesis

Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. This study shows that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of JNK, Notch, and JAK/STAT signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis (Woodfield, 2013).

While it is well established how de-regulated signaling pathways in ESCRT-II mutant clones mediate non-cell autonomous interactions with neighboring non-mutant cells to contribute to hyperplastic overgrowth and increased cell survival, it was largely unknown which signaling pathways trigger neoplastic transformation autonomously. To address this question, predominantly mutant eye-antennal imaginal discs were generated in which competitive interactions are eliminated so that it was possible to examine the autonomous results of de-regulated signaling (Woodfield, 2013).

Overall, it appears that the same signaling pathways that are induced in mosaic clones are also activated in predominantly mutant tissues. However, two results of this study are noteworthy. First, it is surprising that JNK activity is strongly induced in tissues predominantly mutant for ESCRT-II genes. This is surprising because JNK signaling was believed to be induced by cell competition from neighboring non-mutant cells in mosaic tissues. However, non-mutant tissue is largely eliminated by the ey-FLP/cl method and thus competitive interactions are eliminated. Therefore, it is not known how JNK signaling is induced in these tissues. Nevertheless, JNK signaling is critical for the overgrowth phenotype of predominantly ESCRT-II mutant eye discs as inhibition of this pathway partially blocks cell proliferation. Second, de-regulation of the JAK/STAT signaling pathway is critical for the neoplastic transformation of vps22 mutant discs. Loss of JAK/STAT signaling dramatically normalizes the neoplastic phenotype of vps22 mutant cells. In addition to JNK and JAK/STAT activity, Notch activity was also found to be increased in discs predominantly mutant for ESCRT-II genes. Therefore, a genetic requirement of Notch signaling was tested for neoplastic transformation of ESCRT-II mutant cells. However, loss of Notch was inconclusive because even the wild-type control discs did not grow when Notch was inhibited (Woodfield, 2013).

Interestingly, although ESCRT-II mutant tissues undergo neoplastic transformation, they also show high levels of apoptosis. Animals with predominantly mutant eye-antennal imaginal discs die as headless pharate pupae, a phenotype likely caused by the apoptosis of the imaginal discs before the adult stage. Reduction of JNK signaling in vps22, vps25, or vps36 mutant discs leads to lower levels of apoptosis, supporting a role for JNK signaling in the cell death of the predominantly mutant tissues. More excitingly, JNK also controls proliferation in these tissues, as shown by the reduction of proliferation seen when JNK signaling was down-regulated. This observation is consistent with previous findings that JNK can induce non-cell autonomous proliferation and that apoptosis-induced proliferation is mediated by JNK activity. While inhibition of JNK signaling reduces proliferation in predominantly mutant ESCRT-II mutant discs, it does not affect other aspects of the neoplastic phenotype (Woodfield, 2013).

The role of JAK/STAT signaling in these mutants is complex. In mutant clones of ESCRT-II mosaic discs, Notch-induced secretion of the JAK/STAT ligand Upd triggers non-cell autonomous proliferation. However, autonomous de-regulated JAK/STAT signaling observed in predominately mutant discs is critical for the neoplastic transformation of vps22 mutants. In vps22 Stat92E double mutant discs, organization of cellular architecture is definitively rescued with the layout of the tissue closely resembling that of a wild-type eye-antennal imaginal disc. In addition, apical-basal polarity markers are localized more-or-less correctly in these tissues, indicating that epithelial polarity is more intact. Finally, differentiation in the posterior portion of the eye disc is preserved when JAK/STAT signaling is inhibited. Thus, de-regulation of JAK/STAT signaling in vps22 mutant discs contributes to the cellular disorganization and the lack of differentiation seen in the tissues, which is consistent with a previous study that implicated JAK/STAT signaling in cell cycle control, cell size, and epithelial organization in tsg101 mutant tissues (Woodfield, 2013).

It was recently shown that cells with strong gain of JAK/STAT activity transform into supercompetitors and eliminate neighboring cells with normal JAK/STAT activity by cell competition. However, in mosaic discs, a supercompetitive behavior of ESCRT-II mutant cells has not been observed. In fact, these mutant cells are eliminated by apoptosis. Only if apoptosis is blocked in these cells, is a strong overgrowth phenotype with neoplastic characteristics observed. Thus, apoptosis can serve as a tumor suppressor mechanism to remove cells with potentially malignant JAK/STAT activity (Woodfield, 2013).

How endosomal trafficking specifically regulates JAK/STAT signaling and, thus, how blocking trafficking leads to increases in signaling pathway activity are interesting questions to answer in the future. It is possible that, like endocytic regulation of the Notch receptor, the endosomal pathway tightly regulates Domeless (Dome), the JAK/STAT pathway receptor. It has been shown previously that Dome is trafficked through the endocytic machinery and that this trafficking of Dome can affect the downstream output of the JAK/STAT signaling pathway. It is also possible that Notch-induced Upd secretion causes autocrine JAK/STAT signaling in these mutants. However, technical problems (knocking down Notch function both in wild-type and mutant tissue causes general problems in tissue growth) prevented examination of this possibility (Woodfield, 2013).

It will be important to examine how de-regulated JAK/STAT signaling in ESCRT-II mutants causes neoplastic transformation. JAK/STAT signaling is known to be an oncogenic pathway in Drosophila and in humans but its downstream targets that promote tumorigenesis are not yet clear. JAK/STAT signaling may be feeding into other pathways that promote tumorigenesis, such as dpp signaling, or may be targeting other proteins involved in transformation, such as Cyclin D (Woodfield, 2013).

A number of studies have implicated genes that function in endocytosis and endosomal protein sorting as tumor suppressors in human cancers. Most well known is Tsg101, as early studies showed that downregulation of Tsg101 (see Drosophila TSG101) promotes the growth of mouse 3T3 fibroblasts in soft aga. When these cells were injected into nude mice, they formed metastatic tumors. However, later studies have shown conflicting results, and it is still unclear if Tsg101 functions as a tumor suppressor in metazoans. Importantly, a number of studies have shown changes in expression of ESCRT components in human cancer cells, including changes in expression of ESCRT-I components Tsg101 and Vps37A and ESCRT-III components Chmp1A and CHMP3. Since the primary proteins that function in endocytosis and endosomal trafficking are conserved from yeast to humans, it is likely that these findings in Drosophila may have important implications for human disease (Woodfield, 2013).

ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo

Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. This study shows that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, this study shows that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. It is concluded that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo (Eikenes, 2015).

Cytokinesis is the final step of cell division that leads to the physical separation of the two daughter cells. It is tightly controlled in space and time and proceeds in multiple steps via sequential specification of the cleavage plane, assembly and constriction of the actomyosin-based contractile ring (CR), formation of a thin intercellular bridge and finally abscission that separates the two daughter cells. Studies in a variety of model organisms and systems have elucidated key machineries and signals governing early events of cytokinesis. However, the mechanisms of the final abscission step of cytokinesis are less understood, especially in vivo in the context of different cell types in a multi-cellular organism (Eikenes, 2015).

During the recent years key insights into the molecular mechanisms and spatiotemporal control of abscission have been gained using a combination of advanced molecular biological and imaging technologies. At late stages of cytokinesis the spindle midzone transforms to densely packed anti-parallel microtubules (MTs) that make up the midbody (MB) and the CR transforms into the midbody ring (MR, diameter of ~1-2 μm). The MR is located at the site of MT overlap and retains several CR components including Anillin, septins (Septins 1, 2 and Peanut in Drosophila melanogaster), myosin-II, Citron kinase (Sticky in Drosophila) and RhoA (Rho1 in Drosophila) and eventually also acquires the centralspindlin component MKLP1 (Pavarotti in Drosophila). In C. elegans embryos the MR plays an important role in scaffolding the abscission machinery even in the absence of MB MTs (Eikenes, 2015).

Studies in human cell lines, predominantly in HeLa and MDCK cells, have shown that components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins play important roles in mediating abscission. Abscission occurs at the thin membrane neck that forms at the constriction zone located adjacent to the MR. An important signal for initiation of abscission is the degradation of the mitotic kinase PLK1 (Polo-like kinase 1) that triggers the targeting of CEP55 (centrosomal protein of 55 kDa) to the MR. CEP55 interacts directly with GPP(3x)Y motifs in the ESCRT-associated protein ALIX (ALG-2-interacting protein X) and in the ESCRT-I component TSG101, thereby recruiting them to the MR. ALIX and TSG101 in turn recruit the ESCRT-III component CHMP4B, which is followed by ESCRT-III polymerization into helical filaments that spiral/slide to the site of abscission. The VPS4 ATPase is thought to promote ESCRT-III redistribution toward the abscission site. Prior to abscission ESCRT-III/CHMP1B recruits Spastin that mediates MT depolymerization at the abscission site. ESCRT-III then facilitates membrane scission of the thin membrane neck, thereby mediating abscission (Eikenes, 2015).

Cytokinesis is tightly controlled by the activation and inactivation of mitotic kinases at several steps to ensure its faithful spatiotemporal progression. Cytokinesis conventionally proceeds to completion via abscission, but is differentially controlled depending on the cell type during the development of metazoan tissues. For example, germ cells in species ranging from insects to humans undergo incomplete cytokinesis leading to the formation of germline cysts in which cells are interconnected via stable intercellular bridges. How cytokinesis is modified to achieve different abscission timing in different cell types is not well understood, but molecular understanding of the regulation of the abscission machinery has started giving some mechanistic insight (Eikenes, 2015).

The Drosophila female germline represents a powerful system to address mechanisms controlling cytokinesis and abscission in vivo. Each Drosophila female germline stem cell (fGSC) divides asymmetrically with complete cytokinesis to give rise to another fGSC and a daughter cell cystoblast (CB). Cytokinesis during fGSC division is delayed so that abscission takes place during the G2 phase of the following cell cycle (about 24 hours later). The CB in turn undergoes four mitotic divisions with incomplete cytokinesis giving rise to a 16-cell cyst in which the cells remain interconnected by stable intercellular bridges called ring canals (RCs). One of the 16 cells with four RCs will become specified as the oocyte and the cyst becomes encapsulated by a single layer follicle cell epithelium to form an egg chamber. Drosophila male GSCs (mGSCs) also divide asymmetrically with complete cytokinesis to give rise to another mGSC and a daughter cell gonialblast (GB). Anillin, Pavarotti, Cindr, Cyclin B and Orbit are known factors localizing at RCs/MRs and/or MBs during complete cytokinesis in fGSCs and/or mGSCs. It has been recently reported that Aurora B delays abscission and that Cyclin B promotes abscission in Drosophila germ cells and that mutual inhibitions between Aurora B and Cyclin B/Cdk-1 control the timing of abscission in Drosophila fGSCs and germline cysts. However, little is known about further molecular mechanisms controlling cytokinesis and abscission in Drosophila fGSCs (Eikenes, 2015).

This study has characterize the roles of ALIX and the ESCRT-III component Shrub during cytokinesis in Drosophila fGSCs. ALIX and Shrub are required for completion of abscission in fGSCs. They co-localize during this process, and their direct interaction is required for abscission with normal kinetics. This study thus shows that a complex between ALIX and Shrub is required for abscission in fGSCs and provide evidence of an evolutionarily conserved functional role of the ALIX/ESCRT-III pathway in mediating cytokinetic abscission in the context of a multi-cellular organism (Eikenes, 2015).

Loss of ALIX or/and Shrub function or inhibition of their interaction delays abscission in fGSCs leading to the formation of stem cysts in which the fGSC remains interconnected to chains of daughter cells via MRs. As abscission eventually takes place a cyst of e.g. 2 germ cells may pinch off and subsequently undergo four mitotic divisions to give rise to a germline cyst with 32 germ cells. Consistently, loss of ALIX or/and Shrub or interference with their interaction caused a high frequency of egg chambers with 32 germ cells during Drosophila oogenesis. It was also found that ALIX controls cytokinetic abscission in both fGSCs and mGSCs and thus that ALIX plays a universal role in cytokinesis during asymmetric GSC division in Drosophila. Taken together this study provides evidence that the ALIX/ESCRT-III pathway is required for normal abscission timing in a living metazoan tissue (Eikenes, 2015).

The results together with findings in other models underline the evolutionary conservation of the ESCRT system and associated proteins in cytokinetic abscission. Specifically, ESCRT-I or ESCRT-III have been implicated in abscission in a subset of Archaea (ESCRT-III), in A. thaliana (elch/tsg101/ESCRT-I) and in C. elegans (tsg101/ESCRT-I). In S. cerevisiae, Bro1 (ALIX) and Snf7 (CHMP4/ESCRT-III) have also been suggested to facilitate cytokinesis. In cultured Drosophila cells, Shrub/ESCRT-III mediates abscission and in human cells in culture ALIX, TSG101/ESCRT-I and CHMP4B/ESCRT-III promote abscission. ALIX and the ESCRT system thus act in an ancient pathway to mediate cytokinetic abscission (Eikenes, 2015).

Despite the fact that an essential role of ALIX in promoting cytokinetic abscission during asymmetric GSC division was found in the Drosophila female and male germlines, strong bi-nucleation directly attributed to cytokinesis failure was found in Drosophila alix mutants in the somatic cell types that were examined. This might have multiple explanations. One possibility is that maternally contributed alix mRNA may support normal cytokinesis and development. Whereas ALIX and CHMP4B depletion in cultured mammalian cells causes a high frequency of bi- and multi-nucleation it is also possible that cells do not readily become bi-nucleate upon failure of the final step of cytokinetic abscission in the context of a multi-cellular organism. Consistent with the observations of a high frequency of stem cysts upon loss of ALIX and Shrub in the germline, Shrub depletion in cultured Drosophila cells resulted in chains of cells interconnected via intercellular bridges/MRs due to multiple rounds of cell division with failed abscission. Moreover, loss of ESCRT-I/tsg101 function in the C. elegans embryo did not cause furrow regression. These and the current observations suggest that ALIX- and Shrub/ESCRT-depleted cells can halt and are stable at the MR stage for long periods of time and from which cleavage furrows may not easily regress, at least not in these cell types and in the context of a multi-cellular organism. It is also possible that redundant mechanisms contribute to abscission during symmetric cytokinesis in somatic Drosophila cells. Further studies should address the general involvement of ALIX and ESCRT-III in cytokinetic abscission in somatic cells in vivo (Eikenes, 2015).

Different cell types display different abscission timing, intercellular bridge morphologies and spatiotemporal control of cytokinesis. In fGSCs it was found that ALIX and Shrub co-localize throughout late stages of cytokinesis and abscission. In human cells ALIX localizes in the central region of the MB, whereas CHMP4B at first localizes at two cortical ring-like structures adjacent to the central MB region and then progressively distributes also at the constriction zone where it promotes abscission. ALIX and CHMP4B are thus found at discrete locations within the intercellular bridge as cells approach abscission in human cultured cells. In contrast, ESCRT-III localizes to a ring-like structure during cytokinesis in Archaea, resembling the Shrub localization at MRs was observed in Drosophila fGSCs. Moreover, ALIX and Shrub are present at MRs for a much longer time (from G1/S) prior to abscission (in G2) in fGSCs than in human cultured cells. Here, ALIX and CHMP4B are increasingly recruited about an hour before abscission and then CHMP4B acutely increases at the constriction zones shortly (~30 min) before the abscission event (Eikenes, 2015).

How may ALIX and Shrub be recruited to the MR/MB in Drosophila cells in the absence of CEP55 that is a major recruiter of ALIX and ultimately CHMP4/ESCRT-III in human cells? Curiously, a GPP(3x)Y consensus motif was detected within the Drosophila ALIX sequence (GPPPGHY, aa 808-814) resembling the CEP55-interacting motif in human ALIX (GPPYPTY, aa 800-806). Whether Drosophila ALIX is recruited to the MR/MB via a protein(s) interacting with this motif or other domains is presently uncharacterized. Accordingly, alternative pathways of ALIX and ESCRT recruitment have been reported, as well as suggested in C. elegans, where CEP55 is also missing. Further studies are needed to elucidate mechanisms of recruitment and spatiotemporal control of ALIX and ESCRT-III during cytokinesis in fGSCs and different cell types in vivo (Eikenes, 2015).

This study found that the direct interaction between ALIX and Shrub is required for completion of abscission with normal kinetics in fGSCs. This is consistent with findings in human cells in which loss of the interaction between ALIX and CHMP4B causes abnormal midbody morphology and multi-nucleation. Following ALIX-mediated recruitment of CHMP4B/ESCRT-III to cortical rings adjacent to the MR in human cells, ESCRT-III extends in spiral-like filaments to promote membrane scission. Due to the discrete localizations of ALIX and CHMP4B during abscission in human cells ALIX has been proposed to contribute to ESCRT-III filament nucleation. In vitro studies have shown that the interaction between ALIX and CHMP4B may release autoinhibitory intermolecular interactions within both proteins and promote CHMP4B polymerization. Specifically, ALIX dimers can bundle pairs of CHMP4B filaments in vitro [65]. Moreover, in yeast, the interaction of the ALIX homologue Bro1 with Snf7 (CHMP4 homologue) enhances the stability of ESCRT-III polymers. There is a high degree of evolutionary conservation of ALIX and ESCRT-III proteins and because ALIX and Shrub co-localize and interact to promote abscission in fGSCs it is possible that ALIX can facilitate Shrub filament nucleation and/or polymerization during this process (Eikenes, 2015).

The current findings indicate that accurate control of the levels and interaction of ALIX and Shrub ensure proper abscission timing in fGSCs. Their reduced levels or interfering with their complex formation caused delayed abscission kinetics. How cytokinesis is modified to achieve a delay in abscission in Drosophila fGSCs and incomplete cytokinesis in germline cysts is not well understood. Aurora B plays an important role in controlling abscission timing both in human cells and the Drosophila female germline. During Drosophila germ cell development Aurora B contributes to mediating a delay of abscission in fGSCs and a block in cytokinesis in germline cysts. Bam expression has also been proposed to block abscission in germline cysts. It will be interesting to investigate mechanisms regulating the levels, activity and complex assembly of ALIX and Shrub and other abscission regulators at MRs/MBs to gain insight into how the abscission machinery is modified to control abscission timing in fGSCs (Eikenes, 2015).

Intercellular bridge MTs in fGSC-CB pairs were degraded in G1/S when the fusome adopted bar morphology. Abscission in G2 thus appears to occur independently of intercellular bridge MTs in Drosophila fGSCs. This has also been described in C. elegans embryonic cells where the MR scaffolds the abscission machinery as well as in Archaea that lack the MT cytoskeleton]. In mammalian and Drosophila S2 cells in culture, on the other hand, intercellular bridge MTs are present until just prior to abscission (Eikenes, 2015).

It is interesting to note a resemblance of the stem cysts that appeared upon loss of ALIX and Shrub function to germline cysts in that the MRs remained open for long periods of time similar to RCs. Some modification of ALIX and Shrub levels/recruitment may thus contribute to incomplete cytokinesis in Drosophila germline cysts under normal conditions. Because stem cysts were detected in the case when ALIX weakly interacted with Shrub it is also possible that inhibition of their complex assembly/activity may contribute to incomplete cytokinesis in germline cysts. Abscission factors, such as ALIX and Shrub, may thus be modified and/or inhibited during incomplete cytokinesis in germline cysts. Such a scenario has been shown in the mouse male germline where abscission is blocked by inhibition of CEP55-mediated recruitment of the abscission machinery, including ALIX, to stable intercellular bridges. Altogether these data thus suggest that ALIX and Shrub are essential components of the abscission machinery in Drosophila GSCs, and it is speculated that their absence or inactivation may contribute to incomplete cytokinesis. More insight into molecular mechanisms controlling abscission timing and how the abscission machinery is modified in different cellular contexts will give valuable information about mechanisms controlling complete versus incomplete cytokinesis in vivo (Eikenes, 2015).

In summary, this study reports that a complex between ALIX and Shrub is required for completion of cytokinetic abscission with normal kinetics during asymmetric Drosophila GSC division, giving molecular insight into the mechanics of abscission in a developing tissue in vivo (Eikenes, 2015).

The ESCRT machinery regulates the secretion and long-range activity of Hedgehog

The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading. Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. This study shows that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT). In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, ESCRT activity in Hh-producing cells is required for long-range signalling. Evidence is provided that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction (Matusek, 2014).

The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. This study describes rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. It is proposed that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat (Neyen, 2016).

PGRP-LC has a clear role as the major signaling receptor sensing Gram-negative bacteria in flies, but its contribution to the resolution phase once bacteria are killed and release polymeric PGN has remained elusive. This study has uncovered a regulatory isoform of LC (rLC) that adjusts the immune response to the level of threat. rLC specifically downregulates IMD pathway activation in response to polymeric PGN, a hallmark of efficient bacterial killing. The data are consistent with a model whereby the presence of rLC leads to efficient endocytosis of LC and termination of signaling via the ESCRT pathway. Trafficking-mediated shutdown of LC-dependent signaling ensures that LC receptors are switched off once the balance is tipped in favor of ligands signifying dead bacteria, allowing Drosophila to terminate a successful immune response. Failure to do so results in over-signaling, leading to the death of the host despite bacterial clearance. Consistent with this model, defects were found in endosome maturation and in the formation of MVBs enhance immune activation and prevent immune resolution. In addition to regulating LC signaling via the ESCRT machinery, rLC can also inhibit LC signaling by forming signaling-incompetent rLC-LC heterodimers or rLC-rLC homodimers (Neyen, 2016).

Recent evidence from vertebrates also implicates the ESCRT machinery in suppressing spurious NF-κB activation: the TNFR superfamily member lymphotoxin-β receptor, which activates a signaling cascade that is functionally similar to IMD signaling, is degraded in an ESCRT-dependent manner in zebrafish and human cells. Thus ESCRT-mediated clearance of receptors upstream of NF-κB seems well conserved throughout evolution (Neyen, 2016).

Internalization of receptor-ligand complexes raises the question of whether peptidoglycan is fully degraded in the endolysosomal compartment or fragmented and released into the cytosol for sensing by PGRP-LE, as is the case for peptidoglycan sensing by cytoplasmic NOD2 receptors in mammalian cells. Mechanistic coupling of LC-dependent peptidoglycan endocytosis and PGRP-LE-dependent cytosolic sensing of exported peptidoglycan fragments would help explain the partial cooperation between the two receptors (Neyen, 2016).

Molecularly, rLC is characterized by a cytosolic PHD domain predicted to bind to phosphoinositides. The PHD domain targets rLC were found to be distinct membrane domains but it cannot be excluded that this localization relies on additional protein-protein interactions. Furthermore, the PHD domain also mediates binding of rLC to the cytosolic regulator Pirk and the ubiquitin ligase DIAP2. The combined capability to control membrane localization and to recruit downstream signaling modulators is reminiscent of the 'sorting-signaling adaptor paradigm' that is emerging for mammalian PRRs. Sorting adaptors are cytosolic signaling components with phosphoinositide-binding domains that are selectively recruited to defined subcellular locations and thereby shape the signaling output of the receptors they interact with. In vertebrate immune signaling, bacterial sensing modules (for example, TLRs), lipid-binding sorting modules (for example, TIRAP or TRIF) and signaling modules (for example, MyD88 and TRAM) are carried on separate molecules and assemble via transient interactions. Drosophila MyD88 combines sorting and signaling functions in a single molecule, bypassing the need for TIRAP. Notably, rLC merges features of sensing and signaling receptors and sorting adaptors into a single molecule. The fact that Drosophila rLC has no immediate homologs in vertebrates with PGN-sensing and PGN-signaling pathways suggests evolutionary uncoupling of sensing and sorting domains, possibly to increase the spectrum of signaling by combinatorial recruitment of adaptors to sensing receptors (Neyen, 2016).

Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB

Axonal retrograde transport of signalling endosomes from the nerve terminal to the soma underpins survival. As each signalling endosome carries a quantal amount of activated receptors, it was hypothesized that it is the frequency of endosomes reaching the soma that determines the scale of the trophic signal. This study shows that upregulating synaptic activity markedly increased the flux of plasma membrane-derived retrograde endosomes (labelled using cholera toxin subunit-B: CTB) in hippocampal neurons cultured in microfluidic devices, and live Drosophila larval motor neurons. Electron and super-resolution microscopy analyses revealed that the fast-moving sub-diffraction-limited CTB carriers contained the TrkB neurotrophin receptor, transiently activated by synaptic activity in a BDNF-independent manner. Pharmacological and genetic inhibition of TrkB activation selectively prevented the coupling between synaptic activity and the retrograde flux of signalling endosomes. TrkB activity therefore controls the encoding of synaptic activity experienced by nerve terminals, digitalized as the flux of retrogradely transported signalling endosomes (Wang, 2016).

Whether raising synaptic activity also promotes an increase in the flux of CTB retrograde transport was examined in vivo, using live Drosophila melanogaster larval preparations. In addition to allowing measurement. of the effect of activity on retrograde carrier trafficking in a complementary model system, this approach also facilitated tracing the effect of presynaptic activity on the complete transport of CTB from the neuromuscular junction to the soma located in the central nervous system of the larvae (see CTB axonal retrograde carrier flux is controlled by synaptic activity in D. melanogaster motor neurons. In the fibers of neurons from wild-type larvae a low, albeit consistent, rate of CTB-positive retrograde carriers was detected. To analyse the flux of retrograde carriers under conditions of high activity, transgenic larvae were employed expressing dominant-negative forms of Ether-è-go-go (Eag) and Shaker (Sh) voltage-gated potassium channels in motor neurons, upregulating synaptic transmission. Consistent with the data in primary hippocampal neurons, increased presynaptic activity at the neuromuscular junction of the eag, Sh mutant larvae also resulted in an increase in the frequency of retrograde CTB carriers. Furthermore, the net transport of CTB could be quantified by measuring the accumulation of fluorescent CTB in the somata located in the ventral nerve cord. These data confirmed that presynaptic activity controls the transport of CTB retrograde carriers as well as their accumulation in the cell bodies located in the central nervous system (Wang, 2016).

This study demonstrates that the level of activity at the presynapse in hippocampal neurons, whether it be low or high activity, can induce changes in the frequency of CTB-positive retrograde carriers undergoing transport from the nerve terminal to the soma. Greater than 50% of CTB carriers are TrkB-positive signalling endosomes, and there is coupling between the level of presynaptic activity and the flux of signalling endosomes undergoing retrograde axonal transport. Given that the amount of activated TrkB per signalling endosome has been shown to be quantal in nature, it is likely that it is the number of signalling endosomes reaching the cell body that is responsible for monitoring the trophic response of each neuron. Importantly, it was demonstrated that the transient TrkB activation that occurs when presynaptic activity is increased controls the activity-dependent increase in the flux of signalling endosomes in a BDNF-independent manner. This suggests that TrkB activation may play an active role in generating signalling endosomes with a retrograde fate independent of BDNF secretion. Finally, SIM and morphometric analyses reveal a high number of sub-diffraction-limited CTB- and TrkB-positive carriers that may be involved in delivering the survival message (Wang, 2016).

CTB is a widely used neuroanatomical tracer with the ability to undergo retrograde transport in neurons. CTB was found to enter nerve terminals in an activity-dependent manner, and following a 2-4 h chase, is found in retrograde axonal carriers that co-localize extensively with TrkB. Importantly, TrkB staining in resting conditions produced a low and surface-localized pattern, which is in sharp contrast with that observed in the CTB/TrkB vesicular carriers following stimulation. Considering that internalization of activated TrkB is elicited by neuronal activity in hippocampal neurons, this suggests that the retrograde carriers containing both TrkB and CTB are signalling endosomes, destined to deliver a survival signal from the terminal to the soma. The data are also consistent with the previous demonstration that CTB can be co-transported in the same retrograde carriers as the neurotrophin receptor p75NTR and TrkB receptors. It has been speculated that activated neurotrophin receptors internalized into terminals through different pathways may converge in the retrograde traffic and be packaged into 'common' compartments, presumably signalling endosomes, which then undergo microtubule-dependent retrograde trafficking to induce signalling cascades and gene expression in the soma (Wang, 2016).

The signalling endosome model has been proposed to describe the propagation of activated Trk signals from the axon terminal to the neuronal soma. Although the presence of signalling endosomes is widely accepted, the precise nature(s) and regulation of these organelles remain unknown. In particular, it is not known how presynaptic activity translates into an increase in signalling endosome-encoded cell survival. Recent studies using super-resolution microscopy have shown that individual signalling endosomes appear to contain a limited number of signalling receptors, suggesting that retrograde survival signalling could be quantal in nature. Consistent with this idea, recent analysis has demonstrated that signalling endosomes have a fixed number of phosphorylated tyrosine receptor kinases. The current findings suggest that it is the flux of signalling endosomes reaching the cell body that encodes the level of presynaptic activity, and translates this into a survival signal. The data reveal a clear increase in the frequency of retrograde carriers delivering both CTB and TrkB from the nerve terminal to the cell soma following a short, transient burst of activity, whereas BoNT/A pretreatment not only completely blocked this increased flux despite equivalent stimulation but also lowered the basal level of retrograde CTB flux in resting conditions. These results strongly support the hypothesis that the soma of a neuron with lower synaptic activity receives fewer signalling endosomes than that of a cell with high synaptic activity, with ramifications for survival (Wang, 2016).

Previous studies have clearly established that there is constitutive delivery of retrogradely transported neurotrophins and their receptors in different types of primary neurons. This study is the first to reveal a coupling between synaptic activity and the number of retrograde signalling endosomes in both hippocampal neurons and motor neurons from D. melanogaster larvae, suggesting that this coupling represents a conserved regulatory mechanism (Wang, 2016).

Genetic manipulations in Drosophila allowed motor neuron activity to be chronically increase, thereby demonstrating a correlation with increased retrograde signalling endosome transport. Retrograde axonal transport has previously been reported in Drosophila larval motor neurons. However, this process is not regulated by the activation of TrkB receptors in flies, but by neurotrophin-type factors such as BMP (Gbb) p150(Glued) and Toll. Investigating the respective contributions of each of these mechanisms will be required to unravel the mechanism(s) controlling the coupling between synaptic activity and the flux of retrogradely transported signalling endosomes (Wang, 2016).

Clathrin-mediated endocytosis and bulk endocytosis are the two main modes for synaptic vesicle retrieval. On the basis of the current data, internalized CTB is sorted into both synaptic vesicles and larger endosomes following high K+-induced synaptic activation. These are therefore likely to be the source of CTB-positive signalling endosomes. Indeed, it is noted that, following a 4 h chase, the number of CTB-labelled endosomes present in nerve terminals was clearly reduced, suggesting that they could contribute to the generation of retrograde carriers leaving the terminals. More work is needed to pinpoint the actual contribution of the different presynaptic endocytic pathways in the process of generating signalling endosomes and recycling synaptic vesicles. Using electron microscopy and super-resolution SIM on the unidirectional axon bundles, a high proportion was found of CTB-positive retrograde carriers with a diameter <150 nm, indicating that these may constitute a significant source of the signalling endosomes that reach the soma. Interestingly, it was also found that a number of these small CTB retrograde endosomes closely aligned with each other to form consecutive structures. These were reminiscent of single-molecule quantum dot BDNF-labelled compartments, previously described as elongated multiple vesicular bodies. Several different types of organelles contain endocytosed neurotrophin receptors, including electron-lucent endosomes ranging from 50 nm to >200 nm in diameter and multivesicular bodies. The results further confirm this morphological heterogeneity of signalling endosomes. Interestingly, they also show that the sub-diffraction-limited pool of retrograde carriers is increased by a pulse of stimulation. A future challenge will be to fully define these compartments, including their cargo, their regulation and their potential role in neuronal survival (Wang, 2016).

The activity-dependent increase in the flux of CTB retrograde carriers is similar to that observed with autophagosomes undergoing axonal retrograde transport. Genetic and pharmacological inhibition of TrkB prevented the activity-dependent increase in signalling endosomes but did not affect the number of autophagosomes undergoing retrograde transport. This result suggests a limited cross-link between these two pathways, and indicates the existence of a TrkB-dependent sorting mechanism that is selective for the generation of retrograde signalling endosomes and is upregulated by synaptic activity (Wang, 2016).

Although TrkB activation was required to couple synaptic activity with the flux of retrograde CTB carriers, this study found that BDNF collators (anti-BDNF blocking antibody and TrkB-Fc), as well as exogenously applied BDNF, failed to affect this coupling. These results suggest that BDNF may not be required to promote this pathway. First, it can be speculated that increased neuronal activity alone promotes the secretion of alternative TrkB activators, such as NT4. In addition, considering that two different BDNF receptors, p75NTR and TrkB, have been reported to have opposing actions in neurons, the observation that BDNF collators have no effect on retrograde transport or the phosphorylation of TrkB could be explained by the fact that these collators alleviate an inhibitory effect of p75NTR signalling on TrkB activation. Third, it is possible that TrkB receptors could be indirectly transactivated by adenosine or by the MAPK pathway, similar to other tyrosine kinase receptors. However, further experiments are required to pinpoint the precise cascade of molecular events leading to the encoding of the level of synaptic activity by the flux of retrograde signalling endosomes in a BDNF-independent and TrkB activation-dependent pathway (Wang, 2016).

The data show that internalized CTB is sorted into both synaptic vesicles and larger endosomal structures reminiscent of bulk endosomes. Bulk endosomes are therefore likely to contribute to the sorting events that lead to the generation of CTB-positive signalling endosomes. Indeed, the data demonstrate that, in response to stimulation, the number of nerve terminals containing bulk endosomes more than doubles, as does the number of endosomes per terminal. More strikingly, after a chase of 4 h, the number of bulk endosomes returns to control levels, suggesting that sorting has taken place and/or that these large endosomes have undergone retrograde trafficking. In support of this view larger CTB-positive structures inside the axon were observed by electron microscopy. Bulk endocytosis is therefore likely to be a key element of this pathway, not only by budding small synaptic-like vesicles in nerve terminals, but also by generating large endosomes that undergo retrograde trafficking. More work is needed to pinpoint the actual contribution of bulk endocytosis in the process of generating signalling endosomes and recycling synaptic vesicles. The fact that synaptic activity increases the flux of CTB retrograde carriers advocates for the presence of a number of regulatory mechanisms that effectively couple these two pathways. Whether both recycling synaptic vesicles and signalling endosomes bud from the same bulk endosomes will require further investigation (Wang, 2016).

In summary, this study has uncovered a coupling between synaptic activity, trophic signalling and the generation of axonal retrograde signalling endosomes, which contain TrkB receptors and nerve terminal-derived CTB as cargo, and have characterized their kinetic and morphological nature. The data reveal a novel role of presynaptic activity in controlling the flux of retrograde signalling endosomes that will eventually reach the neuronal cell body and deliver cell survival signals. These results suggest that the flux of signalling endosomes undergoing axonal transport constitutes a digital output of the level of synaptic activity experienced by nerve terminals. How the flux of signalling endosomes reaching the cell body is decoded to pursue or halt a survival signal warrants further investigation (Wang, 2016).

Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release

Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density, and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen, 162 new mutations were tested for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. This study shows that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. Evidence is provided that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define roles for Rab3-GAP and Rab3 in synaptic homeostasis and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity (Müller, 2011).

The function of Rab3-GAP and Rab3 have been analyzed extensively, both biochemically and genetically, in systems ranging from yeast to the mammalian central nervous system, and there are several (~10 on average) copies of Rab3-GTP on an individual synaptic vesicle. It has also been shown that Rab3 binds to several presynaptic proteins, most often in its GTP-bound form. Rab3-GAP is required to promote hydrolysis of Rab3-GTP to Rab3-GDP. It is unknown precisely when and where Rab3-GAP acts upon Rab3-GTP, but evidence suggests that this interaction may occur at the synapse. For example, Rab3-GTP is on the vesicle and delivered to the synapse, where it is found bound to the active zone associated protein RIM. Biochemical data indicate that clathrin-coated vesicles lack Rab3-GTP. In combination, these data place Rab3-GAP activity at or near the release site (Müller, 2011).

The data presented in this study are consistent with a model in which Rab3-GTP acts, directly or indirectly, to inhibit the progression of synaptic homeostasis at a late stage of vesicle release, and that Rab3-GAP functions to inactivate this action of Rab3-GTP. Loss of Rab3-GAP was shown to block both the rapid induction and sustained expression of synaptic homeostasis. Rab3 mutations alone do not block synaptic homeostasis and synaptic homeostasis proceeds normally in the rab3–rab3-GAP double mutant. Genetically, these data indicate that the presence of Rab3 is required for the block of synaptic homeostasis observed in the rab3-GAP mutant. Thus, Rab3 is likely to be the cognate GTPase for Rab3-GAP. Furthermore, in genetic terms, Rab3 functions to oppose the progression of synaptic homeostasis and, when it is removed, homeostasis proceeds (Müller, 2011).

The possibility is considered that homeostasis proceeds in the absence of Rab3 because another, redundant Rab takes the place of Rab3. In yeast membrane trafficking, there is evidence for semiredundant Rab function. However, this seems to be an exception because Rabs are hypothesized to have unique binding affinities for downstream effector proteins that are essential for their ability to define discrete membrane domains within membrane trafficking and secretory systems. In C. elegans, Rab3 and Rab27 are both involved in synaptic vesicle release and they can be activated by a common exchange factor. However, based upon available genetic data, these Rabs do not appear to function redundantly during release. In Drosophila, loss of Rab3 causes a dramatic change in active zone size and organization (Graf, 2009). Thus, a redundant Rab would have to selectively and completely replace Rab3 function during synaptic homeostasis without rescuing synapse development, and this seems unlikely. Finally, since Rab3 is required for the complete block of synaptic homeostasis observed in the rab3-GAP mutant, it is concluded that Rab3 itself participates in mechanisms that determine whether or not synaptic homeostasis will proceed (Müller, 2011).

Next, the possibility is considered that Rab3 accumulates in a GTP-bound form at the synapse in the rab3-GAP mutant background, and this accumulation could block homeostatic plasticity. This model is attractive because it could explain why loss of Rab3 does not block homeostatic plasticity. However, a direct test of this model failed to provide supporting evidence. A constitutively active rab3 transgene was expressed in the rab3 mutant background. This experiment should mimic the accumulation of Rab3-GTP in a rab3-GAP mutant background. It was found that the constitutively active rab3 transgene (rab3CA) is trafficked to the NMJ and localizes in a manner that is indistinguishable from overexpressed wild-type Rab3. Furthermore, rab3CA has activity at the synapse because it rescues the defects in active zone organization that are caused by loss of rab3. In addition, constitutively active Rab3A was shown to biochemically interact with Rab3-GAP. However, the expression of rab3CA did not disrupt synaptic homeostasis. Therefore, aberrant accumulation of Rab3-GTP is not the cause of impaired synaptic homeostasis in the rab3-GAP mutant and another model should therefore be considered (Müller, 2011).

Another activity of Rab3-GAP that could be relevant to synaptic homeostasis is its ability to physically bind Rab3-GTP. It is known that Rab3-GTP can bind several synaptic proteins including RIM and Rabphillin. Moreover, this study provides evidence that Rab3's GTPase activity is not limiting during synaptic homeostasis. Therefore, it is proposed that Rab3-GAP competes for Rab3-GTP binding with another protein. If this other protein inhibits synaptic homeostasis when bound to the synaptic vesicle, then displacement by Rab3-GAP binding would be a required step for synaptic homeostasis to proceed. This model can explain all of the experimental data. First, Rab3-GAP would be necessary for synaptic homeostasis. Second, synaptic homeostasis would proceed normally in the rab3 mutant because the homeostatic inhibitor would no longer localize to the synaptic vesicle. Third, overexpression of rab3CA in the rab3 mutant background would not block synaptic homeostasis because Rab3-GAP would still be able to compete for Rab3-GTP binding, displace the homeostatic inhibitor, and allow homeostatic plasticity to proceed (Müller, 2011).

This model is consistent with a conserved function of Rab proteins throughout the membrane trafficking and secretory pathways of organisms ranging from yeast to mammals. Rab proteins, in their GTP-bound state, function to nucleate the assembly of 'effector' protein complexes that define membrane microdomains. However, throughout the literature, more is known about the assembly of these Rab-dependent complexes than is known about their disassembly. The model assumes that the binding of Rab3-GAP to Rab3 and the Rab3CA mutant protein is sufficient to disrupt effector binding (including the proposed homeostatic inhibitor). It is generally believed that Rab-GAPs interact with their cognate GTPases with lower affinity than the effector proteins. However, although Rab3-GAP has a relatively low affinity for Rab3A, Rab3-GAP effectively competes with an effector (Rabphillin) for binding to Rab3A and this is true even when Rab3A harbors the Q81L mutation. These data support the possibility that Rab3-GAP could compete for effector binding and disrupt a Rab3-GTP dependent scaffold. This is not the only manner in which Rab3-GAP differs from other Rab-GAPs. Rab3-GAP is somewhat unique in that it does not contain additional protein-protein interaction motifs. Thus, unlike IQ-GAP proteins for instance, it seems unlikely that Rab3-GAP has unique functions that are independent of Rab3, consistent with the double-mutant analysis of rab3 and rab3-GAP (Müller, 2011).

Finally, the possibility is considered that the rab3-GAP mutation could create a ceiling effect where baseline transmission is normal but release cannot be potentiated under any condition. Several pieces of data argue against this possibility. First, by elevating extracellular calcium, quantal content can be increased in the rab3-GAP mutant, indicating that there is no restriction on the absolute number of quanta that can be released. Second, during a stimulus train (20 Hz, 0.4 mM extracellular calcium), the rab3-GAP mutant plateaus at a higher EPSP amplitude compared to wild-type. Again, there is no evidence for a ceiling effect in rab3-GAP. Finally, it has been demonstrated that mutations that cause a severe defect in baseline transmission can still undergo homeostatic compensation. Taken together, these data argue against a simple ceiling effect and support the conclusion that Rab3-GAP is directly involved in the mechanisms of synaptic homeostasis (Müller, 2011).

A question that cannot be addressed is which step in the model is modified during the induction of synaptic homeostasis. One interesting possibility is that the interaction of Rab3-GTP with the homeostatic repressor is regulated. For example, if this interaction is stabilized, then homeostatic plasticity would be opposed and, conversely, if the interaction is weakened, then homeostasis would be allowed to proceed. A recent study in C. elegans provided evidence that an unknown retrograde signal, from muscle to motoneuron, causes increased expression of YFP-Rab3 at the presynaptic terminal. Although no evidence was found for a similar phenomenon at the Drosophila NMJ during synaptic homeostasis, these data support the possibility that the Rab3/Rab3-GAP signaling complex could be a downstream, regulated target of a homeostatic, retrograde signal at the NMJ (Müller, 2011).

Interpreting the current data requires consideration of a recent study examining the effects of a rab3 mutation on synapse organization at the Drosophila NMJ (Graf, 2009). In the Graf study, it was discovered that a rab3 mutation causes a dramatic accumulation of both the active zone-associated protein Bruchpilot (Brp, T-bars, the Drosophila homolog of CAST/ELKS) and presynaptic calcium channels at a subset of active zones (Graf, 2009). Based on these and other data it was suggested that Rab3 promotes the nucleation of new active zones, and without this activity, active zones coalesce (Graf, 2009). It was possible to clearly dissociate any morphological reorganization of the NMJ from a blockade of synaptic homeostasis. The rab3 mutants have altered NMJ morphology, but normal synaptic homeostasis, whereas rab3-GAP mutants have normal NMJ morphology and a defect in synaptic homeostasis) (Müller, 2011).

It is also important to consider why rab3 mutants do not show excessive homeostatic compensation. It is predicted that Rab3 and Rab3-GAP will not control the magnitude of the homeostatic response, just whether or not it is allowed to proceed. Additional negative feedback signaling mechanisms would be responsible for determining the magnitude of the homeostatic response. This would explain why no excessive homeostatic compensation was observed in the absence of Rab3. Thus, Rab3 and Rab3-GAP provide an additional layer of control on synaptic homeostasis, ensuring that modulation of release probability only occurs when, and perhaps where, appropriate (Müller, 2011).

Ultimately, it would be important to understand how presynaptic vesicle release is modulated during homeostatic plasticity. It is known from previously published data that a homeostatic increase in vesicle release is due to a change in presynaptic release probability without a change in active zone number. Mechanistically, the full functionality of presynaptic calcium channels is necessary for synaptic homeostasis. However, it remains unknown whether synaptic homeostasis involves a change in calcium channel number versus calcium channel function. Given these prior data, one possibility is that the homeostatic signaling system, identified in this study, acts upon presynaptic calcium channels to prevent a change in calcium influx. In this respect, the involvement of RIM and RIM binding protein are intriguing since RIM binds to Rab3-GTP and has been proposed to influence calcium-channel function (Müller, 2011 and references therein).

Rab3-GAP is the first protein to be implicated in the homeostatic modulation of presynaptic release that directly interacts with a resident synaptic vesicle protein. This fact, and analysis of baseline synaptic transmission in the rab3-GAP mutant raise the possibility that the homeostatic modulation of presynaptic release also includes mechanisms that are independent of increased calcium influx. For example, a defect was observed in presynaptic release probability in the rab3-GAP mutant that occurs only when recording is performed in low extracellular calcium. This defect could reveal a function of Rab3-GAP during vesicle release, or it could reflect the activity of the proposed homeostatic repressor on baseline synaptic transmission. One possibility that could explain the decrease in release probability is that synaptic vesicles reside at a greater physical distance from the calcium channel in the rab3-GAP mutant. When recording in low extracellular calcium, the calcium microdomains at the active zone would not effectively trigger the release of these more distant vesicles. This model would suggest that enhanced coupling of the synaptic vesicle and the calcium channel is part of the homeostatic modulation of presynaptic release. By extension, the action of the homeostatic repressor would be to prevent a tight association of the synaptic vesicle with the calcium channel. It is interesting to speculate that the homeostatic repressor could be Rabphillin. It has been shown that Rabphillin can compete with Rab3-GAP for binding to Rab3-GTP. Rabphillin has two C2 domains that could confer calcium-dependence to this protein-protein interaction and, by extension, homeostatic plasticity. Ultimately, a molecular change that influences the functionality of the calcium sensor for vesicle fusion cannot be ruled out. Regardless, the data identify a homeostatic mechanism that functions at a late stage of vesicle release to modulate presynaptic release probability (Müller, 2011).

In combination with a previously published genetic screen, thirteen mutations have now been identified that disrupt the expression of synaptic homeostasis without severely altering baseline synaptic transmission. Among these genes are rab3-GAP and dysbindin. The mutant phenotypes forrab3-GAP and dysbindin are remarkably similar. In both cases, loss of function mutations have little effect on baseline transmission under standard recording conditions (0.4 mM extracellular calcium). However, decreasing extracellular calcium reveals a significant decrease in release probability. In agreement, an increase was also observed in short-term synaptic facilitation in both mutations. Furthermore, neither mutation has an effect on synapse morphology or active zone number. In dysbindin mutants, these effects were shown to be downstream or independent of presynaptic calcium influx. It is tempting to place Dysbindin into the proposed model for homeostatic plasticity. One possibility is that Dysbindin functions to stabilize the close association of synaptic vesicles with the presynaptic calcium channel. The absence of Dysbindin would therefore phenocopy the rab3-GAP mutant but function through a different set of molecular interactions on the synaptic vesicle. The similarity between the phenotypes of dysbindin and rab3-GAP are also interesting because dysbindin has been linked to schizophrenia in human. The intriguing possibility that Dysbindin interacts with Rab3-Rab3-GAP signaling will be the subject of future studies (Müller, 2011).

Cell-free reconstitution of multivesicular body (MVB) cargo sorting

The signaling activity of cell surface localized membrane proteins occurs primarily while these proteins are located on the plasma membrane but is, in some cases, not terminated until the proteins are degraded. Following internalization and movement through the endocytic pathway en route to lysosomes, membrane proteins transit a late endosomal organelle called the multivesicular body (MVB). MVBs are formed by invagination of the limiting membrane of endosomes, resulting in an organelle possessing a limiting membrane and containing internal vesicles. The molecular machinery that regulates the separation of membrane proteins destined for degradation from those resulting in surface expression is not well understood. This study reconstituted an endosomal sorting event under cell-free conditions. Advantage was taken of the itinerary of a prototypical membrane protein, the Epidermal growth factor receptor (EGFR) and a biochemical monitor was designed for cargo movement into internal MVB vesicles that is generally modifiable for other membrane proteins. Since is it not known how internal vesicle formation is related to cargo sorting, morphological examination using transmission electron microscopy (TEM) allows separate monitoring of vesicle formation. This study determined that MVB sorting is dependent on cytosolic components, adenosine triphosphate (ATP), time, temperature, and an intact proton gradient. This assay reconstitutes the maturation of late endosomes and allows the morphological and biochemical examination of vesicle formation and membrane protein sorting (Giraud, 2015).

The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation

Developmental axon pruning is essential for wiring the mature nervous system, but its regulation remains poorly understood. This study shows that the endosomal-lysosomal pathway regulates developmental pruning of Drosophila mushroom body γ neurons. The UV radiation resistance-associated gene (Uvrag) functions together with all core components of the phosphatidylinositol 3-kinase class III (PI3K-cIII; see Phosphotidylinositol 3 kinase 59F) complex to promote pruning via the endocytic pathway. By studying several PI3P binding proteins, this study found that Hrs, a subunit of the ESCRT-0 complex, required for multivesicular body (MVB) maturation, is essential for normal pruning progression. Thus, the existence of an inhibitory signal that needs to be downregulated is hypothesized. Finally, the data suggest that the Hedgehog receptor, Patched, is the source of this inhibitory signal likely functioning in a Smo-independent manner. Taken together, this in vivo study demonstrates that the PI3K-cIII complex is essential for downregulating Patched via the endosomal-lysosomal pathway to execute axon pruning (Issman-Zecharya, 2014).

Neuronal remodeling is an essential step of nervous system development in both vertebrates and invertebrates. One mechanism used to remodel neuronal circuits is by the elimination of long stretches of axons in a process known as axon pruning. With a few exceptions, the current dogma is that axon pruning of long stretches of axons occurs via local axon degeneration while axon pruning of short stretches occurs via retraction. While in some cases remodeling is directly affected by experience or neural activity, in cases of stereotypical pruning the identity of the axon that is destined to be pruned does not depend on experience or neural activity. Because of mechanistic similarities to Wallerian degeneration and dying back neurodegenerative diseases, understanding the molecular mechanisms of axon pruning should result in a broader insight into axon fragmentation and elimination during development and in disease (Issman-Zecharya, 2014).

The neuronal remodeling of the Drosophila mushroom body (MB) during development is a unique model system to study the molecular aspects of axon pruning. The stereotypic temporal and spatial occurrence of MB axon pruning combined with mosaic analyses provide a platform to perform genetic screens and molecular dissections of these processes in unprecedented resolution. The MB is comprised of three types of neurons that are sequentially born from four identical neuroblasts per hemisphere. Out of the three MB neuronal types, only the γ neurons undergo axon pruning, indicating that the process is cell-type specific. During the larval stage, γ neurons project a bifurcated axon to the dorsal and medial lobes. At the onset of metamorphosis, the dendrites of the γ neurons as well as specific parts of the axons are eliminated by localized fragmentation in a process that peaks at about 18 hr after puparium formation. Subsequently, γ neurons undergo developmental axon regrowth, which is distinct from initial axon outgrowth, to occupy the adult specific lobe (Issman-Zecharya, 2014).

Axon pruning of MB γ neurons depends on the cell-autonomous expression of the nuclear steroid hormone receptor, ecdysone receptor B1 (EcR-B1). The expression of EcR-B1 is regulated by at least three distinct pathways: the cohesin complex, the TGF-β pathway, and a network of nuclear receptors comprised of ftz-f1 and Hr39. While expression of EcR-B1 is required for pruning, it is not sufficient to drive ectopic pruning either in γ neurons or in other MB neurons that do not undergo remodeling. This raises two possible nonmutually exclusive scenarios: (1) additional molecules are required to initiate pruning and (2) an inhibitory signal needs to be attenuated in the MB for pruning to occur. Additionally, the ubiquitin pathway is also cell-autonomously required in γ neurons for pruning, but the target that must be ubiquitinated remains unknown. Thus, while understanding of the cellular sequence of events culminating in the elimination of specific axonal branches is quite detailed, understanding of the molecular mechanisms remains incomplete (Issman-Zecharya, 2014).

In a forward genetic screen, this study identified a cell-autonomous role for the UV radiation resistance-associated gene (UVRAG) in MB γ neuron pruning. UVRAG was originally identified based on its ability to confer UV resistance to nucleotide excision repair deficient cells. It was later shown to function as a tumor suppressor gene deleted in various types of cancers including colon and gastric carcinomas. UVRAG interacts with Atg6 (also known as Beclin1), another tumor suppressor gene, and together they promote autophagy in vitro. Their tumor suppression capabilities were first attributed to their autophagy-promoting function. However, a mutant form of UVRAG isolated from colon carcinomas promoted autophagy normally in cell culture. Both UVRAG and Atg6 are subunits in the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex, involved in autophagy and endocytosis. Recent studies have found that UVRAG mediates endocytosis in an Atg6-dependent manner suggesting that as part of the PI3K-cIII complex, both proteins regulate various aspects of vesicle trafficking. Two studies have recently identified new and seemingly unrelated functions for UVRAG in regulating DNA repair in response to UV-induced damage and ER to Golgi trafficking. Finally, an in vivo study has shown that UVRAG affects organ rotation in Drosophila by regulating Notch endocytosis in what seemed to be an Atg6-independent manner. A unifying understanding of the various aspects of UVRAG physiological function in vivo is still lacking. Likewise, although the PI3K-cIII complex has been extensively studied and implicated in autophagy, cytokinesis and endocytosis, its physiological roles during the normal course of development are not known (Issman-Zecharya, 2014).

This study reports that UVRAG and the PI3K-cIII complex mediate the endosome-lysosome degradation of Ptc to promote axon pruning. Furthermore, the results suggest that Ptc represses pruning via a Smo- and Hh-independent manner. This study provides evidence for the existence of a pruning inhibitory pathway originating at the membrane of MB neurons (Issman-Zecharya, 2014).

This study shows that the endosomal-lysosomal pathway is cell-autonomously required for developmental axon pruning of mushroom body (MB) γ neurons. Genetic loss-of-function experiments indicate that UVRAG, a tumor suppressor gene previously linked to both endocytosis and autophagy, promotes pruning as part of the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex and that UVRAG is required in MB neurons for the formation of phosphatidylinositol 3-phosphate (PI3P). The ESCRT-0 complex, which is recruited to the PI3 moiety on endosomal membranes, is required for pruning, indicating that endosome to multivesicular body maturation is critical for the normal progression of axon pruning and suggesting that it involves receptor downregulation. Genetic loss-of-function and gain-of-function experiments suggest that downregulation of the Hedgehog receptor Patched (Ptc) by the endocytic machinery is instrumental in promoting pruning. Finally, the results suggest that Ptc inhibits pruning in a smo-independent and likely also hh-independent manner (Issman-Zecharya, 2014).

A recent study suggested that UVRAG is required for Notch endocytosis during organ rotation in Drosophila in an Atg6-independent manner. While the current study shows that Atg6 is required for pruning, these seemingly contradicting results can be easily explained by specific allele differences. The Atg600096 allele, used in the previous study, is a P element insertion about 100 bp upstream of the Atg6 gene that does not necessarily create a null allele. Indeed, this study could also not see any effect of this allele on axon pruning. This study used an Atg61 null allele created by homologous recombination resulting in a strong effect on pruning. Furthermore, the data clearly show that the entire PI3K-cIII complex is required for axon pruning (Issman-Zecharya, 2014).

The PI3K-cIII complex has been implicated in a wide variety of membrane trafficking processes ranging from autophagy to endocytosis to cytokinesis. How the PI3K-cIII is regulated to participate in these different processes and its physiological roles in vivo are not well understood. While its role in promoting autophagy is supported by several studies, deleting the catalytic unit, Vps34, in sensory neurons does not affect autophagy, but rather endocytosis. Whether this is a common feature of PI3K-cIII function in neurons remains to be further elucidated. One attractive hypothesis is that the PI3K-cIII function is determined by its complex composition. Indeed, it appears that in vitro, UVRAG and Atg14 are mutually exclusive subunits defining two distinct populations of the PI3K-cIII complex (Funderburk, 2010; Itakura, 2009). The current study is consistent with these findings, suggesting that UVRAG may define an endocytosis-specific PI3K-cIII complex at least in neurons. The full spectrum of the various PI3K-cIII complexes physiological roles in vivo remains to be further studied (Issman-Zecharya, 2014).

The PI3K-cIII complex phosphorylates PI to form PI3P on endosomal membranes. Indeed, this study found that UVRAG is essential for efficient PI3P formation and that PI3P is abundant throughout development. It is thus hypothesized that a PI3P binding protein mediates the effect of UVRAG and the PI3K-cIII complex on axon pruning. This study has identified Hrs, a subunit of the ESCRT-0 complex and a PI3P binding protein, as required for axon pruning. The role of ESCRT-0 in MVB maturation led to a hypothesis that the endolysosomal pathway is required to downregulate a signal that originates at the plasma membrane. While signaling can still occur in the early endosome, it is terminated at the MVB (Issman-Zecharya, 2014).

What is the identity of this transmembrane protein? Using genetic loss-of-function and gain-of-function experiments, it is suggested that Patched (Ptc) is at least one of the transmembrane proteins that is responsible for mediating the PI3K-cIII pruning defect. Strikingly, mutating ptc on the background of a Atg6 mutant significantly suppressed its pruning defect. Furthermore, overexpression of Ptc in WT brains resulted in a weak to mild pruning defect, depending on the Gal4 driver. Finally, overexpressing Ptc on the background of an endosomal defect significantly exacerbated the pruning defect. Together, these data suggest that Ptc mediates an inhibitory signal that needs to be attenuated for the normal progression of pruning. Interestingly, Ptc inactivation by endocytosis followed by lysosomal degradation was proposed before as a mechanism to activate the Hh pathway. What is the nature of this signal? Ptc is known to be the Hedgehog (Hh) receptor. Binding of Hh to Ptc relieves the Ptc-induced suppression of another transmembrane protein, Smoothened (Smo). Once derepressed, Smo initiates the intracellular Hh signal that culminates in the expression of specific nuclear transcription factors. Therefore this study tested the role of Smo and Hh in developmental axon pruning and, to surprisingly, demonstrated that both molecules seem to be irrelevant for pruning. Overexpressing Ptc mutant transgenes within MB neurons to identify the domains that are important for pruning inhibitions confirmed that Smo inhibition was not required to inhibit pruning. In contrast, the results suggest that the ligand binding domain is important. Because the results suggest that Hh is not required for pruning inhibition, it will be interesting to investigate in the future what other ligands might bind to Ptc. In this regard it is interesting to mention that a recent study has shown that Ptc is a lipoprotein receptor. The precise mechanism of Ptc action in MB neurons remains to be further elucidated in future studies (Issman-Zecharya, 2014).

This study has uncovered a role for the endocytic machinery in downregulating an inhibitory signal that is dependent on Ptc during MB axon pruning. A recently published study has shown that the Rab5/ESCRT endocytic pathways are required to downregulate neuroglian (Nrg) to promote dendrite pruning of sensory neurons in Drosophila. Both studies highlight that a combination of both promoting and inhibitory signals during developmental pruning is likely important to provide fail-safe mechanisms to regulate the process in a temporal, spatial, and cell-type specific resolution (Issman-Zecharya, 2014).

The lysosomal enzyme receptor protein (LERP) is not essential, but is implicated in lysosomal function in Drosophila melanogaster

The lysosomal enzyme receptor protein (LERP) of Drosophila is the ortholog of the mammalian cation-independent mannose 6-phosphate (Man 6-P) receptor, which mediates trafficking of newly synthesized lysosomal acid hydrolases to lysosomes. However, flies lack the enzymes necessary to make the Man 6-P mark, and the amino acids implicated in Man 6-P binding by the mammalian receptor are not conserved in LERP. Thus, the function of LERP in sorting of lysosomal enzymes to lysosomes in Drosophila is unclear. This study analyzed the consequence of LERP depletion in S2 cells and intact flies. RNAi-mediated knockdown of LERP in S2 cells had little or no effect on the cellular content or secretion of several lysosomal hydrolases. A novel Lerp null mutation, LerpF6, was generated that abolishes LERP protein expression. Lerp mutants have normal viability and fertility and display no overt phenotypes other than reduced body weight. Lerp mutant flies exhibit a 30-40% decrease in the level of several lysosomal hydrolases, and are hypersensitive to dietary chloroquine and starvation, consistent with impaired lysosome function. Loss of LERP also enhances an eye phenotype associated with defective autophagy. These findings implicate Lerp in lysosome function and autophagy (Hasanagic, 2015).

FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, Drosophila models were generated of FIG4-related diseases. Fig4 null mutant flies are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS (Bharadwaj, 2015).

The GATOR2 component Wdr24 regulates TORC1 activity and lysosome function

TORC1 (see Drosophila Tor) is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. The genome of Drosophila contains a single Sea2/Wdr24 homolog encoded by the gene CG7609 that shares 25% identity and 44% similarity to yeast Sea2 and 37% identity and 54% similarity to the human homolog WDR24. This study defines the in vivo role of the GATOR2 component Wdr24 in Drosophila. Wdr24 was shown to have both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, it was shown that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, it was found that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. Wdr24 was also shown to promotes lysosome acidification and autophagic flux in mammalian cells. Taken together these data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes (Cai, 2016).

In metazoans multiple conserved signaling pathways control the integration of metabolic and developmental processes. TORC1 is an evolutionarily conserved multi-protein complex that regulates metabolism and cell growth in response to an array of upstream inputs including nutrient availability, growth factors and intracellular energy levels. The catalytic component of TORC1 is the serine/threonine kinase Target of Rapamycin (TOR). When nutrients are abundant, TORC1 activity promotes translation, ribosome biogenesis as well as other pathways associated with anabolic metabolism and cell growth. However, when nutrients or other upstream activators are limiting, TORC1 activity is inhibited triggering catabolic metabolism and autophagy (Cai, 2016).

The Seh1 associated/GTPase-activating protein toward Rags (SEA/GATOR) complex is a newly identified upstream regulator of TORC1 that can be divided into two putative sub-complexes GATOR1 and GATOR2 (Bar-Peled, 2013; Dokudovskaya, 2011; Panchaud, 2013). The GATOR1 complex, known as the Iml1 complex or the Seh1 Associated Complex Inhibits TORC1 (SEACIT) in yeast, inhibits TORC1 activity in response to amino acid limitation. SEACIT/GATOR1 contains three proteins Npr2/Nprl2, Npr3/Nprl3 and Iml1/DEPDC5. Recent evidence, from yeast and mammals, indicates that the components of the SEACIT/GATOR1 complex function through the Rag GTPases to inhibit TORC1 activity. Notably, Nprl2 and DEPDC5 are tumor suppressor genes while mutations in DEPDC5 are a leading cause of hereditary focal epilepsies (Cai, 2016).

The GATOR2 complex, which is referred to as Sevh1 Associated Complex Activates TORC1 (SEACAT) in yeast, activates TORC1 by opposing the activity of GATOR1. The SEACAT/GATOR2 complex is comprised of five proteins, Seh1, Sec13, Sea4/Mio, Sea2/WDR24, and Sea3/WDR59. Computational analysis indicates that multiple components of the GATOR2 complex have structural features characteristic of coatomer proteins and membrane tethering complexes. In line with the structural similarity to proteins that influence membrane dynamics, in Drosophila the GATOR2 subunits Mio and Seh1 localize to multiple endomembrane compartments including lysosomes, the site of TORC1 regulation, and autolysosomes. In metazoans, members of the Sestrin and Castor family of proteins bind to and inhibit the GATOR2 complex in response to leucine and arginine starvation respectively. This interaction is proposed to inhibit TORC1 activity through the derepression of the GATOR1 complex. However, how GATOR2 opposes GATOR1 activity, thus allowing for the robust activation of TORC1, remains unknown. Additionally, the role of the GATOR2 complex in the regulation of both the development and physiology of multicellular animals remains poorly defined (Cai, 2016).

Recent evidence from Drosophila indicates that the requirement for the GATOR2 complex may be context specific in multicellular animals. In Drosophila, null alleles of the GATOR2 components mio and seh1 are viable but female sterile. Surprisingly, somatic tissues from mio and seh1 mutants exhibit little if any reductions in cell size and have nearly normal levels of TORC1 activity. In contrast, TORC1 activity is dramatically decreased in ovaries from mio and seh1 mutant females. This decrease in TORC1 activity is accompanied by the activation of catabolic metabolism in the female germ line, a dramatic reduction in egg chamber growth and difficulties maintaining the meiotic cycle. Thus, there is a surprising tissue specific requirement for the GATOR2 components Mio and Seh1 during oogenesis. However, the in vivo role of the other members of the GATOR2 complex in the regulation of cellular metabolism remains undefined (Cai, 2016).

This study defines the in vivo requirement for the GATOR2 component Wdr24 in Drosophila. Wdr24 was found to have two distinct functions. First, Wdr24 is a critical effector of the GATOR2 complex that promotes TORC1 activity and cellular growth in a broad array of tissues. Second, Wdr24 is required for the TORC1 independent regulation of lysosome function and autophagic flux. Notably, two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. Taken together these data support the model that multiple components of the GATOR2 complex have both TORC1 dependent and independent roles in the regulation of cellular metabolism (Cai, 2016).

This study describes a dual role for the GATOR2 component Wdr24 in the regulation of TORC1 activity and lysosome dynamics. Wdr24 is a critical effector of the GATOR2 complex that promotes TORC1 activity in both germline and somatic tissues. This lies in contrast to the GATOR2 components Mio and Seh1, which have a limited role in the regulation of TORC1 activity in many cell types. Surprisingly, a second function of Wdr24 was identified that is independent of TORC1 status, the regulation of lysosome acidification and autophagic flux. Taken together these data support the model that the GATOR2 complex regulates both the response to amino acid starvation and lysosome function (Cai, 2016).

Whole animal studies often reveal tissue-specific and/or metabolic requirements for genes that are not readily observed in cell culture. In mammalian and Drosophila tissue culture cells, RNAi based depletions of the GATOR2 components Mio, Seh1, Wdr59, and Wdr24 result in decreased TORC1 activity in return to growth assays (Bar-Peled, 2013, Wei, 2014). These data have resulted in the model that all components of the GATOR2 complex are generally required for TORC1 activation (Wei, 2014). However, the characterization of mio and seh1 null mutants in Drosophila, demonstrated that Mio and Seh1 are critical for the activation of TORC1 and inhibition of autophagy in the female germ line, but play a relatively small role in the regulation of TORC1 activity and autophagy in somatic tissues under standard culture conditions. Thus, the requirement for at least a subset of GATOR2 complex components is tissue and/or context specific (Cai, 2016).

This study reports that the GATOR2 component Wdr24 is required for the full activation of TORC1 in both germline and somatic cells of Drosophila. Consistent with the global down-regulation of TORC1 activity in the absence of Wdr24, wdr24 mutant adults are notably smaller than controls and are female sterile. Depleting the GATOR1 components nprl2 and nprl3 in the wdr24 mutant background rescued the low TORC1 activity, growth defects, and female sterility of wdr24 mutants. Thus, the GATOR2 component Wdr24 is required to oppose GATOR1 activity in both germline and somatic cells of Drosophila. From these results it is proposed that Wdr24 is a key effector of the GATOR2 complex required for the full activation of TORC1 in most cell types (Cai, 2016).

There are several potential models to explain the differential requirement for individual GATOR2 proteins in Drosophila. First, there may be tissue specific requirements for individual GATOR2 subunits. In this model the different phenotypes observed in the seh1 and mio versus wdr24mutants reflects a qualitative difference in the requirement for these proteins in different tissues. However, an alternative model is favored in which Wdr24 is the core effector of GATOR2 activity, with Mio and Seh1 functioning primarily as positive regulators of GATOR2 activity. In this second model, the differential phenotypes observed in the seh1 and mio versus wdr24 mutants reflects a quantitative difference in the requirement for GATOR2 activity in different tissues. The distinction between these two models awaits the identification of the molecular mechanism of Wdr24 and GATOR2 action (Cai, 2016).

A novel TORC1 independent role has been identified for Wdr24 in the regulation of lysosome dynamics and function. In wdr24 mutants, the down-regulation of TORC1 activity and the accumulation of autolysosomes occur independent of nutrient status. It was initially hypothesized that in the absence of the GATOR2 component Wdr24, the deregulation of the GATOR1 complex results in low TORC1 activity, triggering the constitutive activation of autophagy and the accumulation of autolysosomes. Surprisingly, however, epistasis analysis determined that the accumulation of lysosomes could be decoupled from both the chronic inhibition of TORC1 activity and the activation of autophagy. Raising TORC1 activity in the wrd24 mutant background, by depleting either components of the GATOR1 or TSC complex, failed to rescue the accumulation of abnormal lysosomal structures. Notably, it was determined that two additional members of the GATOR2 complex, Mio and Seh1, also regulate lysosomal behavior independent of both GATOR1 and the down-regulation of TORC1 activity. From these data it is inferred that multiple components of the GATOR2 complex have a TORC1 independent role in the regulation of lysosomes (Cai, 2016).

An increased number of autolysosomes is often associated with reduced autophagic flux due to diminished lysosomal degradation. Consistent with reduced autophagic flux, in Drosophila wrd24-/- mutants accumulated enlarged autolysosomes filled with undegraded material. Moreover, lysosomes in the wrd24-/- mutants failed to quench the GFP fluorescence of a GFP-mCherry-Atg8a protein. These phenotypes are consistent with decreased lysosomal pH and degradative capacity. In order to examine in detail the role of Wdr24 in the regulation of lysosome function a wrd24-/- knockout HeLa cell line was generated that recapitulated the phenotypes observed in Drosophila wrd24-/- mutants. Specifically, wrd24-/- HeLa cells had have decreased TORC1 activity and accumulate a large number of autolysosomes. Using multiple assays it was determined that wrd24-/- lysosomes had reduced degradative capacity and autophagic flux and thus accumulate proteins that are normally degraded by lysosomal enzymes such as p62, LC3II and Cathepsin D. Additionally, it was determined that wrd24-/-lysosomes have increased pH relative to wild-type cells, again consistent with reduced lysosomal function. Taken together these data confirm that Wdr24 plays a key role in the regulation of lysosomal activity (Cai, 2016).

This study shows that components of the GATOR2 complex function in the regulation of TORC1 activity and in the TORC1 independent regulation of lysosomal dynamics and autophagic flux. These two functions suggest that the GATOR2 complex may regulate cellular homeostasis by coordinating TORC1 activity with the dynamic regulation of lysosomes during periods of nutrient stress. Intriguingly, several recent reports describe a very similar dual function for the RagA/B GTPases in both mice and zebrafish (Kim, 2014; Shen,2016). RagA/B play a critical role in the activation of TORC1 in the presence of amino acids (Kim, 2008; Sancak, 2008). Surprisingly, however, TORC1 activity was not found to be significantly decreased in cardiomyocytes of RagA/B knockout mice (Cai, 2016).

Nevertheless, the RagA/B mutant cardiomyocytes have decreased autophagic flux and reduced lysosome acidification. From published data, it was concluded that the RagA/B GTPases regulate lysosomal function independent of their role in the regulation of TORC1 activation in some cell types. Similarly, RagA is required for proper lysosome function and phagocytic flux in microglia. Notably, Mio, a component of the GATOR2 complex is found associated with RagA (Bar-Peled, 2013). Thus, in the future it will be important to determine if components of the GATOR2 complex function in a common pathway with the Rag GTPases to regulate lysosomal function (Cai, 2016).

In Saccharomyces cerevisiae single mutants of wrd24/sea2/ and wdr59/sea3 do not exhibit defects in TORC1 regulation but do have defects in vacuolar structure. Moreover, several recently identified genes that regulate the GATOR2-GATOR1-TORC1 pathway in response to amino acid limitation are restricted to metazoans. These data make it tempting to speculate that the ancestral function of the GATOR2 complex maybe the regulation of lysosome/vacuole function and autophagic flux. Indeed, the finding that GATOR2 components regulate lysosome dynamics is particularly intriguing in light of the observation that GATOR2 complex is comprised of proteins with characteristics of coatomer proteins and membrane tethering complexes. Notably, the GATOR2 complex components Mio, Seh1 and Wdr24 localize to lysosomes and autolysosomes. Similarly, these proteins associate with the vacuolar membrane in budding yeast. Thus, going forward it will be important to examine if the GATOR2 complex acts directly on lysosomal membranes to regulate their structure and/or function. More broadly, future studies on the diverse roles of the SEACAT/GATOR2 complex will further understanding of the complex relationship between cellular metabolism and the regulation of endomembrane dynamics in both development and disease (Cai, 2016).

Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction

This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).

Mucolipidosis type IV (MLIV) and Batten disease are untreatable lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV arises from loss-of-function mutations in the gene encoding TRPML1, an endolysosomal cation channel belonging to the TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished axonal development in the cortex and corpus callosum, the causes of which remain unknown (Wong, 2015).

To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml1) flies have led to insight into the mechanisms of neurodegeneration and lysosomal storage (Wong, 2015).

This study reports that trpml1 larvae exhibit diminished synaptic growth at the NMJ, a well-studied model synapse. Lysosomal function supports Rag GTPases and MTORC1 activation, and this is essential for JNK phosphorylation and synapse development (Wong, 2015).

Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/ MTORC1 and JNK activation, suggesting that alterations in neuronal signaling are similar in different LSDs and are evolution- arily conserved. Interestingly, the NMJ defects in the two fly LSD models were suppressed by the administration of a high-protein diet and a drug that is currently in clinical trials to treat certain forms of cancer. These findings inform a pharmacotherapeutic strategy that may suppress the neurodevelopmental defects observed in LSD patients (Wong, 2015).

This study shows that lysosomal dysfunction in Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).

Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather, MTORC1 promotes NMJ growth via a MAP kinase cascade culminating in JNK activation. Therefore, decreasing lysosomal function or Rag/MTORC1 activation in hiwND8 suppressed the associated synaptic overgrowth. However, the 'small-bouton' phenotype of hiwND8 was independent of MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both rheb expression and hiw loss result in Wnd-dependent elevation in bouton numbers, the supernumerary boutons in each case show distinct morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).

Biochemical analyses revealed that both JNK phosphorylation and its transcriptional output correlated with the activity of MTORC1, which are consistent with prior observations that cln3 overexpression promotes JNK activation and that tsc1/tsc2 deletion in flies result in increased JNK-dependent transcription. These findings point to the remarkable versatility of MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).

Using an in vitro kinase assay, this study demonstrates that Wnd is a target of MTORC1. Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1, which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent with this notion, overexpression of Atg1 in the Drosophila neurons has been shown to promote JNK signaling and NMJ synapse overgrowth via Wnd) (Wong, 2015).

This study also found that developmental JNK activation in axonal tracts of the CC and pJNK levels in cortical neurons were compromised in a mouse model of Batten disease. Thus, the signaling deficits identified in Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd) and JNK signaling are critical for axonal development in the mouse CNS. Therefore, decreased neuronal JNK activation during development might underlie the thinning of the axonal tracts observed in many LSDs (Wong, 2015).

Although the findings of this study demonstrate a role for an amino-acid- responsive cascade in the synaptic defects associated with lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings were reminiscent of an elegant study that showed that the growth of Drosophila neuroblasts is uncoupled from dietary amino acids owing to the function of ALK, which suppresses the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with trpml1. Although these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).

Although LSDs result in lysosomal dysfunction throughout the body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption of lysosomal degradation leads to severe shortage of free amino acids in neurons, regardless of the quantity of dietary proteins, thus explaining the exquisite sensitivity of neurons to lysosomal dysfunction (Wong, 2015).

Coordination of autophagosome-lysosome fusion and transport by a Klp98A-Rab14 complex

Degradation of cellular material by autophagy is essential for cell survival and homeostasis, and requires intracellular transport of autophagosomes to encounter acidic lysosomes through unknown mechanisms. This study identified the PX domain-containing kinesin Klp98A as a novel regulator of autophagosome formation, transport and maturation in Drosophila. Depletion of Klp98A caused abnormal clustering of autophagosomes and lysosomes at the cell center and reduced the formation of starvation-induced autophagic vesicles. Reciprocally, overexpression of Klp98A redistributed autophagic vesicles toward the cell periphery. These effects were accompanied by reduced autophagosome-lysosome fusion and autophagic degradation. In contrast, depletion of the conventional kinesin heavy chain caused a similar mislocalization of autophagosomes without perturbing their fusion with lysosomes, indicating that vesicle fusion and localization are separable, independent events. Klp98A-mediated fusion required the endolysosomal GTPase Rab14, which interacted and colocalized with Klp98A and required Klp98A for normal localization. Thus, Klp98A coordinates the movement and fusion of autophagic vesicles by regulating their positioning and interaction with the endolysosomal compartment (Mauvezin, 2016).

Lorincz, P., Toth, S., Benko, P., Lakatos, Z., Boda, A., Glatz, G., Zobel, M., Bisi, S., Hegedus, K., Takats, S., Scita, G. and Juhasz, G. (2017). Rab2 promotes autophagic and endocytic lysosomal degradation. J Cell Biol. PubMed ID: 28483915

Rab2 promotes autophagic and endocytic lysosomal degradation

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. This study shows that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster. This study demonstrates that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. RAB2A is also required for autophagosome clearance in human breast cancer cells. In conclusion, Rab2 has been identified as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes (Lorincz, 2017).

The two main pathways of lysosomal degradation are endocytosis and autophagy. Double-membrane autophagosomes (generated in the main pathway of autophagy) and endosomes can fuse with each other to generate amphisomes, and mature into degradative endo- and autolysosomes, respectively, by ultimately fusing with lysosomes. One of the main regulators of intracellular trafficking and vesicle fusions are Rab small GTPases. Active, GTP-bound Rab proteins recruit various effectors including tethers and molecular motors, of which Rab7 is the only known direct regulator of both autophagosome-lysosome and endosome-lysosome fusions (Lorincz, 2017).

The tethering complex homotypic fusion and vacuole protein sorting (HOPS) was identified in yeast, and it simultaneously binds two yeast Rab7 (Ypt7) molecules on its opposing ends. In animal cells, Rab7 binds to RILP, ORPL1, FYCO1, and PLEKHM1 to recruit dyneins and HOPS and ensure the fusion of late endosomes and autophagosomes with lysosomes. This way, HOPS could cross-link two Rab7-positive membranes to prompt tethering and fusio. Rab7 is present on lysosomes, autophagosomes, and endosomes, but it is not clear whether another Rab is involved in degradative auto- and endolysosome formation, which also requires transport of hydrolases from the Golgi (Lorincz, 2017).

Rab2 is known to control anterograde and retrograde traffic between the ER and Golgi. A recent biochemical screen identified Rab2 as a direct binding partner of HOPS, and active Rab2 was found to localize to Rab7-positive vacuoles in cultured Drosophila melanogaster cells. This study proposes an updated model in which Rab7 and Rab2 coordinately promote the HOPS-dependent degradation of autophagosomes and endosomes via fusion of these as well as biosynthetic vesicles with lysosomes (Lorincz, 2017).

Rab2 is highly conserved among higher eukaryotes, including Drosophila melanogaster and humans. The HOPS subunits Vps39 and Vps41 directly bind to Ypt7/Rab7 in yeast, whereas their interaction may be indirect in mammalian cells. No binding was detected between Drosophila Rab7 and Vps39 or Vps41, whereas GTP-locked Rab7 bound to its known effector PLEKHM1 in yeast two-hybrid (Y2H) experiments. Vps39 directly bound Rab2GTP in both Y2H and recombinant protein pull-down experiments, and Rab2GTP immunoprecipitated endogenous Vps16A (another HOPS subunit) from fly lysates. Consistently, it has been reported that recombinant mammalian RAB2A pulls down Vps39 but not Vps41 from cell lysates, and human HOPS subunits did not show Rab7 binding in Y2H experiments (Lorincz, 2017).

To address whether Rab2 functions in autophagy and endocytosis, rab2 was knocked out by imprecise excision of a transposon from the 5' UTR. The resulting rab2d42 allele carries a 2,047-bp deletion, which removes most of the protein coding sequences of both predicted Rab2 isoforms and eliminates protein expression. Rab2 mutant animals die as L2/L3-stage larvae, and their viability is fully rescued by expression of YFP-Rab2 (Lorincz, 2017).

Larval fat cells are widely used for autophagy analyses because of their massive autophagic potential. Numerous Lysotracker Red (LTR)-positive vesicles appear upon starvation, which represent newly formed autolysosomes with likely increased v-ATPase-mediated acidification in these cells. LTR dot number and size (and signal intensity as a likely consequence) decreased in rab2-null cells compared with controls, which was rescued by expression of YFP-Rab2. RNAi knockdown of Rab2 in GFP-marked fat cell clones also impaired starvation-induced punctate LTR staining compared with surrounding GFP-negative cells (Lorincz, 2017).

A 3xmCherry-Atg8a reporter that labels all autophagic structures via retained fluorescence of mCherry inside autolysosomes revealed increased number and decreased size of such vesicles in both starved rab2 RNAi and mutant fat cells. A dLamp-3xmCherry reporter of late endosomes and lysosomes showed similar changes in rab2 RNAi or mutant fat cells of starved animals. Tandem tagged mCherry-GFP-Atg8a reporters are commonly used to follow autophagic flux, because GFP is quenched in lysosomes, whereas mCherry signal persists. Knockdown of rab2 prevented the quenching of GFP that is seen in starved control fat cells: dots positive for both GFP and mCherry accumulated, raising the possibility that Rab2 promotes autophagosome-lysosome fusion, similar to HOPS. Colocalization of 3xmCherry-Atg8a with the lysosomal hydrolase cathepsin L (CathL) was examined. The overlap of these markers of autophagic and lysosomal structures strongly decreased in rab2 mutant fat cells compared with controls, and rab2 RNAi also impaired endogenous CathL-positive vesicle formation, suggesting that formation of degradative autolysosomes requires Rab2 (Lorincz, 2017).

These phenotypes resembled the autophagosome-lysosome fusion defect of mutants for the autophagosomal SNARE syntaxin 17, HOPS, and Rab7. Accordingly, ultrastructural analysis of starved fat cells revealed accumulation of double-membrane autophagosomes and small dense structures likely representing amphisomes, similar to HOPS mutants. Recently, rab2 RNAi was reported to cause accumulation of autophagosomes in Drosophila muscles and enlarged amphisomes in fat cells. Autophagosome accumulation in our rab2-null mutant fat cells is likely caused by a complete loss-of-function condition (Lorincz, 2017).

Western blots detected increased levels of the selective autophagy cargo p62/Ref2p, along with both free and lipidated autophagosome-associated forms of Atg8a in starved rab2 mutants. Basal autophagic degradation was also impaired in rab2 mutants, based on increased numbers of endogenous Atg8a and p62 dots in well-fed conditions (Lorincz, 2017).

The importance of Rab2 for autophagic degradation was confirmed in human cells. Knockdown of RAB2B had no effect on endogenous LC3 structures in breast cancer cells, whereas RAB2A or combined siRNA treatment caused accumulation of autophagic vesicles. LC3 accumulated within Lamp1-positive structures upon RAB2A knockdown, which likely represent amphisomes unable to mature into autolysosomes in these cells, consistent with the recently reported role of Rab2 homologs for degradation of autophagic cargo in mouse embryonic fibroblasts (Lorincz, 2017 and references therein).

To analyze the possible involvement of Drosophila Rab2 in endosomal degradation, dissected nephrocytes were incubated with fluorescent avidin for 5 min. Trafficking of this endocytic tracer was clearly perturbed in rab2 mutant cells, similar to vps41/lt and rab7 mutants. Loss of HOPS leads to enlargement of late endosomes. Similarly, Rab7 endosomes are enlarged in rab2 mutant nephrocytes compared with control or rescued cells. Importantly, fluorescent avidin was trapped in Rab7 endosomes and failed to reach CathL-positive lysosomes after a 30-min chase in rab2 mutants. LTR staining showed the presence of acidic vacuoles in rab2 mutant nephrocytes, which probably include the enlarged late endosomes in rab2 mutant nephrocytes, based on ultrastructural analysis . Aberrant late endosomes accumulated in mutant cells, which were apparently unable to fuse with neighboring acid phosphatase-positive lysosomes. Of note, the number of acid phosphatase-positive lysosomes also decreased in mutant nephrocytes, suggesting that Rab2 promotes both endosome-lysosome fusion and biosynthetic transport to lysosomes (Lorincz, 2017).

GTP-locked, constitutively active Rab2GTP redistributes from the Golgi onto Rab7 vacuoles in cultured Drosophila cells. Similarly, Rab2GTP colocalized with endogenous Rab7 in starved fat cells, unlike wild-type Rab2. Rab2GTP appeared as large pronounced rings around LTR-positive autolysosomes in starved fat cells, unlike wild-type Rab2. Similarly, Rab2GTP formed rings around lysosomes and autophagic structures marked by dLamp-3xmCherry and 3xmCherry-Atg8a, respectively. Of note, small Rab2GTP dots often closely associated with large Rab2GTP rings in these experiments, raising the possibility that Rab2 vesicles fuse with autolysosomes. Finally, wild-type Rab2 or Rab2GTP modestly overlapped with autophagosomes marked by endogenous Atg8a (Lorincz, 2017).

These localization and loss-of-function data pointed to Rab2 as a positive regulator of autolysosome formation. Indeed, fat and midgut cells expressing Rab2GTP contained enlarged and brighter 3xmCherry-Atg8a autophagic structures and dLamp-3xmCherry lysosomes compared with surrounding control cells, suggesting that Rab2 controls autolysosome size. Increased lysosomal input or a block of degradation can cause enlargement of autolysosomes. Systemic expression of Rab2GTP did not impair the viability of animals, and Western blots of starved L3 larval lysates revealed no changes in p62 and Atg8a levels, suggesting that autophagic degradation proceeds normally in cells expressing Rab2GTP. Thus, Rab2GTP may increase autolysosome size by accelerating fusions with other vesicles. Importantly, expression of GTP-locked, active Rab7 did not increase the size of autophagic structures. Rab7 is required for autophagosome-lysosome fusion, and its knockdown prevents the formation of large, bright 3xmCherry-Atg8a-positive autolysosomes: these cells contain only small, faint autophagosomes. Similarly, only small, faint 3xmCherry-Atg8a dots appeared in Rab2GTP-expressing fat cells undergoing Rab7 RNAi, indicating that Rab2-dependent fusions also require Rab7 and there is no functional redundancy between them (Lorincz, 2017).

Eye pigment granules are lysosome-related organelles. Changes in lysosomal transport often lead to eye discoloration caused by pigment granule alterations, such as in HOPS mutants. Rab2GTP expression led to a slight darkening of eyes and appearance of enlarged pigment granules, consistent with the role of Rab2 in promoting lysosomal fusions (Lorincz, 2017).

Several homo- and hetero-typic fusions occur during endosome and autophagosome maturation into degradative lysosomes. Known metazoan factors acting at lysosomal fusions include HOPS and EPG5 tethers and Rab7 together with its effectors. Because biosynthetic transport to lysosomes also requires input from Golgi, the role of Golgi-associated Rab2 in various lysosomal fusions fits well into this picture. Consistently, Rab2 promotes breakdown of phagocytosed apoptotic bodies and lysosome-related acrosome biogenesis (Lorincz, 2017).

Accumulation of unfused autophagosomes and enlarged late endosomes in rab2 mutants resembles the fusion defect of rab7 mutant cells. The decreased function of lysosomes in rab2 mutants is unlikely to account for these fusion defects, because we have shown that autophagosome-lysosome fusion proceeds and gives rise to enlarged, nondegrading autolysosomes in fat cells with perturbed acidification or biosynthetic transport to lysosomes (Lorincz, 2017).

The role of Rab2 in the fusion of lysosomes with other vesicles is also supported by the autolysosomal localization of its active form and by its binding to the Vps39-containing end of HOPS, the tethering complex required for autophagosomal, endosomal, and biosynthetic transport to lysosomes. Consistently, Rab2 recruits HOPS to Rab7-positive vesicles in cultured Drosophila cells. Expression of Rab2GTP increases degradative autolysosome and pigment granule size, suggesting that it is rate limiting during these fusion reactions, unlike Rab7. This is supported by low levels of wild-type Rab2 on these organelles, unlike wild-type Rab7 that is abundant on autophagosomes, late endosomes, and lysosomes. Consistent with this, it has been recently shown that expression of RAB2AGTP also increases Rab7 vesicle size in human cells. Based on binding of Rab2 to one end of HOPS, an updated model is proposed of lysosomal fusions in animal cells. It is hypothesized that GTP-loaded Rab2 is transported on Golgi-derived carrier vesicles toward Rab7 positive vesicles, and its interaction with Vps39 promotes fusions. Vps41 located on the other end of HOPS may bind Rab7 vesicles via adaptors such as PLEKHM1. These interactions help the tethering and fusion of autophagic, endocytic, and lysosomal vesicles to generate degrading compartments. Lysosomal membranes may contain active Rab2 for only a short period of time, and it likely dissociates upon GTP hydrolysis to limit organelle size. Rab asymmetry is also observed during homotypic vacuole fusion in yeast: GTP-bound Ypt7/Rab7 is necessary on only one of the vesicles, and its nucleotide status is irrelevant on the opposing membrane. Importantly, Rab7 directly interacts with both ends of HOPS in the absence of a Rab2 homolog in yeast. This difference may explain why yeast cells contain one large vacuole instead of the many smaller lysosomes seen in animal cells. Collectively, these data indicate that Rab2 and Rab7 coordinately promote autophagic and endosomal degradation and lysosome function (Lorincz, 2017).

A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila

Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. The deubiquitinating enzyme UBPY/USP8 has been identified as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, it was hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. This study blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. Inhibition of endosomal trafficking at different steps in vivo was observed to be systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. These results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo (Jacomin, 2016).

Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Lipocalin Apolipoprotein D (ApoD) is an extracellular lipid binding protein endowed with antioxidant capacity. This study performed a comprehensive analysis of ApoD intracellular traffic and demonstrates its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. ApoD was shown to be endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins (see Glial Lazarillo). These results set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases (Pascua-Maestro, 2017).

Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis

How multicellular organisms maintain immune homeostasis across various organs and cell types is an outstanding question in immune biology and cell signaling. In Drosophila, blood cells (hemocytes) respond to local and systemic cues to mount an immune response. While endosomal regulation of Drosophila hematopoiesis is reported, the role of endosomal proteins in cellular and humoral immunity is not well-studied. This study demonstrated a functional role for endosomal proteins in immune homeostasis. The ubiquitous trafficking protein ADP Ribosylation Factor 1 (ARF1) and the hemocyte-specific endosomal regulator Asrij differentially regulate humoral immunity. Asrij and ARF1 play an important role in regulating the cellular immune response by controlling the crystal cell melanization and phenoloxidase activity. ARF1 and Asrij mutants show reduced survival and lifespan upon infection, indicating perturbed immune homeostasis. The ARF1-Asrij axis suppresses the Toll pathway anti-microbial peptides (AMPs) by regulating ubiquitination of the inhibitor Cactus. The Imd pathway is inversely regulated- while ARF1 suppresses AMPs, Asrij is essential for AMP production. Several immune mutants have reduced Asrij expression, suggesting that Asrij co-ordinates with these pathways to regulate the immune response. This study highlights the role of endosomal proteins in modulating the immune response by maintaining the balance of AMP production. Similar mechanisms can now be tested in mammalian hematopoiesis and immunity (Khadilkar, 2017).

A balanced cellular and humoral immune response is essential to achieve and maintain immune homeostasis. In Drosophila, aberrant hematopoiesis and impaired hemocyte function can both affect the ability to fight infection and maintain immune homeostasis. Endosomal proteins are known to regulate Drosophila hematopoiesis. This study shows an essential function for endosomal proteins in regulating immunity (Khadilkar, 2017).

Altered hemocyte number and distribution as a result of defective hematopoiesis, can also lead to immune phenotypes like increased melanization or phagocytosis. This study shows that perturbation of normal levels of endocytic molecules ARF1 or Asrij leads to aberrant hematopoiesis, affecting the circulating hemocyte number. This in turn leads to an impaired cellular immune response. The aberrant hematopoietic phenotypes with pan-hemocyte tissue-specific depletion of ARF1 using e33cGal4 or HmlGal4 are comparable to the phenotypes observed in the case of asrij null mutant. Hence this study has compared Gal4-mediated ARF1 knockdown to asrij null mutant (Khadilkar, 2017).

In addition, it was also shown that ARF1 and Asrij have a direct role in humoral immunity by regulating AMP gene expression. This is likely to be a contribution from the hemocyte compartment which is primarily affected upon perturbation of Asrij or ARF1. It is well established that hemocytes, apart from acting as the cellular arm of the immune response, also act as sentinels and relay signals to the immune organs that mount the humoral immune response. Hemocytes have been shown to produce ligands like Spaetzle and upd3 that activate immune pathways and induce anti-microbial peptide secretion from the fat body or gut. Asrij or ARF1 could also be affecting the production of such ligand molecules thereby affecting the target immune-activation pathways (Khadilkar, 2017).

Considering the involvement of Asrij and ARF1 in both the arms of immune response, a model is proposed for the role of the ARF1-Asrij axis in maintaining immune homeostasis that can be used for testing additional players in the process (Khadilkar, 2017).

It is known that ARF1 is involved in clathrin coat assembly and endocytosis and has a critical role in membrane bending and scission. In this context it is also intriguing to note that ARF1, like Asrij, does not seem to have an essential role in phagocytosis. This suggests that hemocytes could be involved in additional mechanisms beyond phagocytosis in order to combat an infection (Khadilkar, 2017).

Both ARF1 and Asrij control hemocyte proliferation as their individual depletion leads to an increase in the total and differential hemocyte counts. Also, both mutants have higher crystal cell numbers due to over-activation of Notch as a result of endocytic entrapment. This suggests that increased melanization accompanied by increase in phenoloxidase activity upon ARF1 or Asrij depletion is a consequence of aberrant hematopoiesis and not likely due to a cellular requirement in regulating the melanization response. Constitutive activation of the Toll pathway or impaired Jak/Stat or Imd pathway signaling in various mutants also leads to the formation of melanotic masses. Thus the phenotypes seen on Asrij or ARF1 depletion could either be due to the defective hematopoiesis which directly affects the cellular immune response or leads to a mis-regulation of the immune regulatory pathways (Khadilkar, 2017).

Regulation of many signaling pathways, including the immune regulatory pathways takes place at the endosomes. For example, endocytic proteins Mop and Hrs co-localize with the Toll receptor at endosomes and function upstream of MyD88 and Pelle, thus indicating that Toll signalling is regulated by endocytosis. This study shows that loss of function of the ARF1-Asrij axis leads to an upregulation of some AMP targets of the Toll pathway. Upon depletion of ARF1-Asrij endosomal axis, increased ubiquitination of Cactus, a negative regulator of the Toll pathway, was found in both hemocytes and fat bodies. This suggests non-autonomous regulation of signals by the ARF1-Asrij axis, which is in agreement with an earlier model of signalling through this route. Thus the endosomal axis may systemically control the sorting and thereby degradation of Cactus, which in turn promotes the nuclear translocation of Toll effector, Dorsal. This could explain the significant increase in Toll pathway reporter expression such as Drosomycin-GFP. Interestingly the effect of ARF1 depletion on the Toll pathway is more pronounced than that of Asrij depletion. This is not surprising as ARF1 is a ubiquitous and essential trafficking molecule that regulates a variety of signals. This suggests that ARF1 is likely to be involved with additional steps of the Toll pathway and may also interact with multiple regulators of AMP expression (Khadilkar, 2017).

ARF1 and Asrij show complementary effects on IMD pathway target AMPs. While ARF1 suppresses the production of IMD pathway AMPs, Asrij has a discriminatory role. Asrij seems to promote transcription of AttacinA and Drosocin, whereas it represses Cecropin. However in terms of AMP production only Drosocin and Diptericin are affected, but not to the extent of ARF1. In addition, Relish shows marked nuclear localization in fat body cells of hemocyte-specific arf1 knockdown larvae whereas there is no significant difference in the localization in Asrij depleted larval fat bodies. This indicates that ARF1-Asrij axis exerts differential control over the Imd pathway. Thus ARF1 causes strong generic suppression of the Imd pathway while the role of Asrij could be to fine tune this effect. Mass spectrometric analysis of purified protein complexes indicates that ARF1 and Imd interact. Hence it is very likely that ARF1 regulates Imd pathway activation at the endosomes. Whether this interaction involves Asrij or not remains to be tested and will give insight into modes of differential activation of immune pathways (Khadilkar, 2017).

This analysis shows that Asrij is the tuner for endosomal regulation of the humoral immune response by ARF1 and provides specialized tissue- specific and finer control over AMP regulation. This is in agreement with earlier data showing that Asrij acts downstream of ARF127. Since ARF1 is expressed in the fat body it could communicate with the hemocyte- specific molecule, Asrij, to mediate immune cross talk (Khadilkar, 2017).

As reduced Asrij expression is seen in Toll and Jak/Stat pathway mutants such as Rel E20 and Hop Tum1, it is likely that these effectors also regulate Asrij, setting up a feedback mechanism to modulate the immune response. Earlier work has shown that ARF1-Asrij axis modulates different signalling outputs like Notch by endosomal regulation of NICD (Notch Intracellular Domain) transport and activity and JAK/STAT by endosomal activation of Stat92e. Further, ARF1 along with Asrij regulates Pvr signaling in order to maintain HSC's. ARF1 acts downstream of Pvr. Surprisingly, Asrij levels are downregulated in the Pvr mutant. Hence it is likely that the ARF1-Asrij axis regulates trafficking of the Pvr receptor, which then also regulates Asrij levels thus providing feedback regulation. While active modulation of signal activity and outcome at endosomes could be orchestrated by ARF1 and Asrij, their activities in turn need to be modulated. The data suggest that targets of Asrij endosomal regulation may in turn regulate Asrij expression at the transcript level. Further, upon Gram positive infection in wild type flies, asrij transcript levels decrease with a concomitant increase in suppressed AMPs such as Cecropin. This indicates additional regulatory loops such as that mediated by the IMD pathway effector NFκB may regulate asrij transcription. Using bioinformatics tools, presence of binding sites for NFκβ and Rel family of transcription factors are seen in the upstream regulatory sequence (1kb upstream) of asrij and arf1. Hence, feedback regulation is proposed of Asrij and ARF1 by the effectors of the Toll and Imd pathway respectively. This is reflected in the regulation of Asrij expression by these pathways. This also implies multiple modes of regulation of asrij and arf1, which are likely important in its role as a tuner of the generic immune response, thereby allowing it to discriminate between AMPs that were thought to be uniformly regulated, such as those downstream of IMD. Thus this analysis gives insight into additional complex regulation of the Drosophila immune response that can now be investigated further (Khadilkar, 2017).

Asrij and ARF1 being endocytic proteins are likely to interact with a number of molecules that regulate different cell signalling cascades. Due to endosomal localization, molecular interactions may be favored that further translate into signalling output. Hence, it is not surprising that Asrij and ARF1 genetically interact with multiple signalling pathways and can aid crosstalk to regulate important developmental and physiological processes like hematopoiesis or immune response. It is quite likely that Asrij and ARF1 are themselves also part of different feedback loops or feed-forward mechanisms as their levels need to be tightly regulated. Evidence for this is found with respect to the Toll, JAK/STAT and Pvr pathway as described earlier. Hence it is proposed that the Asrij-ARF1 endosomal signalling axis genetically interacts with various signalling components thereby regulating blood cell and immune homeostasis (Khadilkar, 2017).

AMP transcript level changes upon ARF1 or Asrij depletion also correspond to reporter-AMP levels seen after infection. This suggests that although ARF1 is known to have a role in secretion, mutants do not have an AMP secretion defect. Hence aberrant regulation of immune pathways on perturbation of the ARF1-Asrij axis is most likely due to perturbed endosomal regulation (Khadilkar, 2017).

ARF1 has a ubiquitous function in the endosomal machinery and is well-positioned to regulate the interface between metabolism, hematopoiesis and immunity in order to achieve homeostasis. Along with Asrij and other tissue-specific modulators, it can actively modulate the metabolic and immune status in Drosophila. In this context, it is interesting to note that Asrij is a target of MEF253, which is required for the immune-metabolic switch in vivo. Thus Asrij could bring tissue specificity to ARF1 action, for example, by modulating insulin signalling in the hematopoietic system (Khadilkar, 2017).

It is likely that in Asrij or ARF1 mutants, the differentiated hemocytes mount a cellular immune response and perish as in the case of wild type flies where immunosenescence sets in with age and the ability of hemocytes to combat infection declines. Since their hematopoietic stem cell pool is exhausted, they may fail to replenish the blood cell population, thus compromising the ability to combat infections. Alternatively, mechanisms that downregulate the inflammatory responses and prevent sustained activation may be inefficient when the trafficking machinery is perturbed. This could result in constitutive upregulation thus compromising immune homeostasis (Khadilkar, 2017).

In summary, this study shows that in addition to its requirement in hematopoiesis, the ARF1-Asrij axis can differentially regulate humoral immunity in Drosophila, most likely by virtue of its endosomal function. ARF1 and Asrij bring about differential endocytic modulation of immune pathways and their depletion leads to aberrant pathway activity and an immune imbalance. In humans, loss of function mutations in molecules involved in vesicular machinery like Amphyphysin I in which clathrin coated vesicle formation is affected leads to autoimmune disorders like Paraneoplastic stiff-person syndrome. Synaptotagmin, involved in vesicle docking and fusion to the plasma membrane acts as an antigenic protein and its mutation leads to an autoimmune disorder called Lambert-Eaton myasthenic syndrome. Mutations in endosomal molecules like Rab27A, β subunit of AP3, SNARE also lead to immune diseases like Griscelli and Hermansky-Pudlak syndrome. Mutants of both ARF1 and Asrij are likely to have drastic effects on the immune system. Asrij has been associated with inflammatory conditions such as arthritis, thyroiditis, endothelitis and tonsillitis, whereas the ARF family is associated with a wide variety of diseases. ARF1 has been shown to be involved in mast cell degranulation and IgE mediated anaphylaxis response. Generation and analysis of vertebrate models for these genes such as knockout and transgenic mice will provide tools to understand their function in human immunity (Khadilkar, 2017).

Lysosomal degradation is required for sustained phagocytosis of bacteria by macrophages

Clearance of bacteria by macrophages involves internalization of the microorganisms into phagosomes, which are then delivered to endolysosomes for enzymatic degradation. These spatiotemporally segregated processes are not known to be functionally coupled. This study shows that lysosomal degradation of bacteria sustains phagocytic uptake. In Drosophila and mammalian macrophages, lysosomal dysfunction due to loss of the endolysosomal Cl- transporter ClC-b/CLCN7 delayed degradation of internalized bacteria. Unexpectedly, defective lysosomal degradation of bacteria also attenuated further phagocytosis, resulting in elevated bacterial load. Exogenous application of bacterial peptidoglycans restored phagocytic uptake in the lysosomal degradation-defective mutants via a pathway requiring cytosolic pattern recognition receptors and NF-κB. Mammalian macrophages that are unable to degrade internalized bacteria also exhibit compromised NF-κB activation. These findings reveal a role for phagolysosomal degradation in activating an evolutionarily conserved signaling cascade, which ensures that continuous uptake of bacteria is preceded by lysosomal degradation of microbes (Wong, 2017).

Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster

In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulate both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. This study shows that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H+-ATPase pump (V-ATPase) subunits. Remarkably, it was also found that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, that will form sensory organs in the adult, appear to possess a distinctive endo-lysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endo-lysosomal function and disrupts proneural patterning. Similar to these findings in Drosophila, in human breast epithelial cells, it was observed that the impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endo-lysosomal compartment and depletion of TFEB reduces ligand-independent N signaling activity. These data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate (Tognon, 2016).

Class III phosphatidylinositol-3-OH kinase controls epithelial integrity through endosomal LKB1 regulation

The molecular mechanisms underlying the interdependence between intracellular trafficking and epithelial cell polarity are poorly understood. This study shows that inactivation of class III phosphatidylinositol-3-OH kinase (CIII-PI3K), which produces phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes, disrupts epithelial organization. This is caused by dysregulation of endosomally localized LKB1, also known as STK11, which shows delocalized and increased activity accompanied by dysplasia-like growth and invasive behaviour of cells provoked by JNK pathway activation. CIII-PI3K inactivation cooperates with Ras(V12) to promote tumour growth in vivo in an LKB1-dependent manner. Strikingly, co-depletion of LKB1 reverts these phenotypes and restores epithelial integrity. The endosomal, but not autophagic, function of CIII-PI3K controls polarity. The CIII-PI3K effector, WD repeat and FYVE domain-containing 2 (WDFY2), was identified an LKB1 regulator in Drosophila tissues and human organoids. Thus, this study defines a CIII-PI3K-regulated endosomal signalling platform from which LKB1 directs epithelial polarity, the dysregulation of which endows LKB1 with tumour-promoting properties (O'Farrell, 2017).

Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification

This study shows that Mask, an Ankyrin-repeat and KH-domain containing protein, plays a key role in promoting autophagy flux and mitigating degeneration caused by protein aggregation or impaired ubiquitin-proteasome system (UPS) function. In Drosophila eye models of human tauopathy or amyotrophic lateral sclerosis diseases, loss of Mask function enhanced, while gain of Mask function mitigated, eye degenerations induced by eye-specific expression of human pathogenic MAPT/TAU or FUS proteins. The fly larval muscle, a more accessible tissue, was then used to study the underlying molecular mechanisms in vivo. Mask was found to modulate the global abundance of K48- and K63-ubiquitinated proteins by regulating macroautophagy/autophagy-lysosomal-mediated degradation, but not UPS function. Indeed, upregulation of Mask compensated the partial loss of UPS function. It was further demonstrated that Mask promotes autophagic flux by enhancing lysosomal function, and that Mask is necessary and sufficient for promoting the expression levels of the proton-pumping vacuolar (V)-type ATPases in a TFEB-independent manner. Moreover, the beneficial effects conferred by Mask expression on the UPS dysfunction and neurodegenerative models depend on intact autophagy-lysosomal pathway. These findings highlight the importance of lysosome acidification in cellular surveillance mechanisms and establish a model for exploring strategies to mitigate neurodegeneration by boosting lysosomal function (Zhu, 2017).

Misfolded protein aggregates in and outside of cells in the central nervous system are pathological hallmarks of many neurodegenerative disorders including Alzheimer (AD), Parkinson (PD), Huntington (HD) diseases and amyotrophic lateral sclerosis (ALS). Interestingly, many of the aggregated proteins (such as MAPT (TAU) and APP for Alzheimer disease, SNCA/α-synuclein for Parkinson disease, HTT (Huntingtin) for Huntington disease, FUS, SOD1 and TARDBP/TDP-43 for ALS) can serve as seeds for 'prion-like' spreading of the aggregation within and among cells. It is not entirely clear whether these aggregates are the causes or the results of progressive and cell-type-specific neurodegeneration. However, mounting evidence suggests that clearance and prevention of these toxic protein aggregates are beneficial for meliorating degeneration (Zhu, 2017).

Two major pathways collaborate in regulating intracellular protein degradation: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system. Under the normal conditions, UPS serves as the primary route for rapid protein turnover while autophagy mainly degrades long-lived proteins and large cellular organelles under basal conditions and can be robustly induced in face of stresses such as starvation, organelle damage or accumulation of misfolded proteins. However when it comes to degradation of damaged proteins in diseased states, autophagy has been shown to play at least an equally important role as UPS.5. Many of the neurodegenerative disease-related proteins are delivered to autophagic vacuoles and degraded by the autophagy pathway. Meanwhile, impairment of autophagy in the mouse brain causes neurodegeneration associated with ubiquitin-positive protein aggregation. These data suggest that UPS and autophagy are both indispensable in maintaining cellular protein homeostasis. Furthermore, recent studies indicate that UPS and autophagy pathways coordinate with each other to prevent accumulation of toxic protein aggregates, so that enhanced activity of one pathway can compensate if the other is compromised (Zhu, 2017).

Both UPS and autophagy degradation systems are complex processes consisting of chains of sequential events orchestrated by a large group of proteins. To understand their coordinated action, it is necessary to identify novel players that are necessary and sufficient to mediate the compensatory function between the two systems. This study shows Mask, a conserved protein with Ankyrin repeats and a KH domain, as a novel and critical player in such a context. Initially identified as a modulator of receptor tyrosine signaling during Drosophila development (Smith, 2002), Mask has recently been shown to function as a cofactor of the Hippo pathway effector Yorkie and together they regulate target gene transcription with another transcription cofactor (Scalloped) during cell proliferation (Sansores-Garcia, 2013; Sidor, 2013). The human ortholog of Mask, ANKHD1, is highly expressed in several cancer cell lines. Loss of mask function rescues the mitochondrial defects and muscle degeneration observed with pink1 and park mutants (Zhu, 2015). This study shows that in MAPT- and FUS-induced eye degeneration fly models, loss of Mask function enhances degeneration, while gain of Mask function suppresses degeneration. By enhancing V-type ATPase expression, Mask promotes lysosome acidification and autophagic flux; Mask is necessary and sufficient to mediate a compensatory effect for partial loss of UPS function, to increase clearance of ubiquitinated proteins, and to protect against degeneration induced by aggregation-prone mutations (Zhu, 2017).

Autophagy, an evolutionarily conserved cellular mechanism that preserves metabolic homeostasis during nutrient unavailability, is traditionally regarded as a self-eating degradative process with limited selectivity. However, mounting evidence suggests that both micro- and macro-autophagy can play cytoprotective roles to specifically target damaged and toxic organelles and proteins for clearance under pathological conditions. The mechanism of selective autophagy is unclear. There is some evidence that autophagy receptors can recognize ubiquitin-dependent and ubiquitin-independent signals for selective degradation. Autophagy is a multistep process including nucleation, autophagosome formation and fusion with lysosomes and each step can be regulated to enhance degradation of damaged cellular components. Research has emerged showing TFEB is a potent regulator of the autophagy-lysosomal pathway whose activation can promote lysosomal function and mitigate disease in a range of neurodegenerative disorders. This study shows that Mask acts in a TFEB-independent manner to boost the expression of V-ATPase subunits. This study provides novel evidence that lysosome function is not only required for the normal clearance of ubiquitinated and misfolded proteins, but its activity can also be boosted potential through enhanced lysosomal acidification, to mitigate cellular degeneration caused by toxic protein aggregation (Zhu, 2017).

Mask is well positioned to regulate lysosome-mediated clearance of ubiquitinated and misfolded proteins. As a positive regulator of several V-type ATPase V1 subunits expression, Mask function is necessary and sufficient to promote lysosomal acidification and autophagosome degradation in a cell-autonomous manner. When the UPS function is impaired, increased Mask expression is sufficient to increase autophagic flux, which in turn compensates the partial loss of the proteasome-mediated degradation. Interestingly, even when UPS function is intact, levels of Mask activity impact the abundance of UPS-dependent (K48) and -independent (such as K63) ubiquitin-conjugated proteins, suggesting that autophagy and lysosome-mediated degradation plays an important role for basal protein homeostasis. Under pathological conditions such as UPS inactivation or excessive accumulation of disease proteins, upregulation of Mask activity substantially suppressed the cellular degeneration phenotypes in both muscles and photoreceptors, potentially through Mask-mediated increase of autophagy and lysosome activities and subsequent degradation of harmful protein aggregates, as suggested by the current biochemical and genetic analyses. In support of this notion, upregulation of Mask promotes autophagic flux in larval muscles, adult eyes and adult brains (Zhu, 2017).

This work in the Drosophila model organism yielded new insight into Mask-mediated cellular protective mechanisms that regulate lysosomal function in normal and stressed conditions caused by misfolding-prone disease proteins or impaired UPS. Such mechanisms may provide a therapeutic approach for the treatment of a group of neurodegenerative disorders caused by intracellular inclusions (Zhu, 2017).

The ABC transporter Snu and the extracellular protein Snsl cooperate in the formation of the lipid-based inward and outward barrier in the skin of Drosophila

Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, this study found that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu (CG9990) and the extracellular protein Snsl (CG2837). In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. These data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates (Zuber, 2017).


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