InteractiveFly: GeneBrief

Rab5: Biological Overview | References


Gene name - Rab5

Synonyms -

Cytological map position - 22E1-22E1

Function - signaling

Keywords - fusion of endocytic vesicles with endosomes, synapse, tracheal maturation, regulation of JAK/STAT activity, endosomal transport to promotes wingless signaling, promotes F-actin reorganization in oogenesis

Symbol - Rab5

FlyBase ID: FBgn0014010

Genetic map position - 2L:2,359,546..2,365,362 [-]

Classification - Rab5-related subfamily

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Norman, M., Vuilleumier, R., Springhorn, A., Gawlik, J. and Pyrowolakis, G. (2016). Pentagone internalises glypicans to fine-tune multiple signalling pathways. Elife [Epub ahead of print]. PubMed ID: 27269283
Summary:
Tight regulation of signalling activity is crucial for proper tissue patterning and growth. This study investigates the function of Pentagone (Pent), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. Pent was shown to internalise the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and the study proposes that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, a requirement of Pent was found to be required for Wg signalling. These data propose a novel mechanism by which morphogen signalling is regulated.

Fu, Y., Zhu, J. Y., Zhang, F., Richman, A., Zhao, Z. and Han, Z. (2017). Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes. Cell Tissue Res [Epub ahead of print]. PubMed ID: 28180992
Summary:
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.
Wang, Y., Zhang, H., Shi, M., Liou, Y.C., Lu, L. and Yu, F. (2017). Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons. Development [Epub ahead of print]. PubMed ID: 28420712
Summary:
Pruning, whereby neurons eliminate their exuberant neurites, is central for the maturation of the nervous system. In Drosophila, sensory neurons, ddaCs, selectively prune their larval dendrites without affecting their axons during metamorphosis. However, it is unknown whether the secretory pathway plays a role in dendrite pruning. This study shows that the small GTPase Arf1, an important regulator of secretory pathway, is specifically required for dendrite pruning of ddaC/D/E sensory neurons but dispensable for apoptosis of ddaF neurons. Analyses of the GTP and GDP-locked forms of Arf1 indicate that the cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. Sec71 was identified as a guanine nucleotide exchange factor for Arf1 that preferentially interacts with its GDP-bound form. Like Arf1, Sec71 is also important for dendrite pruning, but not apoptosis, of sensory neurons. Arf1 and Sec71 are interdependent for their localizations on Golgi. Finally, Sec71/Arf1-mediated trafficking process is a prerequisite for Rab5-dependent endocytosis to facilitate endocytosis and degradation of the cell adhesion molecule Neuroglian
Tang, Y., Geng, Q., Chen, D., Zhao, S., Liu, X. and Wang, Z. (2017). Germline proliferation is regulated by somatic endocytic genes via JNK and BMP signaling in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 28315838
Summary:
Signals derived from the microenvironment contribute greatly to tumorigenesis. This study used Drosophila testis as a model system to address this question, taking the advantage of the ease to distinguish germline and somatic cells and to track the cell numbers. In an EMS mutagenesis screen, Rab5, a key factor in endocytosis, was identified for its non-autonomous role in germline proliferation. The disruption of Rab5 in somatic cyst cells, which escort the development of germline lineage, induced the over-proliferation of under-differentiated but genetically wild-type germ cells. This non-autonomous effect was mediated by the transcriptional activation of Dpp (the fly homolog of BMP) by examining the Dpp-reporter expression and knocking down Dpp to block germline overgrowth. Consistently, the protein levels of Bam, the germline pro-differentiation factor normally accumulated in the absence of BMP/Dpp signaling, decreased in the over-proliferating germ cells. Further, it was discovered that JNK signaling pathway operated between Rab5 and Dpp, because simultaneously inhibiting JNK pathway and Rab5 in cyst cells prevented both dpp transcription and germline tumor growth. Additionally, it was found that multiple endocytic genes, such as avl, TSG101, Vps25, or Cdc42, were required in the somatic cyst cells to restrict germline amplification. These findings indicate that when the endocytic state of the surrounding cells are impaired, genetically wild-type germ cells overgrow. This non-autonomous model of tumorigenesis provides a simple system to dissect the relation between tumor and its niche.

BIOLOGICAL OVERVIEW

During constitutive endocytosis, internalized membrane traffics through endosomal compartments. At synapses, endocytosis of vesicular membrane is temporally coupled to action potential-induced exocytosis of synaptic vesicles. Endocytosed membrane may immediately be reused for a new round of neurotransmitter release without trafficking through an endosomal compartment. Using GFP-tagged endosomal markers, an endosomal compartment in Drosophila neuromuscular synapses was monitored. In conditions in which the synaptic vesicles pool is depleted, the endosome is also drastically reduced and recovers only from membrane derived by dynamin-mediated endocytosis. This suggests that membrane exchange takes place between the vesicle pool and the synaptic endosome. The small GTPase Rab5 is required for endosome integrity in the presynaptic terminal. Impaired Rab5 function affects endo- and exocytosis rates and decreases the evoked neurotransmitter release probability. Conversely, Rab5 overexpression increases the release efficacy. Therefore, the Rab5-dependent trafficking pathway plays an important role for synaptic performance (Wucherpfennig, 2003).

At the presynaptic terminal, Ca2+-triggered neurotransmitter (NT) release by exocytosis is immediately followed by the local recycling of the synaptic vesicle (SV) membrane. SV recycling is necessary to preserve the plasma membrane surface area, to sustain the population of SVs, and to maintain the molecular diversity of the vesicle versus the plasma membrane (Wucherpfennig, 2003).

There are at least two distinct recycling mechanisms: 'kiss and run'. During kiss and run, SVs make brief contact with the plasma membrane forming a transient porelike structure through which the NT is released. In contrast, clathrin-mediated endocytosis occurs after complete fusion of the SV with the plasma membrane. New vesicles are subsequently reformed through a complex process initiated by the formation of an invagination at the plasma membrane mediated by clathrin and its adaptors. In lamprey, snake, and fly neuromuscular synapses, the invagination of the membrane into pits occurs at distinct 'centers of endocytosis' surrounding the active zones of exocytosis. Subsequently, amphiphysin, dynamin, and endophilin are thought to lead to the formation of a clathrin-coated, endocytic vesicle. The subsequent steps are still a matter of debate and it is controversial whether endocytic vesicles mature directly into SVs, recycle through an intermediate endosomal compartment before they become SVs, or whether both pathways are used under different conditions of synaptic demand (Wucherpfennig, 2003 and references therein).

In nonneuronal cells, it is well established that endocytic vesicles fuse with endosomes in a process mediated by the small GTPase Rab5 (Bucci, 1992; Horiuchi, 1997). Through the recruitment of several effector molecules, Rab5 has been suggested to form a specialized membrane domain (Rab5 domain) at the early endosome (Sonnichsen, 2000; De Renzis, 2002). Based on this, Rab5 has been used as a marker for early endosomes. Active Rab5 recruits two phosphatidylinositol-3-kinases (PI[3]-kinases), p85α/p110ß and VPS34/p150, which trigger a local enrichment of phosphatidylinositol-3-phosphate (PI[3]P) in the endosomal membrane (Christoforidis, 1999). PI(3)P specifically binds to the FYVE zinc-finger domain of endosomal factors such as the Rab5 effectors EEA1 and Rabenosyn-5, which ultimately mediate endocytic vesicle tethering and fusion with the endosome (Stenmark, 1995; Simonsen, 1998; Lawe, 2000; Nielsen, 2000). Consistently, blocking the PI(3)-kinases with antibodies or wortmannin impairs the association of FYVE domain proteins with the endosome and, thereby, blocks endosomal trafficking (Mills, 1998; Simonsen, 1998). Furthermore, it has been shown that the FYVE domain binds to PI(3)P only when inserted in a lipid bilayer (Misra, 1999; Sankaran, 2001) and that the localization of a myc-tagged tandem repeat of the FYVE domain (myc-2xFYVE) is restricted to early endosomes and the internal membrane of multivesicular bodies (Gillooly, 2000). Therefore, 2xFYVE is a bona fide marker for the PI(3)P-containing endosomes (Wucherpfennig, 2003).

Rab5 has been found on SVs (de Hoop, 1994; Fischer von Mollard, 1994), suggesting that SVs have the capacity to fuse with an endosomal compartment. Furthermore, both neuroendocrine PC12 cells and hippocampal neurons contain synaptic-like vesicles that traffic in a Rab5-dependent manner through endosomal compartments at least in the absence of synaptic transmission. However, it is unclear whether these Rab5-dependent endocytic pathways act only during nourishment and cell signaling in neurons, or also function in the recycling and maturation of SVs during synaptic transmission (Wucherpfennig, 2003).

In favor of SV recycling through the endosome, it has recently been suggested that a neuron-specific isoform of the AP3 clathrin adaptor complex from brain cytosol is required for SV budding from PC12 cell endosomes. Furthermore, FM1-43 styryl dye recycling experiments in the Drosophila neuromuscular junction (NMJ) uncovered two recycling pathways, a rapid and a slower one, suggesting the existence of an endosome-dependent pathway. However, in rat hippocampal neurons in culture, FM1-43 experiments suggested that SVs retain their identity through the endocytic cycle, implying that the SV membrane does not traffic through an intermediate endosome. In addition, SV recycling in neurons is likely too rapid to allow for constitutive trafficking through an intermediate endosomal compartment. However, not all vesicles participate in the endo-exo recycling at any given time. Remaining vesicles may have sufficient time to exchange membrane with the endosome (Wucherpfennig, 2003 and references therein).

A Rab5-positive, PI(3)P-containing endosomal compartment is present at the presynaptic terminal of Drosophila. As in nonneuronal cells, this compartment depends on Rab5 function. The endosome is depleted under conditions where SVs are depleted, and the endosome is replenished by membrane derived by dynamin-mediated endocytosis. Rab5 also influences the synaptic efficacy: impairment of Rab5 function decreases the NT release probability and the recycling SV pool size, whereas overexpression of Rab5 increases the release probability. A working model suggests that membrane exchange between the vesicle pool and the presynaptic endosome occurs and is of functional importance for the efficiency of SVs to fuse with the plasma membrane during Ca2+-triggered endocytosis (Wucherpfennig, 2003).

The mechanism of SV recycling has since long been a matter of debate. It has been proposed that vesicles internalized by clathrin-mediated endocytosis traffic through an intermediate endosomal compartment to become mature SVs. However, in cultured hippocampal neurons, endocytic vesicle membrane does not intermix with an internal intermediate compartment. The current study presents evidence that Rab5-dependent membrane exchange between vesicles and the endosome at the synapse can occur. Furthermore, a Rab5-mediated trafficking step determines, in a rate-limiting manner, the synaptic performance (Wucherpfennig, 2003).

It has been established that Rab5 is involved in the fusion of endocytic vesicles with their target endosomal compartment (Bucci, 1992; Stenmark, 1994). In addition Rab5 has been implicated in the budding of endocytic vesicles from the plasma membrane (McLauchlan, 1998). The current data are consistent with a key role of Rab5 during both endocytic trafficking steps at the presynaptic terminal. This is because exocytosis and endocytosis are temporally and functionally coupled at the Drosophila NMJ, making it difficult to ascertain the primary basis of an endo- or exocytic/recycling phenotype. Because the ultrastructure of the endosome is grossly disrupted in Rab5 loss- and gain-of-function mutants, the possibility is favored that it is the Rab5-dependent endosomal dynamics that play a key role in the SV cycle (Wucherpfennig, 2003).

The data leaves open the question whether this proposed trafficking step is obligatory during SV recycling or if it involves trafficking of only an SV sub-pool at any given time. However, regardless of what percentage of the SV pool recycles at a given time through the endosome, at the steady state, this recycling pathway must play a key role for the synaptic performance of the full SV pool, because synaptic efficacy increases or decreases in a rate-limiting manner depending on the levels of Rab5 function (Wucherpfennig, 2003).

Interfering with Rab5 function using the dominant-negative version of Rab5 causes a reduction in the number of released quanta during synaptic transmission, whereas elevated levels of Rab5 increase the quantal content. Morphological and electrophysiological analysis of these Rab5 mutants shows that the changes in synaptic performance are not due to a change in the readily releasable pool size, but are rather due to a change in the release probability of the SVs (Wucherpfennig, 2003).

How could the membrane exchange between vesicles and the endosome affect the SV release probability? It is well established in cultured mammalian cells that the Rab5 endosome functions as a sorting station where endocytic cargo is targeted either toward recycling or degradation (Zerial, 2001). A similar scenario may take place within the presynaptic terminal. The protein and lipid composition of the SV membrane could be controlled at the endosome by sorting out aged components and replacing them with newly synthesized ones. This, in turn, will be likely of consequence for SV function, particularly for the efficacy of regulated exocytosis and, therefore, for the SV release probability. Furthermore, sorting of alternative vesicular proteins could occur at the endosome. In this context, it has been recently found that the ratio of different Synaptotagmin isoform (Synaptotagmin I vs. Synaptotagmin IV) in SVs affects the efficacy of Ca2+-triggered exocytosis (Wucherpfennig, 2003).

Based on the findings that enhancement or reduction of Rab5 function lead to a parallel increase or decrease in the SV release probability, it is suggested that a Rab5-mediated membrane exchange between vesicles and the endosome affects the synaptic strength in a rate-limiting manner (Wucherpfennig, 2003).

Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy

The small GTPase Rab5 promotes recruitment of the Ccz1-Mon1 guanosine exchange complex to endosomes to activate Rab7, which facilitates endosome maturation and fusion with lysosomes. How these factors function during autophagy is incompletely understood. This study shows that autophagosomes accumulate due to impaired fusion with lysosomes upon loss of the Ccz1-Mon1-Rab7 module in starved Drosophila fat cells. In contrast, autophagosomes generated in Rab5 null mutant cells normally fuse with lysosomes during the starvation response. Consistent with that, Rab5 is dispensable for the Ccz1-Mon1-dependent recruitment of Rab7 to PI3P-positive autophagosomes, which are generated by the action of the Atg14-containing Vps34 PI3 kinase complex. Finally, Rab5 was found to be required for proper lysosomal function. Thus, the Ccz1-Mon1-Rab7 module is required for autophagosome-lysosome fusion, whereas Rab5 loss interferes with a later step of autophagy: the breakdown of autophagic cargo within lysosomes (Hegedus, 2016).

Autophagy ensures the lysosomal degradation of self-material, including cytosol and organelles. During the main pathway, double-membrane autophagosomes serve as the transport vesicles. Endocytosis delivers plasma membrane, including transmembrane receptors, and exogenous substances taken up from the environment to lysosomes. Thus autophagy and endocytosis converge at the level of lysosomes, where degradation of cargo arriving from both routes takes place (Hegedus, 2016).

A critical event during these transport processes is vesicle maturation: how the newly formed vesicles acquire the molecular characteristics and protein complexes that establish their identity and determine the subsequent vesicle fusion events that often culminate in the lysosomal compartment. Several similarities between endosomes and autophagosomes are known. For example, both autophagosomes and endosomes are positive for phosphatidylinositol-3-phosphate (PI3P) due to localized vacuolar protein sorting 34 (Vps34) PI3 kinase activity, which has been showed to be required for the generation of both types of vesicles in Drosophila larvae. Autophagosomes can also fuse with endosomes to give rise to hybrid organelles termed amphisomes, which then fuse with lysosomes (Hegedus, 2016).

Small GTPases of the Ras-related protein in brain (Rab) family are critical regulators of membrane trafficking in eukaryotic cells. An active, GTP-bound Rab protein binds to various effectors that usually regulate vesicle motility and fusion with the proper membrane compartment. In the endocytic pathway, Rab5 associates with early endosomes and activates a Vps34-containing phosphoinositide 3-kinase complex that generates PI3P on the surface of these vesicles. PI3P-binding domains such as the Fab-1, YGL023, Vps27, and EEA1 (FYVE) domain promote recruitment to early endosomes. Of importance, several proteins, including the vesicle tethers early endosomal antigen 1 (EEA1) and Rabenosin-5, have both FYVE and Rab5-binding domains, indicating that multiple interactions may play a role in the recruitment of effectors. Similarly, the Rab7 guanine nucleotide exchange factor (GEF) monensin sensitivity protein 1 (Mon1)-caffeine, calcium, and zinc 1 (Ccz1) complex binds to both the GTP-bound form of endosomal Rab5 and PI3P. Rab7 is then activated by this complex and promotes fusion of late endosomes and lysosomes (Hegedus, 2016).

Recruitment of the soluble N-methylamaleimide-sensitive factor attachment protein receptor (SNARE) Syntaxin 17 is a critical step in autophagosome maturation because these vesicles acquire fusion competence this way. Interaction of Syntaxin 17 with the homotypic fusion and vacuole protein sorting (HOPS) tethering complex ensures efficient fusion between autophagosomes and lysosomes. HOPS is believed to be a Rab7 effector, and Rab7 was indeed found to promote the formation of degradative autolysosomes in cultured cells, although it remains to be established whether this protein is already present on autophagosomes before the fusion with lysosomes. In theory, the binding of HOPS to lysosomal Rab7 and autophagosomal Syntaxin 17 (and other factors, such as phospholipids) may be sufficient for its tethering activity. In addition, autophagy-related gene 14 (Atg14), a Vps34 kinase complex subunit that is important for autophagosome formation, also functions as a tether and promotes autophagosome-lysosome fusion by directly binding to Syntaxin 17 (Hegedus, 2016 and references therein).

In yeast, the fusion machinery differs somewhat from that of the animal cells because the SNAREs involved are not homologous. Still, autophagosome fusion with the vacuole (the equivalent of the lysosomal system in metazoan cells) requires HOPS, Ypt7/Rab7, and its GEF, the Mon1-Ccz1 complex, and more recently, autophagosome-like structures were found to accumulate in yeast cells lacking the major Rab5 homologue Vps21. Of interest, decreased Rab5 function attenuates the autophagic degradation of the pathogenic, mutant form of huntingtin in cultured human cells. This was attributed to impaired Vps34 lipid kinase activity and reduced formation of the Atg5-Atg12 conjugate, both of which are important for autophagosome formation (Hegedus, 2016).

Thus the role of the Rab5-Ccz1-Mon1-Rab7 axis during autophagy is not clear. This study set out to address this problem in the popular animal model Drosophila. Fruit flies offer certain advantages for such studies. First, there is only a single fly homologue of Rab5 (unlike in mammalian and yeast cells, which both have three different Rab5 proteins). Second, massive induction of autophagy is seen in the fat and liver tissue-like fat cells of starved larvae. Third, it is straightforward to carry out functional studies in mosaic animals, in which mutant cells are surrounded by control cells in the same tissue of the same animal, which reduces variability because one can compare the phenotype of neighboring control and loss-of-function cells. Using this system, Ccz1, Mon1, and Rab7 are shown to be required for autophagosome-lysosome fusion in fat cells of starved animals independent of Rab5. Of interest, Rab5 was found to function downstream of the Rab7 module by controlling a later step of autophagy: degradation of autophagic cargo within lysosomes (Hegedus, 2016).

This study showed that the Rab7 module and Rab5 control different steps of autophagy. Rab7 mediates autophagosome-lysosome fusion together with its GEF, the Ccz1-Mon1 complex. This is likely achieved by the recruitment of Rab7 to autophagosomes in a Ccz1-Mon1-dependent manner. Although Drosophila Mon1 binds to the active, GTP-locked form of Rab5 as in other organisms, Rab5 is dispensable for the fusion of autophagosomes with lysosomes and for Rab7 localization to autophagosomes and autolysosomes. The question is then: what is the signal that recruits Ccz1-Mon1 and Rab7 to autophagic structures? (Hegedus, 2016).

Mon1 and Ccz1 bind to phospholipids, including PI3P, in yeast, and this study found that Drosophila Mon1 has similar features. This raises the possibility that the Ccz1-Mon1 complex is recruited to the PI3P-positive surface of autophagosomes through this interaction. Vps34-dependent PI3P generation is required for autophagosome formation and endosome maturation. Vps34 is activated by Rab5. Of interest, the current data suggest that loss of Rab5 inhibits PI3P generation only on endosomes but not on autophagosomes. Loss of UVRAG but not Atg14 inhibits PI3P generation on endosomes, whereas loss of Atg14 leads to complete inhibition of PI3P-positive autophagosome biogenesis. Thus UVRAG is dispensable for Vps34 activity during autophagosome formation, and its loss causes a defect in autolysosomal degradation. Similarly, Rab5 mutant cells showed accumulation of autophagic cargo due to impaired lysosomal degradation (Hegedus, 2016).

Recently the Rab5-related Vps21 small GTPase was suggested to control the fusion of autophagosome with the vacuole (lysosome) in yeast cells. In this study, clusters of autophagic structures were found to accumulate near the vacuole. However, these vesicles were positive for both the autophagy marker GFP-Atg8 and the vacuolar marker FM4-64, suggesting that some sort of fusion must have occurred in this case, too (Hegedus, 2016).

On the basis of the current results, the following model is proposed of autolysosome formation in fat cells of starved Drosophila larvae (see The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy). PI3P-positive autophagosomes are generated through the action of an Atg14-containing Vps34 PI3 kinase complex. PI3P attracts Ccz1-Mon1, which promotes Rab7 recruitment to autophagosomes. Both PI3P and Rab7 bind to the HOPS tethering complex, and thus these factors promote the tethering of autophagosomes with late endosomes and lysosomes. The membrane fusion is then executed by the Syx17-Snap29-Vamp7 SNARE complex. Autophagic cargo is broken down in autolysosomes, and their full degradative capacity requires the function of Rab5 and the UVRAG-containing Vps34 complex for the proper delivery of lysosomal proteins, likely including both acidic hydrolases and membrane proteins. This is in line with the finding that simultaneous knockdown of all three Rab5 homologues leads to a collapse of the endolysosomal system in mouse liver cells (Hegedus, 2016).

It has already been demonstrated that autophagosome-lysosome fusion is mediated by the HOPS tethering complex and the SNAREs Syx17, Snap29, and Vamp7/8. It is not yet clear how these fusion factors are recruited to the autophagosomal membrane. HOPS is known as a Rab7 effector, and according to the current findings, Rab7 is present on autophagosomes. It is proposed that autophagosomal PI3P recruits the Ccz1-Mon1-Rab7 module to facilitate the loading of HOPS and subsequent tethering of vesicles (Hegedus, 2016).

Vps34 is considered as a bona fide Rab5 effector. Surprisingly, this study found that whereas Rab5 mediates only the generation of PI3P on endosomes mainly through the action of a UVRAG-containing Vps34 complex, it is dispensable for PI3P-positive autophagosome biogenesis, which depends on the Atg14-containing Vps34 complex. Thus the current concept that Vps34 is a Rab5 effector must be revisited: it is true for endocytosis but not applicable for autophagy in fat cells of starved Drosophila larvae (Hegedus, 2016).

A previous study showed that Rab5 promotes autophagy-mediated huntingtin clearance in cultured human cells and Drosophila eyes. Simultaneous small interfering RNA knockdown of all three Rab5 genes (Rab5a, Rab5b, Rab5c) reduced the level of Atg5-Atg12 conjugate and autophagosome formation. Although no perturbations of autophagosome biogenesis and fusion were seen in Rab5 mutant fat cells, these discrepancies may be due to the different models used. In the current experiments, starvation induces a massive wave of autophagy in larval Drosophila fat cells that entirely relies on the activity of the Rab5-independent Atg14-Vps34 PI3 kinase complex. It is possible that during the basal, nonstarved conditions in a previous study, Rab5 can contribute to autophagosome formation. In fact, UVRAG has also been suggested to control autophagosome formation in cultured cells, which is compatible with this model (Hegedus, 2016).

In summary, Rab7 is recruited to autophagosomes by the Ccz1-Mon1 complex to promote autophagosome-lysosome fusion. This study show that autophagosome formation and fusion is independent of Rab5 and the UVRAG-containing Vps34 PI3 kinase complex but requires the action of the Atg14-Vps34 complex. Rab5, similar to UVRAG, is necessary for proper lysosomal function by promoting the trafficking of lysosomal proteins (Hegedus, 2016).

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 (Gui, 2016).

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).

Interplay between Rab5 and PtdIns(4,5)P2 controls early endocytosis in the Drosophila germline

Phosphoinositides have emerged as key regulators of membrane traffic through their control of the localization and activity of several effector proteins. Both Rab5 and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] are involved in the early steps of the clathrin-dependent endocytic pathway, but little is known about how their functions are coordinated. This study investigated the role of PtdIns(4,5)P2 and Rab5 in the Drosophila germline during oogenesis. Rab5 was found to be required for the maturation of early endocytic vesicles. PtdIns(4,5)P2 is required for endocytic-vesicle formation, for Rab5 recruitment to endosomes and, consistently, for endocytosis. Furthermore, a previously undescribed role of Rab5 was revealed in releasing PtdIns(4,5)P2, PtdIns(4,5)P2-binding budding factors and F-actin from early endocytic vesicles. Finally, overexpressing the PtdIns(4,5)P2-synthesizing enzyme Skittles leads to an endocytic defect that is similar to that seen in rab5 loss-of-function mutants. Hence, these results argue strongly in favor of the hypothesis that the Rab5-dependant release of PtdIns(4,5)P2 from endosomes that was discovered in this study is crucial for endocytosis to proceed (Compagnon, 2009).

Regulation of the level of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] within a cell and recruitment of the small GTPase Rab5 to clathrin-coated vesicles (CCVs) are required to promote the formation and maturation of these vesicles. Although these two mechanisms appear to participate in the same step of clathrin-dependant endocytosis, the potential interdependency between them is not known. The existence in Drosophila of a well-characterized endocytic route in the oocyte makes its oogenesis a very attractive model in which to address this issue in a physiological context. The ovarian follicle is composed of a 16-cell germline cyst -- one cell of which is determined as the oocyte -- that is surrounded by follicle cells. The most-studied endocytic process in the oocyte is the uptake of yolk proteins (Yp1-Yp3) via clathrin-mediated endocytosis. From stages 8 to 11, the oocyte intakes, via endocytosis, a large quantity of vitellogenins, which are yolk-protein precursors that are synthesized in the fat body and the follicle cells. After their entry into the oocyte, yolk proteins are stored inside large late endocytic compartments, yolk granules, which will later provide the reserves necessary for embryonic development. This specific accumulation of embryonic reserves inside the oocyte is achieved by the localization of the vitellogenin receptor Yolkless (Yl) at the plasma membrane (PM) of the oocyte during stages 8 to 11. The morphology of the endocytic intermediates in insect oocytes is well described at the ultrastructural level. For instance, it is inside the ovary of Aedes aegypti that coated vesicles were observed for the first time. Furthermore, the succession of these intermediates has also been well described by means of a thermosensitive dominant negative allele of shibire, the Drosophila homolog of dynamin. The vitellogenins are first found in clathrin-coated pits (CCPs) at the oocyte PM; after fission, these CCPs form CCVs. These vesicles then lose their coat and form tubular intermediates that fuse with the forming yolk-protein storage granules, thus filling the lumen of these granules with yolk proteins to form mature storage granules (Compagnon, 2009).

The small GTPase Rab5 is a well-known regulator of early endocytosis in mammals. Rab5 is involved in the budding of CCVs in vitro and also regulates their subsequent maturation by promoting the fusion of early endocytic vesicles (EEVs) with sorting endosomes. This contribution to endocytic-vesicle maturation relies on the recruitment of several effector proteins that trigger a local enrichment in the endosomal membrane of phosphatidylinositol 3-phosphate [PtdIns(3)P] (Christoforidis, 1999; Erdmann, 2007; Hyvola, 2006; Shin, 2005). The Rab5-dependent formation of the PtdIns(3)P-positive endosomal domain on early endosomes participates in the recruitment of endosomal factors regulating various aspects of early-endosome function, such as tethering, fusion and mobility. Another aspect of endocytosis-related phosphatidylinositol (PtdIns) regulation is the control of PtdIns(4,5)P2 levels during early endocytic steps. PtdIns(4,5)P2 plays a crucial role in the selective recruitment of endocytic proteins to the PM for CCV formation. It binds to endocytic clathrin adaptor complexes such as AP2 to initiate the assembly of the coat and also to dynamin, which controls the fission reaction. Consistent with this, lower levels of PtdIns(4,5)P2 impair endocytosis. There is yet another requirement in the regulation of PtdIns(4,5)P2 after CCV formation. The PtdIns(4,5)P2 5-phosphatase activity of Synaptojanin (Synj) is necessary for the hydrolysis of PtdIns(4,5)P2 from EEVs, thereby triggering coat-component shedding (Compagnon, 2009 and references therein).

In Drosophila, Rab5 has been found to localize on PtdIns(3)P-containing early endosomes at the neuromuscular junction, where it is required for synaptic-vesicle recycling. It has also been demonstrated that Rab5 function is required for the formation of PtdIns(3)P-containing early endosomes. Thus, Rab5 is, in Drosophila, a fundamental regulator of the early endocytic pathway, similar to its mammalian homologs (Compagnon, 2009).

This study used complete loss of rab5 function in the germline cyst to study the consequences on the endocytic pathway. Rab5 was found to be required for maturation of the EEV and yolk-protein endocytosis in the oocyte. Using loss of function of skittles (sktl), coding for a type I phosphatidylinositol 4-phosphate 5-kinase, it was shown that PtdIns(4,5)P2 is required for endocytic-vesicle formation, for Rab5 recruitment and accordingly for yolk-protein endocytosis. Furthermore, a previously undescribed role for Rab5 was revealed in controlling the release of PtdIns(4,5)P2, PtdIns(4,5)P2-binding budding factors and F-actin from EEVs. Finally, it was shown that overexpressing the PtdIns(4,5)P2-synthesizing enzyme Sktl first leads to the formation of an abnormal early endocytic compartment containing Rab5, PtdIns(4,5)P2-binding coat component and F-actin, and, second, affects yolk-protein endocytosis. Hence, these results argue strongly in favor of the hypothesis that Rab5-dependent release of PtdIns(4,5)P2 from EEVs is crucial for endocytosis to proceed (Compagnon, 2009).

The first stage of endocytosis, CCV formation, has previously been shown to rely on the presence of PtdIns(4,5)P2 at the PM for coat-component recruitment and fission in mammals (for review see, Di Paolo and De Camilli, 2006). In Drosophila, the situation has been less clear; early studies attempting to address the requirement of PtdIns(4,5)P2 for endocytosis were inconclusive. The current results in sktl mutant oocytes indicate that, when PtdIns(4,5)P2 level is lowered, endocytosis is impaired. Moreover, analysis of endocytic-compartment morphology at the ultrastructural level in this context revealed a depletion of all intracytoplasmic endocytic intermediates. This strongly suggests a conserved requirement of PtdIns(4,5)P2 for the formation of endocytic vesicles in the oocyte. Interestingly, the build-up of coated pits along invaginations of the PM that was observed in sktl mutant oocytes is very similar to what is observed with a dominant-negative allele of dynamin, encoding an essential PtdIns(4,5)P2-binding regulator of fission. Moreover, in sktl mutant oocytes, the subcortical recruitment of Rab5 during vitellogenic stages was affected. These observations suggest that PtdIns(4,5)P2-dependent CCV formation is necessary for Rab5 recruitment (Compagnon, 2009).

Consistent with a role of Rab5 following EEV formation, analysis of endocytic-compartment morphology at the ultrastructural level in the absence of Rab5 showed an accumulation of EEVs, which was associated with a depletion of later endocytic structures. This suggests a conserved requirement of Rab5 for EEV maturation, but does not exclude that Rab5 might be also required for other steps along the endocytic pathway. Surprisingly, loss of function of rab5 impairs the removal of PtdIns(4,5)P2 from the endosomal membrane (Compagnon, 2009).

The importance of PtdIns(4,5)P2 turnover for endocytic-vesicle maturation was demonstrated by the study of Synj, a PtdIns 5-phosphatase, in synaptic termini. In synj knock-out mice or Drosophila mutants, synaptic-vesicle recycling is impaired and CCVs accumulate in thecytoplasm (Cremona, 2001; Verstreken, 2003). This study observed, in mutants with strong loss of synj function, that neither yolk-protein endocytosis nor actin restriction at the cortex were impaired in the oocyte. Nevertheless, it was found that an excess in the PtdIns(4,5)P2-producing enzyme Sktl led to the formation of an abnormal endocytic compartment containing PtdIns(4,5)P2 and, accordingly, a reduction of yolk-protein endocytosis in this context was also observed. The results indicate that, in vivo, besides its known requirement in neuronal cells, PtdIns(4,5)P2 removal from endosomal membranes is also essential for endocytosis to proceed in other cell types. Furthermore, this study found a situation different from that in neurons; removing PtdIns(4,5)P2 from endosomal membrane of oocytes does not require Synj. The finding also echoes the recent observation that Synj is not required for endocytosis in S2 cells (Korolchuk, 2007). Altogether, this suggests that different enzymes could fulfill this function in various cell types. Interestingly, the finding that Rab5 is present on the abnormal endocytic structures induced by Sktl overexpression suggests that, in this context, endocytosis is blocked at the stage when Rab5 is required to proceed further along the endocytic path. Altogether, these observations make the finding of a role of Rab5 in the removal of PtdIns(4,5)P2 from endosomal membrane all the more relevant (Compagnon, 2009).

In the absence of Rab5, PtdIns(4,5)P2, found in the ectopic endosomal compartments, is associated with the PtdIns(4,5)P2-binding factors necessary for coat recruitment and fission, and with F-actin aggregates, hence suggesting that the defective PtdIns(4,5)P2 regulation impairs the dynamics of budding factors and F-actin organization. Although the involvement of Rab5 in these processes independently from its effect on PtdIns(4,5)P2 distribution cannot be ruled out, the interpretation that these phenotypes are a direct consequence of altered PtdIns(4,5)P2 removal is favored for several reasons. First, recent studies using live-cell imaging have shown that there is an intimate connection between the regulation of PtdIns(4,5)P2 levels and coat assembly and/or disassembly (Sun, 2007; Zoncu, 2007). Second, it has been established that the PtdIns(4,5)P2-dependent shut-down of actin polymerization is required for endocytosis to proceed in yeast (Sun, 2007). Third, the phenotypes are reminiscent of those observed when the PtdIns(4,5)P2-synthesizing enzyme Sktl was overexpressed (Compagnon, 2009).

These observations raise the issue of a possible link that could exist between Rab5 and the spatial restriction of PtdIns(4,5)P2. Among the Rab5 effectors known to be involved in PtdIns metabolism, three could directly regulate PtdIns(4,5)P2 levels: the PtdIns 3-kinase p110 is able to use PtdIns(4,5)P2 as a substrate to produce PtdIns(3,4,5)P3, and the PtdIns 5-phosphatases INPP5B and ORCL are able to use both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to produce PtdIns(4)P and PtdIns(3,4)P2, respectively. This study found that PtdIns(3,4,5)P3 accumulates on endosomes along with PtdIns(4,5)P2 in rab52 mutant oocytes. This leads to favoring of the assumption that the accumulation of PtdIns(4,5)P2 that was revealed in this study is more likely to arise from defective PtdIns 5-phosphatase recruitment than defective PtdIns 3-kinase recruitment. Another hypothesis, compatible with the previous assumption, is that Rab5 can also restrict PtdIns(4,5)P2 synthesis by negatively regulating Sktl activity from the endosomes. Two observations are in line with this scenario: (1) Sktl overexpression led to defects that were similar to those observed in rab5 loss-of-function mutants, and (2) Sktl is found on abnormally maturing endosomes in rab52 mutant oocytes. It may thus prove fruitful in the future to search for a Rab5 effector that is able to restrict Sktl localization from endosomes, and to explore the influence of Rab5-recruited PtdIns 5-phosphatase on the regulation of PtdIns(4,5)P2 levels along the endocytic pathway (Compagnon, 2009).

Analysis of integrin turnover in fly myotendinous junctions: Rab5 can regulate the proportion of integrin adhesion complex components that undergo turnover

Transient (short-term) cell adhesion underlies dynamic processes such as cell migration, whereas stable (long-term) cell adhesion maintains tissue architecture. Ongoing adhesion complex turnover is essential for transient cell adhesion, but it is not known whether turnover is also required for maintenance of long-term adhesion. This study used fluorescence recovery after photobleaching to analyze the dynamics of an integrin adhesion complex (IAC) in a model of long-term cell-ECM adhesion, myotendinous junctions (MTJs), in fly embryos and larvae. The turnover of components of the IAC used fluorescently tagged constructs of β position-specific (βPS) integrin (βPS-integrin-YFP) and of the core structural IAC components Talin (Talin-GFP) and Tensin (Tensin-GFP), as well as a viable line with a GFP inserted in the genomic ilk (integrin-linked kinase) gene (ILK-GFP). IAC was found to undergo turnover in MTJs, and this process was found to be mediated by clathrin-dependent endocytosis. Moreover, the small GTPase Rab5 can regulate the proportion of IAC components that undergo turnover. Also, altering Rab5 activity weakened MTJs, resulting in muscle defects. In addition, growth of MTJs was concomitant with a decrease in the proportion of IAC components undergoing turnover. It is proposed that IAC turnover is tightly regulated in long-term cell-ECM adhesions to allow normal tissue growth and maintenance (Yuan, 2010).

This is the first study of the turnover of integrin adhesions in live animals. The MTJs analyzed are long-lasting cell-ECM adhesions that form during late embryonic stages and last throughout larval life (about 5 days at room temperature). Although MTJs grow and undergo remodeling at larval stages, they must nonetheless support ongoing muscle attachment during this time. Overall, these results show considerable IAC dynamics in the MTJs. The lowest levels of IAC turnover measured were in 3rd instar larval muscles and even at that stage the mobile fraction of IAC components ranged from as low as 5% for homozygous talin-GFP to as high as 24% for homozygous integrin-YFP (Yuan, 2010).

Surprisingly, it was found that a significant proportion of the βPS-integrin in MTJs is mobile. Previous studies in cell culture suggested that integrins are mostly immobile within the range of the life-time of focal contacts (10 to 30 minutes), whereas other components of the IAC are highly dynamic and have a half-life on the order of 2-7 minutes. In the MTJs, the proportion of βPS-integrin that is mobile is in line with other components of the IAC, such as talin, tensin and ILK. Although this suggests that some differences exist between the turnover mechanisms of stable and transient adhesions, major mechanistic similarity was found between turnover in MTJs and focal contacts. For instance, both processes require dynamin-mediated endocytosis and are regulated by the Rab family of small GTPases. This study establishes the MTJ as a useful model to analyze turnover in the context of stable cell-ECM adhesion (Yuan, 2010).

Mobile fractions of various IAC components were measured to assess their dynamics at the MTJs. In the case of integrins, the mobile fraction could be a measurement of turnover (assembly and disassembly) of the IAC or, alternatively, of lateral diffusion. FRAP experiments on whole and partial MTJs demonstrate that lateral mobility is not a significant factor contributing to the integrin dynamics that were measured. For the cytoplasmic components of the IAC, the mobile fractions could measure one or more of three processes: turnover, the assembly and disassembly of the IAC; diffusion of IAC molecules within the cytoplasm; or exchange, the process in which cytoplasmic IAC components bind to and depart from the already assembled adhesion complex. For example, a recent study found that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion and a fast-diffusing cytoplasmic pool. Although it is likely that all three processes listed could contribute to the dynamics of various IAC cytoplasmic components, it is proposed that the mobile fraction observed in the MTJ is mainly due to IAC assembly and disassembly, rather than diffusion and exchange. This is suggested based on two observations. First, the fluorescence recovery of IAC components reaching their mobile fractions was measured in the range of minutes and seconds rather than milliseconds. Studies in cell culture show that the dynamics of IAC components near the adhesion site are dominated by binding kinetics rather than by free diffusion and occur on a similar timescale. Second, if the mobile fraction of ILK represented only the binding kinetics of ILK with other IAC components, then an increase in the stability of integrin at the MTJ would not reduce the mobile fraction of ILK. However, it was observed that the mobile fractions of both ILK and βPS-integrin significantly decline upon blockage of endocytosis. Nevertheless, it is still possible that ILK undergoes exchange; this might account for some of the 20% of the ILK protein that remained in the mobile fraction when clathrin-mediated endocytosis was inhibited (Yuan, 2010).

Rab5 concentrates at MTJs and can regulate the size of the mobile fraction of IAC molecules that are undergoing turnover. This is consistent with published results showing that other Rab proteins, such as Rab21, regulate adhesion. In migrating cells, overexpression of Rab21 leads to increased integrin adhesion, whereas decreased expression of Rab21 leads to reduced adhesion. Intriguingly, MTJ defects are conferred by the expression of either Rab5-DN, which decreases the mobile fraction, or Rab5-CA, which increases the mobile fraction. It is not clear why the expression of either DN or CA versions of Rab5 gave rise to a nearly identical phenotype. However, the findings are consistent with previous work in flies showing that overexpression of integrins gives rise to muscle-detachment phenotypes identical to those found in integrin null mutants. By extension, a small reduction or a small increase in the amount of immobile ECM-ligand-bound integrin conferred by expression of Rab5-DN or Rab5-CA could lead to a similar muscle defect. These observations emphasize the importance of precisely regulating the level of Rab5 activity at the MTJ for the maintenance of muscle attachment. It is likely that maintenance of the MTJ necessitates a careful balance between the process of integrin internalization and IAC disassembly, and the process of integrin trafficking to the MTJ and IAC assembly. Any deviation from the required balance between adhesion complex assembly and disassembly leads to muscle detachment (Yuan, 2010).

At the end of muscle morphogenesis (stage 16 of embryogenesis), IACs in muscles exhibit high rates of turnover similar to those observed in migrating cells. One possible explanation is that, because muscle morphogenesis involves dynamic processes, such as cell migration and tissue rearrangement, it requires extensive IAC turnover. The high levels of turnover observed at the immediate conclusion of muscle morphogenesis are therefore a lingering after-effect of this phase of myogenesis. Another likely explanation is that a certain amount of turnover persists in the newly formed MTJ to allow growth and remodeling to take place during larval development. Moreover, it is predicted that the substantial levels of turnover observed in late embryonic and early larval stages are generally unsustainable in mature MTJs. Furthermore, it is conjectured that a gradual reduction in the level of turnover, similar to observations in the MTJs, is a general feature of cell adhesion complexes undergoing the transformation from a transient to a stable and long-lasting adhesion (Yuan, 2010).

In addition to supporting stabilization of the adhesion junctions, it is speculated that the reduction in the proportion of integrin and IAC components that undergo turnover plays an active role in MTJ growth. Shifting a greater proportion of the integrins in MTJs from the mobile to the immobile fraction could result in an increase in the size and overall strength of the MTJs, so that they can support the strain placed on muscle-tendon attachment as muscles grow. The question arises as to whether MTJs in adults, which form during pupal stages and last even longer, also exhibit IAC turnover. Adult muscles do not undergo further growth, but could potentially undergo remodeling of the MTJs, for example in response to increased mechanical stress. Integrin turnover in the adult might also contribute to the repair of MTJs in response to accrued mechanical damage. Because of the presence of an exoskeleton in the adults, it is not currently possible to analyze integrin turnover using FRAP, but the data show that depletion of integrin and other IAC components in adult muscles gives rise to muscle defects, consistent with ongoing adhesion complex turnover (Yuan, 2010).

Based on these data, it is proposed that, in order to maintain the MTJs, the level of IAC turnover in the fully assembled muscle must be limited to within a specific range. This level of turnover necessitates equilibrium between IAC disassembly and IAC assembly. There are three generalized models for the turnover of the IAC: in one case, the entire complex is disassembled and assembled as a set unit; the second is that some of the IAC remains assembled and that only integrin molecules are internalized; the third is a mixture of both. The experiments show that an increase or decrease in the mobile fraction of integrin is correlated with a similar increase or decrease in the turnover of other IAC components. Especially striking in this regard are the coordinated developmental changes in the mobile fractions of individual IAC components that occur during larval stages. This suggests that the turnover of multiple IAC components is co-regulated, which makes it unlikely that only integrins recycle while the rest of the complex remains intact (Yuan, 2010).

Previous work has implicated focal adhesion kinase (FAK) and Src family kinases in regulating the dynamics of integrin-mediated adhesion. However, expressing a dominant-negative version of Src in fly muscles induced early muscle defects, whereas disrupting FAK did not affect IAC turnover. An important future goal is to identify the mechanism by which turnover is controlled in order to gain further insight into how IAC dynamics are modulated during development (Yuan, 2010).

It is hypothesized that modulating the levels of integrin turnover in the context of a stable long-term adhesive contact, such as the MTJ, provides a way for tissues to respond to changes in the external environment without wholesale disassembly and assembly of the adhesive contact. The ongoing existence of MTJs in a dynamic state enables expansion, contraction, remodeling and changes in the molecular components of the adhesion complex. This provides a flexibility that is vitally important for long-term tissue maintenance (Yuan, 2010).

A novel function for the Rab5 effector Rabenosyn-5 in planar cell polarity

In addition to apicobasal polarization, some epithelia also display polarity within the plane of the epithelium. To what extent polarized endocytosis plays a role in the establishment and maintenance of planar cell polarity (PCP) is at present unclear. This study investigated the role of Rabenosyn-5 (Rbsn-5), an evolutionarily conserved effector of the small GTPase Rab5, in the development of Drosophila wing epithelium. It was found that Rbsn-5 regulates endocytosis at the apical side of the wing epithelium and, surprisingly, a novel function was discovered of this protein in PCP. At early stages of pupal wing development, the PCP protein Flamingo (Fmi) redistributes between the cortex and Rab5- and Rbsn-5-positive early endosomes. During planar polarization, Rbsn-5 is recruited at the apical cell boundaries and redistributes along the proximodistal axis in an Fmi-dependent manner. At pre-hair formation, Rbsn-5 accumulates at the bottom of emerging hairs. Loss of Rbsn-5 causes intracellular accumulation of Fmi and typical PCP alterations such as defects in cell packing, in the polarized distribution of PCP proteins, and in hair orientation and formation. These results suggest that establishment of planar polarity requires the activity of Rbsn-5 in regulating both the endocytic trafficking of Fmi at the apical cell boundaries and hair morphology (Mottola, 2010).

This study uncovered a novel role of Rbsn-5 in the establishment of PCP during pupal wing development, and it was further demonstrated that the PCP protein Fmi undergoes endocytic trafficking in a process that is dependent on Rbsn-5 and required for the establishment of PCP (Mottola, 2010).

Rbsn-5 shares with its mammalian orthologue Rabenosyn-5 several structural features, as well as the function of molecular coordinator of endocytosis and recycling. First, the inhibition of fluid-phase endocytosis observed in rbsn34 cells is consistent with the impairment of early endocytic transport described for both Rabenosyn-5 and Vps45 in mammalian cells, C. elegans and Drosophila. Second, although Rbsn-5 does not bind Rab4, it interacts with EHD/RME1, a protein that is required for recycling cargo from endosomes to the surface. Third, the formation of expanded Rab5-positive endosomes in Rbsn-5 mutant cells phenocopies the endosomal enlargement observed upon inhibition of recycling. Moreover, the accumulation of Fmi in late endocytic compartments, which is also consistent with the requirement of Rbsn-5 (and the yeast orthologue Vac1p) for protein sorting to the degradative pathway, resembles the phenotype previously described for sec5E13 clones in Drosophila oocytes (Mottola, 2010).

The function of Rbsn-5 in endocytic transport is required for the re-distribution of Fmi between endosomes and the apical cell boundaries during the establishment of PCP in the Drosophila wing. Before PD asymmetry is established in the whole tissue, endogenous Fmi is detected on Rbsn-5- and Rab5-positive early endosomes. At later stages, Fmi must recycle back to the plasma membrane because it subsequently localizes to the apical cell boundaries concomitantly with Rbsn-5. Recycling from endosomes to the cell surface is also consistent with the dependence on Fmi for the recruitment of the exocyst subunit Sec5 at the apical cell boundaries. Loss of Rbsn-5 causes intracellular accumulation of Fmi, which correlates with defects in PD polarity and hair orientation. Consistently, Rab5 overexpression, which influences Rbsn-5 redistribution, also alters Fmi trafficking and causes PCP defects. These data therefore indicate that Rbsn-5-dependent trafficking of Fmi is relevant for the establishment of PCP. Clearly, the data do not exclude the possibility that other (e.g. biosynthetic) trafficking events of Fmi might contribute to this process (Mottola, 2010).

Why is Fmi endocytosed and recycled during establishment of PCP in the pupal wing? It has been recently proposed that a combination of polarized secretion, Fmi endocytosis and stabilization of Fmi and Fz to the distal apical cell boundaries might underlie the establishment of cellular asymmetry. In line with this proposal, Rbsn-5-dependent trafficking might be required to remove unstable Fmi (associating only to Fz) from the apical cell boundaries and relocate it in regions of the plasma membrane where it can be stabilized in proximal PCP complexes. Therefore, the weak proximal non-autonomy in trichome orientation observed for rbsn34 clones, which resembles the phenotype of fmi clones rescued with a GFP-tagged Fmi mutant lacking the cytoplasmic domain (Fmiδintra-EGFP), might be explained with the blockade of Fmi at the plasma membrane preferentially bound to Fz:Dsh complexes (Mottola, 2010).

Some defects observed for rbsn34 clones, such as Fmi redistribution as swirls and proximal perturbation of trichome polarity inside mutant clones, together with weak proximal non-autonomy are also reminiscent of defects in Fat and Ds mutant clones. Interestingly, it was observed that big rbsn34 clones could be found only on the distal side of the pupal wing. This might reflect a less important requirement for Rbsn-5 on this side compared with the proximal one. However, whether Rbsn-5 is also involved in the global propagation of PCP signalling via the upstream module Ds-Fat-Fj remains to be determined (Mottola, 2010).

Additionally, Rbsn-5 mutant clones show defects in hair formation and elongation. As endocytosis and actin cytoskeleton remodelling are functionally connected, these defects might be indirect consequences of endocytosis impairment. However, the specific accumulation of Rbsn-5 at the bottom of emerging hairs would be consistent with the idea that Rbsn-5 mediated endocytic/recycling trafficking might actively contribute to outgrowth of wing hairs, possibly by regulating specific membrane delivery (Mottola, 2010).

While preparing this manuscript, a study on the regulation of membrane protein localization by PI3K (III) and Rabenosyn-5 in Drosophila wing cells reported (Abe, 2009) that loss-of-function mutation of Rbsn-5 does not affect Fmi localization and hair formation and orientation. The discrepancy with the current data could be explained by the fact that, in that study, the analysis was conducted at 25°C instead of 18°C. Indeed, it was noticed that rbsn34 clones are less healthy and tend to be smaller when grown at higher temperatures. Under these conditions, the intracellular accumulation of Fmi might well be less noticeable and the rate of lysosomal degradation may be higher (Mottola, 2010).

In conclusion, the characterization of Rbsn-5 during Drosophila wing development allowed discovery of a novel function for this Rab5 effector in vivo in a developmental context and provided evidence in favor of a role of the apical endocytic trafficking of Fmi in the establishment of PCP. Future studies will hopefully provide additional molecular links and mechanistic insights into the functional interplay between the endocytic and the PCP machineries (Mottola, 2010).

Planar cell polarity controls directional Notch signaling in the Drosophila leg

The generation of functional structures during development requires tight spatial regulation of signaling pathways. Thus, in Drosophila legs, in which Notch pathway activity is required to specify joints, only cells distal to ligand-producing cells are capable of responding. This study shows that the asymmetric distribution of planar cell polarity (PCP) proteins correlates with this spatial restriction of Notch activation. Frizzled and Dishevelled are enriched at distal sides of each cell and hence localize at the interface with ligand-expressing cells in the non-responding cells. Elimination of PCP gene function in cells proximal to ligand-expressing cells is sufficient to alleviate the repression, resulting in ectopic Notch activity and ectopic joint formation. Mutations that compromise a direct interaction between Dishevelled and Notch reduce the efficacy of repression. Likewise, increased Rab5 levels or dominant-negative Deltex can suppress the ectopic joints. Together, these results suggest that PCP coordinates the spatial activity of the Notch pathway by regulating endocytic trafficking of the receptor (Capilla, 2012).

Spatially coordinated regulation of signaling pathways is essential to generate correct anatomical and functional structures, as exemplified by the Drosophila leg, in which activity of the N pathway is required to specify leg joints. In this case, only cells distal to the stripe of Ser expression appear to be capable of responding to the ligand. This study shows that activity of the core PCP pathway is required in those cells proximal to the domain of Ser expression to prevent them from responding to this N ligand. This regulation correlates with the asymmetric distribution of the core PCP proteins, since this study shows that Fz and Dsh are enriched at the distal side of each cell, which in the non-responding cells faces the neighboring Ser-expressing cells. Conversely, in those cells distal to Ser, Fz and Dsh are depleted from the proximal side, leaving N free to interact with its ligand to promote joint formation. It appears that elimination of core PCP gene function in cells proximal to the Ser-expressing cells is sufficient to alleviate the repression resulting in ectopic N activity and ectopic joint formation. Such regulation of the membrane availability of Notch could equally affect Dl-mediated activation, although Ser appears to be the major ligand responsible in the joints. Other factors are likely to influence proximal repression of N because ectopic joints are also observed in alterations of the EGFR pathway and mutants of defective proventriculus (Capilla, 2012).

It is also noted that the domains of N activation (both normal and ectopic) extend beyond the cells at the interface with Ser. This additional level of regulation has not been investigated, but the results indicate that it is unlikely to be due to a secondary signal emanating from the Ser-interfacing cells because the loss of function clones show complete autonomy, without any 'shadow' of activation adjacent to the clone. An alternative possibility is that the cells make more extensive contacts, as has been seen in other tissues (Capilla, 2012).

PCP regulation of N has been observed in other developmental processes, most notably in photoreceptor fate choice in the Drosophila eye. There, much of the regulation is via effects on levels and activity of the ligand. However, no change was detected in the pattern of N or Ser expression in PCP mutants. Instead, the evidence suggests that regulation involves direct interaction between Dsh and N and that this interaction has consequences on the endocytic trafficking of N, resulting in its inactivation. The interaction requires the amino-terminal portion of the Dsh DIX domain, which is also required for Axin binding in the canonical Wnt pathway, making it difficult to dissect its role in the PCP-mediated N inhibition. Nevertheless, one mutation was generated that reduced interactions with N with minor consequences on Axin binding. Rescue experiments with this mutant form of Dsh indicated that it was less effective in PCP function in the leg joints compared with others (e.g., polarity of leg bristles). These results support the model that a direct interaction between Dsh and N is relevant in the context of joint determination. However, the possibility cannot be fully ruled that the mutation has more generalized effects on Dsh, if the joints are particularly sensitive to the levels of Dsh activity (Capilla, 2012).

Several studies indicate that endocytic sorting of N is involved in its regulation, with either positive or negative effects depending on the particular context. The current findings suggest that regulation of N by PCP in the leg is mediated by interaction with Dsh, and probably involves the control of N endocytic trafficking. This suggests a model whereby the interaction between Dsh and N results in increased endocytosis of the N receptor, so reducing its capability to interact with ligands on neighboring cell. Removal of Fz or Dsh compromises this endocytic trafficking, allowing N to be activated. The interaction between Dsh and N is thus only likely to be relevant under circumstances in which there is a strong localization of Dsh co-incident with an interface between N and ligand-expressing cells (Capilla, 2012).

Previous studies have also suggested a role for Dsh in regulating N and on promoting its endocytosis (Axelrod, 1996; Munoz-Descalzo, 2010). In both instances, these effects were linked to Wg signaling, rather than to the core PCP pathway as in this study. Nevertheless several aspects are consistent with the current results, most notably the direct binding between Dsh and N. Additionally it has been argued that Dsh specifically antagonizes Dx-mediated effects of N, which is compatible with their complementary effects on joint formation. However, it is also evident that the ability of Dsh to inhibit N depends on the developmental context. For example, whereas overexpression of Dsh in the leg is sufficient to inhibit N activation at presumptive joints, overexpression of Dsh at the wing margin is not sufficient to repress N signaling: there are no nicks and cut expression is not inhibited. Interestingly, differences in Dx behavior are also evident in these two contexts. At the wing margin, Deltex mutation DxΔpro acts as a dominant-negative form of Dx, whereas DxΔNBS is inactive. By contrast, in the leg joints DxΔpro behaves as wild-type Dx, whereas DxΔNBS is a dominant negative. It is postulated, therefore, that the subcellular localization of Dsh and the availability of Dx are important for determining the regulation of N trafficking at joints (Capilla, 2012).

The autonomous effect of core PCP mutants was clear when the E(spl)mβ1.5-CD2 N reporter and disco-lacZ was used. However, the consequences on bib-lacZ were more complex. Although larger clones of mutant cells always exhibited autonomous ectopic expression, similar to E(spl)mβ1.5-CD2, some narrow clones exhibited no ectopic expression. It is suggested that this might be due to bib-lacZ having a higher threshold of response, so it would need stronger N activation. The domain of bib-lacZ is narrower than that of the other known reporters, in agreement with this model. Furthermore, some cases were found in which there was a reduction of the normal bib-lacZ expression in the mutant cells, in addition to ectopic expression. This suggests that PCP-mediated distal localization of Dsh would be required not only for inhibition of N in proximal cells, but also for efficient activation of N in distal ones (Capilla, 2012).

Drosophila Strip serves as a platform for early endosome organization during axon elongation

Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. This study showed that Drosophila Strip is a key regulator for neuronal morphogenesis. Strip knockdown disturbs the early endosome clustering, and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip-mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that has important roles in neuronal morphogenesis (Sakuma, 2014).

Endocytic pathways downregulate the L1-type cell adhesion molecule Neuroglian to promote dendrite pruning in Drosophila

Pruning of unnecessary axons and/or dendrites is crucial for maturation of the nervous system. However, little is known about cell adhesion molecules (CAMs) that control neuronal pruning. In Drosophila, dendritic arborization neurons, ddaCs, selectively prune their larval dendrites. This study reports that Rab5/ESCRT-mediated endocytic pathways are critical for dendrite pruning. Loss of Rab5 or ESCRT function (see Hrs) leads to robust accumulation of the L1-type CAM Neuroglian (Nrg) on enlarged endosomes in ddaC neurons. Nrg is localized on endosomes in wild-type ddaC neurons and downregulated prior to dendrite pruning. Overexpression of Nrg alone is sufficient to inhibit dendrite pruning, whereas removal of Nrg causes precocious dendrite pruning. Epistasis experiments indicate that Rab5 and ESCRT restrain the inhibitory role of Nrg during dendrite pruning. Thus, this study demonstrates the cell-surface molecule that controls dendrite pruning and defines an important mechanism whereby sensory neurons, via endolysosomal pathway, downregulate the cell-surface molecule to trigger dendrite pruning (Zhang, 2014).

Endocytic pathways profoundly regulate turnover and homeostasis of various cell-surface adhesion proteins and guidance receptors in the developing nervous systems. Perturbation of endocytic pathways often leads to a variety of neurodegenerative diseases, such as frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, lysosomal storage diseases, and Niemann-Pick disease. In Drosophila, the endolysosomal pathway is activated in neighboring glia to engulf degenerating axon/dendrite fragments for their subsequent breakdown during pruning, suggesting a non-cell-autonomous role. This study reports that Rab5 and the ESCRT complexes, two key endocytic regulators, cell autonomously promote dendrite pruning in ddaC neurons. Consistent with these findings, the endocytic pathways also play a cell-autonomous role in axon pruning of MB γ neurons. How do Rab5/ESCRT-dependent endocytic pathways facilitate dendrite pruning in ddaC neurons at the cellular level? This study has identified a cell-surface adhesion protein, namely the L1-type CAM Nrg, as a target of Rab5/ESCRT-dependent endocytic pathways (Zhang, 2014).

Drosophila Nrg and the mammalian L1-type CAMs regulate axonal growth and guidanc, synaptic stability and function, and axon/dendrite morphogenesis. Mutations in the human L1 CAM gene have been reported to cause a broad spectrum of neuronal disorders. This study has identified the Drosophila L1-type CAM Nrg as the key cell-surface molecule that inhibits dendrite pruning in ddaC neurons. The extracellular domains of the L1-type CAMs can regulate cell-cell adhesion via homophilic and/or heterophilic interactions, whereas their intracellular domains can link the proteins with F-actin/spectrins to stabilize the cytoskeletal architecture. In C. elegans, a ligand-receptor complex of cell adhesion molecules containing the nematode Nrg homolog controls dendrite-substrate adhesion to stabilize and pattern dendritic arbors in certain sensory neurons. In Drosophila, Nrg-mediated cell adhesion plays an essential role in stabilizing synapse growth and maintenance at the larval neuromuscular junction. Likewise, Nrg may also mediate adhesion of the dendrites to their adjacent epidermis to stabilize the dendritic architecture in ddaC sensory neurons, whereas downregulation of Nrg may reduce dendritic adhesion/stability and result in disassembly of dendrites. Consistent with the potential adhesive role, structure-function analysis indicates that the ECD of Nrg is important for its function in stabilizing dendrite and/or inhibiting dendrite pruning in ddaC neurons. The fact that overexpression of the ICD-deleted Nrg protein partially rescued the nrg14 mutant phenotype suggests that, in addition to the adhesion function of the ECD, the ICD of Nrg may recruit cytoskeletal components to stabilize dendritic branches in ddaC neurons. The model is therefore favored that the adhesive role of Nrg is a potential mechanism for inhibiting pruning in ddaC sensory neurons (Zhang, 2014).

Another major class of CAMs, integrins, were shown to regulate dendrite-substrate interactions and anchor ddaC dendritic arbors to the extracellular matrix. However, unlike Nrg, integrins do not accumulate on enlarged endosomes in Rab5 or ESCRT ddaC neurons, implying that integrins are not regulated by Rab5/ESCRT-dependent endocytic pathways in ddaC neurons. Moreover, other cell-surface molecules Robo and N-Cad, albeit regulated by the endolysosomal pathway in motor neurons, photoreceptors, or sensory neurons, are dispensable for normal progression of dendrite pruning in ddaC neurons. Thus, this study highlights an important role of the L1-type CAM Nrg in inhibiting dendrite pruning of ddaC sensory neurons (Zhang, 2014).

Interestingly, loss of nrg function causes precocious dendrite pruning without affecting the axonal integrity and connectivity in ddaC neurons, underscoring a specific requirement of Nrg in stabilizing the dendrites, but not the axons. Downregulation of Nrg may reduce dendritic adhesive properties of ddaC sensory neurons and thereby make the dendritic architecture more susceptible to pruning. It is conceivable that Nrg-independent mechanisms may be utilized to protect the axonal structure from the pruning machinery in ddaC neurons. Moreover, both nrg loss of function and gain of function did not affect axon pruning in MB γ neurons (data not shown), further supporting the conclusion that Nrg plays a specific role in dendrite pruning in ddaC sensory neurons. Future studies may elucidate whether and how Nrg mediates its dendritic adhesive properties to inhibit dendrite pruning (Zhang, 2014).

In summary, this study shows that Rab5/ESCRT-dependent endocytic pathways facilitate dendrite pruning of ddaC neurons by downregulating the Drosophila L1-type CAM Nrg during early metamorphosis. This study also demonstrated the role of the cell-surface adhesion protein Nrg in inhibiting dendrite pruning in ddaC sensory neurons. Thus, this study opens the door for further studies of the functions of cell-surface molecules in the regulation of dendritic adhesion during neuronal remodeling (Zhang, 2014).

Rabaptin-5 and Rabex-5 are neoplastic tumour suppressor genes that interact to modulate Rab5 dynamics in Drosophila melanogaster

Endocytosis plays an important role in the regulation of tumour growth and metastasis. In Drosophila, a number of endocytic neoplastic tumour suppressor genes have been identified that when mutated cause epithelial disruption and over-proliferation. This study characterises the Drosophila homologue of the Rab5 effector Rabaptin-5, and shows that it is a novel neoplastic tumour suppressor. Its ability to bind Rab5 and modulate early endosomal dynamics is conserved in Drosophila, as is its interaction with the Rab5 GEF Rabex5, for which neoplastic tumour suppressor characteristics were also demonstrated. Surprisingly, disruption of apico-basal polarity is not disrupted in Rabaptin-5 and Rabex-5 mutant tissues; instead the tumour phenotype is associated with upregulation of Jun N-terminal Kinase (JNK) and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signalling (Thomas, 2013).

Endocytosis is increasingly recognised as an important mechanism for the growth and metastasis of tumours, both in the clinical setting and in cancer models. The categorisation of endocytic neoplastic tumour suppressor genes in Drosophila began less than ten years ago with the identification of Rab5, avl, Vps25 and Tsg101 and has since expanded with the discovery of at least 14 others, in particular multiple components of the ESCRT complex. Most mutations so far identified are zygotic lethal long before the third instar larval stage and so have been discovered through screens in which homozygous mutant clones were generated that spanned entire epithelial compartments in otherwise heterozygous animals. However, not all chromosome arms have been screened and screens have not been saturating, hence it is likely that many others remain to be found (Thomas, 2013).

This study identified two novel endocytic neoplastic tumour suppressor genes. Rbpn-5 has not been previously characterised in Drosophila, and this work is the first to show that not only does it act as a neoplastic tumour suppressor gene in Drosophila, but that its endocytic functions, initially investigated in mammalian cell culture and in vitro, are conserved in a multi-cellular organism during development. Unlike many of the Drosophila endocytic neoplastic tumour suppressor genes so far identified, Rbpn-5 has a human homologue that has been directly implicated in tumour growth and metastasis. Mammalian Rabaptin-5 controls integrin recycling during migration of invasive tumour cells, has been identified in patients suffering from myelomonocytic leukaemia, is involved in preventing hypoxia in primary kidney and breast tumours and has been shown to interact physically with the Tuberous sclerosis protein Tuberin. Although not all of these functions may be conserved in Drosophila, this work provides a basis for further investigating the mechanisms of Rbpn-5 dependent tumourigenesis in a whole animal system (Thomas, 2013).

Rabex-5 has been previously identified as a tumour suppressor gene in Drosophila (Yan, 2010), but its neoplastic characteristics were not described. Importantly, both Rbpn-5 and Rabex-5 mutants are homozygous viable until late larval stages, and thus may provide a more facile model than other early endocytic tumour suppressor genes for uncovering the mechanistic basis of neoplasia (Thomas, 2013).

This study initially set out to identify new regulators of planar polarity through an RNAi screen. As core planar polarity proteins are known to undergo internalisation and recycling, and Rbpn-5 and Rabex-5 are general endocytic regulators that are functional in epithelial tissues at the time of planar polarisation, it is highly likely that they are playing a role in trafficking of core proteins. Indeed, another Rab5 effector, Rbsn-5, which is also a neoplastic tumour suppressor protein, regulates Fmi localisation. Unlike Rbsn-5, cell-autonomous depletion of Rbpn-5 or Rabex-5 does not significantly alter planar polarity protein levels or localisation, suggesting that some level of endocytosis can occur in the absence of these proteins. One possibility is that Rbpn-5 and Rabex-5 proteins might be highly stable compared with Rbsn-5. Evidence in support of this comes from zygotic mutants, which survive to early pupal stages for Rbpn-5 and Rabex-5, presumably due to perdurance of maternal protein, but die before the second larval instar in the Rbsn-5 background. If this is the case, residual protein activity in clones or RNAi-treated tissue may allow a low level of general endocytosis to occur, which would be sufficient to correctly localise core planar polarity proteins. Another, non-mutually exclusive hypothesis is that a level of endocytosis can occur in the absence of either Rabex-5 or Rbpn-5. There are three other Rab5 GEF homologues in Drosophila, and these may be able to step in if Rabex-5 is absent (Thomas, 2013).

Whatever the reason may be, the lack of defects seen in clones or RNAi, combined with the severe pleiotropic effects on epithelial architecture observed in zygotic mutants, means that analysis of a specific role in planar polarity is extremely complex, and it was decided to focus attention on their tumour suppressor roles (Thomas, 2013).

In mammalian cells, it has been shown that Rabaptin-5 is an effector of Rab5 that promotes early endosome fusion through its interaction with Rabex-5. This study provides the first characterisation of a Rabaptin-5 homologue in a multicellular organism, and show that many of its functions are conserved. Rab5 effectors are defined by several criteria including GTP-dependent binding to Rab5 and the ability to modulate a Rab5-dependent process in response to Rab5 activity. This study shows that Drosophila Rbpn-5 fulfills the criteria of a Rab5 effector protein in the same way as its mammalian counterpart. Firstly this study has demonstrated that Rbpn-5 physically interacts specifically with GTP-bound Rab5, probably through its predicted C-terminal Rab5 binding domain. Secondly, that Rbpn-5 and Rab5 colocalise in Drosophila pupal wings. Thirdly, when Rbpn-5 was depleted using RNAi or in mutant clones, a strong reduction was seen in apical Rab5-positive vesicles indicating that Rbpn-5 is required for Rab5 recruitment. Lastly, if Rbpn-5 is over-expressed, subtle alterations is seen in early endosome structure, which are indicative of a role for Rbpn-5 in promoting early endosome fusion (Thomas, 2013).

Although the Rab5 effector function of Rabaptin-5 is conserved in Drosophila, its Rab4 effector function may not be. Mammalian Rabaptin-5 has been shown to bind Rab4 through a separate N-terminal domain, and the divalent Rab5-Rab4 binding ability allows Rbpn-5 to act as a bridge between the endocytic and fast recycling pathways. It was not possible to recapitulate Rab4 binding, and indeed the N-terminal Rab4 binding site is not highly conserved in Drosophila Rbpn-5. A similar result was found for the Drosophila homologue of another Rab5-Rab4 divalent effector, Rabenosyn-5 , suggesting that the mechanism of transfer of cargo from the endocytic to recycling routes might not be conserved across phyla (Thomas, 2013).

A large number of proteins have been shown to bind Rabaptin-5, but one of the best characterised is Rabex-5. The endogenous GEF activity of Rabex-5 is fairly low, and unless large quantities are overexpressed, the formation of a Rabaptin-5/Rabex5 complex is necessary to promote nucleotide exchange on Rab5. The effect of this is to enhance Rab5 activity, thus facilitating its recruitment of factors involved in tethering and fusion of early endocytic membranes (Thomas, 2013).

This study has provided evidence that the interaction between Rabex-5 and Rbpn-5, and their ability to alter early endosome dynamics is conserved in Drosophila. As with their mammalian counterparts, the two proteins bind in vitro: it is suspected that Rabex-5 binds to a central domain in Rbpn-5, as this is homologous to the Rabex-5 binding site in mammalian Rabaptin-5, and neither N- nor C-termini of Rbpn-5 can bind alone. As in mammalian cells, over-expression of Rabex-5 in Drosophila wings is sufficient to promote early endosome fusion even if Rbpn-5 levels are depleted by RNAi. However, co-expression of Rbpn-5 significantly enhances this, demonstrating that the complex acts synergistically. The dependency on Rabex-5 for Rabaptin-5 localisation to early endosomes is also conserved in Drosophila as this study showed that depletion of Rabex-5 levels using RNAi causes a reduction in Rbpn-5 puncta. This is surprising as not only does Rbpn-5 contain a Rab5 binding domain, but also a FYVE domain (unlike its mammalian homologue), which would be expected to promote its recruitment to early endosomes. It is possible that the FYVE domain is non-functional. Alternatively, its ability to bind PI3P may be somehow inhibited in the absence of Rabex-5, or the protein may be destabilised. Further work would be required to distinguish between these possibilities (Thomas, 2013).

This study found that over-expressing a mutant form of Rabex-5 lacking GEF activity prevents both Rbpn-5 and Rab5 from accumulating in apical puncta. It is surmiseed that the construct is acting in a dominant-negative manner to suppress Rbpn-5 recruitment and/or Rab5 activation via endogenous Rabex-5. In mammalian cells, localisation of all three proteins appears to be highly interdependent, suggesting that it is likely that Rabex-5 recruitment to early endosomes is also compromised in the absence of Rbpn-5 or Rab5. However, in the absence of reagents for visualising endogenous Rabex-5, this remains speculative (Thomas, 2013).

The mechanistic basis of neoplastic tumour formation is complex and much remains unclear. Drosophila models, in particular the classic baso-lateral polarity complex mutants scrib, dlg and lgl, which were discovered many years ago and which, unlike most of the endocytic mutants, are zygotically viable until late larval stages, have been extremely useful in describing the process of tumorigenesis. There are several different pathways and mechanisms that have been investigated. Given that Scrib, Dlg and Lgl regulate apico-basal polarity by inhibiting the apical Crb and Par-3/Bazooka (Baz) complexes, an expansion of the apical domain was proposed as one possible mechanism for tumourigenesis. This hypothesis was reinforced by the finding that over-expressing either Crb or atypical Protein kinase C (aPKC) is sufficient to promote tumourous discs, and that in the endocytic mutants, Crb accumulates massively and apico-basal polarity is disrupted. Crb accumulation is thought to promote the growth aspect of the neoplastic tumour phenotype through misregulation of the Hpo/Wts pathway. However, there is some evidence that Crb is not always strongly misregulated in neoplastic tumours, and it has been speculated that in scrib, dlg and lgl mutants it may be the concurrent disruption of endocytosis, rather than the polarity defect, which is the primary cause of their neoplastic phenotypes (Thomas, 2013).

The results show that neither Rabex-5 nor Rbpn-5 exhibit disruption in apico-basal polarity and there is no accumulation of Crb. It is suggested that these mutants are likely to represent a milder phenotype than the other endocytic mutants studied so far, possibly due to enhanced perdurance of maternal protein which allows the survival of zygotic mutants to early pupal stages. In any case, the lack of an apico-basal polarity defect in Rbpn-5 and Rabex-5 mutants indicates that loss of apico-basal polarity is not strictly required for neoplasia (Thomas, 2013).

Other pathways that have been found to be activated in Drosophila neoplastic tumours include N, JAK/STAT and JNK. Due to the differential ability of N to signal in different endocytic compartments, the pathway is upregulated in ESCRT mutant tumours, but not in early endocytic mutants, despite their accumulation of N protein on the cell surface. As with Crb, accumulation of N was not seen in Rbpn-5 or Rabex-5 mutant discs, suggesting that a certain level of endocytosis is still occurring, and unsurprisingly it was also found that the N pathway is not activated (Thomas, 2013).

In ESCRT mutant clones, activation of the N pathway leads to transcription of the JAK/STAT ligand Upd, which acts non-autonomously on wild-type tissue to induce JAK/STAT signalling and promote neoplasia. The massive upregulation of JAK/STAT signalling that was observed in this study must be activated via an alternate mechanism, as it is both cell autonomous and N-independent. Although autonomous JAK/STAT signalling in neoplastic discs has been documented and shown to contribute to excessive cell size and proliferation, it is unclear how it is activated in endocytic mutants. One explanation that was not possible to test, is that the endocytic block could directly disrupt the trafficking of the JAK/STAT receptor Domeless (Dome), as Dome localisation has been shown to be altered in ESCRT mutant neoplastic tumourous discs, and signalling ability is known to be influenced by the intracellular compartment in which the ligand-receptor complex finds itself. This study showed that a hypomorphic stat92E mutation is able to rescue many of the neoplastic defects, including over-proliferation, inability to differentiate and overall disc shape and structure, suggesting that ectopic activation of JAK/STAT signalling in endocytic mutants is indeed causative for these aspects of tumourigenesis (Thomas, 2013).

The upregulation of the JNK pathway that was observed in the Rbpn-5 and Rabex-5 mutants is likely to also contribute to the over-proliferation phenotype. The JNK pathway is unusual in having both pro-proliferation and pro-apoptotic roles, and was initially thought to be activated only where wild-type tissue abuts clones mutant for neoplastic tumour suppressor genes, promoting apoptosis within mutant cells and their elimination from the tissue. However, more recent work has shown that JNK signalling is also activated in tissues wholly mutant for endocytic neoplastic tumour suppressor genes where it promotes cell proliferation (Thomas, 2013).

It is thought that JNK pathway upregulation in neoplastic tumours is likely to act through the Eiger/TNF ligand receptor complex. Eiger and activated JNK have been shown to accumulate in early endosomes in scrib mutant clones to promote apoptosis, and there are several other pieces of evidence that implicate the endocytic pathway in Eiger regulation. Interestingly, the switch from JNK-mediated apoptosis to proliferation can be mediated by co-expression of the Ras oncogene. For example, expressing Ras in scrib mutant clones produces a highly invasive neoplastic phenotype such that clones that would usually be eliminated instead invade neighbouring wild-type tissue. Given that Ras is ubiquitinated by Rabex-5, and the Ras pathway has been shown to be upregulated in Rabex-5 mutant larvae, it is intriguing to speculate whether this may be contributing in some way to the neoplastic phenotype seen in mutant larval discs, not only in the Rabex-5 background but perhaps more generally in other endocytic neoplastic tumour suppressor mutants. Indeed, as Rabex-5-mediated ubiquitination targets cargo to early endosomes for degradation, it seems highly likely that Ras signalling is not only regulated by ubiquitination but also by endosomal dynamics. Further work will be needed to investigate a potential role for Ras in endocytic neoplastic tumours, to determine the mechanisms of JNK and JAK/STAT activation and to elucidate how differential activity of these pathways is controlled in clones versus whole mutant tissues (Thomas, 2013).

Toward a comprehensive map of the effectors of Rab GTPases

The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, a comprehensive set of Drosophila Rabs was used for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. Over 25 interactions were confirmed for Rab2, Rab4, Rab5, Rab6, Rab7, Rab9, Rab18, Rab19, Rab30, and Rab39. These include tethering complexes, coiled-coiled proteins, motor linkers, Rab regulators, and several proteins linked to human disease (Gillingham, 2014).

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. This study shows that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, it was demonstrated that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, these data demonstrate a fundamental role for a plasma membrane-endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration (Assaker, 2010).

During migration, the cell needs to rearrange its cytoskeleton, its plasma membrane content, and its interaction with other cells. Many of these features can be controlled by vesicular trafficking. For example, Integrins, Cadherins, and other cell-cell or cell-matrix attachment proteins are transmembrane proteins tightly regulated by trafficking. Furthermore, the distribution of proteins and lipids at the plasma membrane is directly controlled by vesicular trafficking, as well as the localization of some actin remodeling proteins. During the process of border cell migration, endocytosis has been shown to regulate the polarity of RTK activity. This paper shows that the endocytic process plays a role in regulating the spatial localization of RTK activity by trafficking through the recycling endosome and by the polarized redelivery of endocytosed material to the plasma membrane (Assaker, 2010).

The key endocytic proteins previously involved in border cell migration - Sprint, Cbl, and Shibire - regulate the polarization of RTK activity during border cell migration. Different possible mechanisms are proposed to explain their action and have not been addressed in this landmark article (Jékely, 2005). Recently, it was shown that both the degradative pathway and the recycling pathway might be involved in this process. Thus, at least two models, which are not mutually exclusive, could explain the role of endocytosis in establishing this polarity. First, active RTKs could be endocytosed and degraded when diffusing away from the leading edge. Second, polarized recycling of endocytosed active RTKs could concentrate these active receptors at the leading edge. From the current experiments it can be concluded that the recycling of active RTKs or of a cofactor at the plasma membrane is necessary for border cell migration. Furthermore, it was demonstrated that the slow recycling route, through the recycling endosome, is used and that polarized redelivery at the plasma membrane is mediated by the exocyst subunit sec15 (Assaker, 2010).

It seems logical to think that RTKs, or active RTKs, are the cargo transported through this endocytic cycle. However, the identity of the protein being recycled remains to be determined. Indeed, there is no indication that RTKs are recycled. Immunofluorescence staining of the EGFR was performed. The signal obtained was diffuse and inconclusive in both control and rab11SN-expressing border cells. In addition, if active RTKs were trafficking through the recycling endosome, the pTyr in endocytic vesicles would be expected to be marked by Rab11. However, such a colocalization has never been observed. These data do not rule out a potential recycling of RTKs, because they could be present in the recycling endosome in quantities below detection levels or in an inactive form. However, the data suggest that the main cargo of this trafficking cycle is of another nature. This cargo could be a plasma membrane diffusion barrier, because polarized cells, such as epithelial cells and neurons, maintain different membrane domains, which rely on such barriers: the tight junctions and the axon hillock, respectively. Moreover, diffusion barriers have been proposed to define plasma membrane domains in migrating cells. These diffusion barriers appear to be linked to the actin cytoskeleton, but their exact nature is unknown. Because E-cadherin is involved in cell migration, it would have been an ideal candidate to play such a role, but it is unaffected by rab11SN expression (Assaker, 2010).

Another possibility is that endocytosis acts indirectly. For example, it might regulate key components of the plasma membrane or of the cytoskeleton. Recent evidence has shown that endocytosis and recycling can play a critical role in creating a positive feedback loop during polarity establishment in the budding yeast. In this particular case, endocytosis is critical for the localization of regulators of small GTPases of the Rho family. Furthermore, mammalian Tiam1, a GDP-to-GTP exchange factor (GEF) for Rac, has been shown to localize to endosomes, leading to the loading of active Rac at the plasma membrane through an endocytic-recycling cycle. Interestingly, a robust genetic interaction has been found between Rab11 and Rac1, which directs border cell migration (Wang, 2010). Until now, two Rac-GEFs, Myoblast city and Elmo, have been involved in border cell migration, but not the Drosophila Tiam1 homolog still life. Further studies will be necessary to determine if Rac1 is the main cargo of the endocytic-recycling cycle that regulates border cell migration (Assaker, 2010).

In the past few years, trafficking via the recycling endosome has been involved in the establishment or rearrangement of cell polarity in various events. In particular, a role for the recycling endosome has been observed when a rapid and dramatic rearrangement of the cell organization is required, including cellularization, cell-cell boundary rearrangement, asymmetric cell division, and cell migration. Trafficking through the recycling endosome is an ideal mechanism to polarize a cell rapidly, because it hijacks material already available in the cell at a new location. Furthermore, it is a very efficient mechanism to reinforce polarity by feedback loops. Similarly the exocyst plays a key role in the majority of these cell polarizations. In the case of cell migration, the recycling endosome may transform the diffuse extracellular gradient of RTK ligand into a robust intracellular polarization of RTK activity that is crucial for directed migration (Assaker, 2010).

There is much evidence that the function of the recycling endosome in the regulation of directed migration is conserved in mammals. The mammalian homologs of Drosophila Rab11 are Rab11A and -B and Rab25. They have been directly implicated in the migration of cancerous cells and in the formation of metastasis, a cell migration event resembling border cell migration. Rab11 effectors are also involved in mammalian cell migration. More specifically, PDGF receptor-dependent cell migration has been shown to be regulated by endocytosis in a mammalian cell culture assay and the recycling endosome has been indirectly implicated in the regulation of migration guided by the EGFR. Given the involvement of the recycling endosome in so many processes, targeting its function to reduce metastasis is unlikely to be efficient. However, identifying the main cargo of this recycling cycle could help identify more specific targets for drugs blocking the formation of metastasis (Assaker, 2010).

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 (reviewed in Bonifacino and Glick, 2004). 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 (Martin-Belmonte, 2007). In zebrafish embryos and cultured human endothelial cells, capillary vessels form through the coalescence and growth of intracellular pinocytic vesicles (Kamei, 2006). 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 (Zhu, 2005). At later stages, zygotic expression of sar1 mRNA is initiated in multiple epithelial tissues (Abrams, 2005). To monitor Sar1 zygotic expression in the trachea, a Sar1-GFP protein trap line (Wilhelm, 2005) 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 (Zhu, 2005) 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 (Olver, 2004). 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 (Abrams, 2005). 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 (Bucci, 1992; Schnatwinkel, 2004; Stenmark, 1994). 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 (Abrams, 2006; Perens, 2005). 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).

The endocytic control of JAK/STAT signalling in Drosophila

Domeless (Dome) is an IL-6-related cytokine receptor that activates a conserved JAK/STAT signalling pathway during Drosophila development. Despite good knowledge of the signal transduction pathway in several models, the role of receptor endocytosis in JAK/STAT activation remains poorly understood. Using both in vivo genetic analysis and cell culture assays, it was shown that ligand binding of Unpaired 1 (Upd1) induces clathrin-dependent endocytosis of receptor-ligand complexes and their subsequent trafficking through the endosomal compartment towards the lysosome. Surprisingly, blocking trafficking in distinct endosomal compartments using mutants affecting either Clathrin heavy chain, rab5, Hrs or deep orange led to an inhibition of the JAK/STAT pathway, whereas this pathway was unchanged when rab11 was affected. This suggests that internalization and trafficking are both required for JAK/STAT activity. The requirement for clathrin-dependent endocytosis to activate JAK/STAT signalling suggests a model in which the signalling 'on' state relies not only on ligand binding to the receptor at the cell surface, but also on the recruitment of the complex into endocytic vesicles on their way to lysozomes. Selective activation of the pool of receptors marked for degradation thus provides a way to tightly control JAK/STAT activity (Devergne, 2007).

Using genetic analysis this study shows that several regulators of the endocytic pathway are required for normal JAK/STAT signalling in vivo. The membrane-bound Dome receptors undergo ligand-dependent internalization in clathrin-coated vesicles, which are then targeted to the sorting endosome via Rab5. The function of Hrs is required for JAK/STAT activation and to direct most of the active receptors to the MVBs, targeting them to the lysosome for degradation (Devergne, 2007).

One important question is to know whether the trafficking of ligand-bound receptors has any effect on signalling. This question was addressed by looking at Stat nuclear localization, which represents a robust readout to assess JAK/STAT activity, and at pnt-lacZ expression (Devergne, 2007).

The effect of Hrs is opposite on the JAK/STAT pathway compared with its effect on other pathways. Indeed, in the egg chamber, Hrs plays a positive role on JAK/STAT activity, whereas it has been shown to downregulate the EGFR, Notch and TGF-β pathways in the same tissue. Interestingly, HRS has been shown to interact with STAM in the same mono-ubiquitylated recognition complex (Lohi, 2001). STAM is a known JAK/STAT activator (Pandey, 2000), suggesting that HRS could control STAT signalling through its interaction with STAM. So, Hrs could play two crucial roles: first, allowing the sequestration and the sorting of the receptor to the lysosome and, second, activating the ligand-receptor complex in collaboration with STAM (Devergne, 2007).

The data challenge the simple view whereby binding of the ligand to the receptor at the membrane would be sufficient to activate the pathway. Indeed, it was found that equally essential is the need of clathrin for the activation of JAK/STAT signalling. Thus, activation can occur only when the ligand-receptor complex is assembled into clathrin-coated vesicles. In this view, activation would proceed in two steps, requiring both binding of the ligand and interaction with clathrin. The role of clathrin could be to concentrate/cluster receptors and/or bring them together with other signal transducers in the endosomal compartment. This finding is in agreement with a recent work showing that, in mammals, clathrin is required to transduce JAK/STAT signals through the IFNα-receptor, but not the IFNγ-receptor, suggesting a conserved function for clathrin in JAK/STAT signalling (Marchetti, 2006). Interestingly, like in mammals, JAK/STAT signalling in Drosophila might be controlled in a cell-type-specific manner by Chc-dependent endocytosis. Indeed, in Drosophila eyes, Vps25 and TSG101 mutations lead to Upd upregulation and JAK/STAT activation in a Notch-dependent manner (Devergne, 2007).

What is the significance of clathrin function and, more generally, of the requirement for internalization, in JAK/STAT signalling? It has been shown for several signalling pathways that internalization brings together membrane receptors and intracellular pathway components in the endosomal compartment, which thus serves as a platform for signalling. The fact that Dome internalization and activation are coupled to degradation has important consequences. Making signalling complexes only active in the endosomal compartment is a powerful mechanism to control the number of active complexes in the cell. Their targeting to the lysosome allows the control of their lifetime as active receptors, providing a temporal -- hence quantitative -- control on signalling (Devergne, 2007).

Activation of JAK/STAT follows an off/on/off model in which two conditions are required for correct JAK/STAT activation: (1) formation of a ligand-receptor complex (as proposed in the classical model), followed by (2) the internalization of the complex via Chc-containing budding vesicles. The sole formation of the ligand-receptor interaction would lead to an inactive complex (off). However, interaction with Chc and subsequent internalization activate the complex (on), thus ensuring that only the complexes targeted for degradation are activated. Arrival of the complex in the MVB/lysosome turns it into the off state (Devergne, 2007).

Internalization is required for proper Wingless signaling in Drosophila melanogaster

The Wingless pathway regulates development through precisely controlled signaling. This study shows that intracellular trafficking in the Wg target cell regulates Wg signaling levels. In Drosophila cells stimulated with Wg media, dynamin or Rab5 knockdown causes reduced reporter (Super8XTOPflash) activity, suggesting that internalization and endosomal transport facilitate Wg signaling. In the wing, impaired dynamin function reduces Wg transcription. However, when Wg production is unaffected, extracellular Wg levels are increased. Despite this, target gene expression is reduced, indicating that internalization is also required for efficient Wg signaling in vivo. When endosomal transport is impaired, Wg signaling is similarly reduced. Conversely, the expression of Wg targets is enhanced by increased transport to endosomes or decreased hepatocyte growth factor-regulated tyrosine kinase substrate- mediated transport from endosomes. This increased signaling correlates with greater colocalized Wg, Arrow, and Dishevelled on endosomes. Since these data indicate that endosomal transport promotes Wg signaling, these findings suggest that the regulation of endocytosis is a novel mechanism through which Wg signaling levels are determined (Seto, 2006).

This analysis has revealed the surprising finding that intracellular transport affects the efficiency of Wg signaling. In cell culture, knockdown of dynamin, a protein essential for clathrin-mediated internalization, reduces the TOPFlash/RL ratio (RL is Renilla luciferase), which is suggestive of decreased Wg signaling. Similarly, Rab5 knockdown causes reduced TOPFlash/RL ratios under most conditions, suggesting that internalization and endosomal transport are important for Wg signaling. Interestingly, transfection with polIII-RL, a control vector used in a recent screen for modifiers of Wg signaling, produces conflicting results for Rab5 compared with other RL controls, indicating that cell culture-based Wg signaling assays are very sensitive to experimental conditions. Thus, although the cell culture results indicate an endocytic regulation of Wg signaling, in vivo validation is critically important (Seto, 2006).

In the wing, further evidence was found that Wg signaling levels are highly dependent on intracellular transport. When endocytosis is altered, ligand levels and signaling levels are uncoupled such that high Wg levels do not necessarily enhance signaling. Therefore, limited usage has been made of the term morphogen gradient, which could refer to either ligand or signaling levels. Instead describe Wg distribution and signaling readouts are described. When internalization is inhibited in a domain that does not affect Wg production, high levels of Wg(ex) were found, likely as a result of reduced degradation. However, Wg target gene expression is diminished, indicating that impaired internalization decreases Wg signaling in vivo as well as in cell culture. When early endosomal transport is impaired, Senseless (Sens) and Distal-less (Dll) expression are also reduced despite abundant Wg levels. In both cases, markers of high signaling levels are especially affected, indicating that intracellular signaling is important to achieve robust Wg signaling levels. The differential decrease also argues that changes in Sens and Dll expression are not merely the result of cell death or global changes in transcription. Further supporting this, normal expression of other genes was found in the wing pouch. Additionally, when endosomal transport is enhanced or when transport from the endosome is impaired, Wg signaling is increased. These data suggest that protein localization to the endosome facilitates Wg signaling. Conversely, increased transport to MVBs decreases the expression of Wg readouts. This causes an adult wing phenotype that can be suppressed by Wg signaling components. Thus, it is proposed that in addition to low levels of cell surface signaling, intracellular Wg signaling is critical for proper signaling levels (Seto, 2006).

Because endocytosis is tightly regulated, intracellular Wg signaling may allow for the rapid modulation of signaling levels. For example, endosomal transport can be regulated merely by changing the GDP/GTP state of Rab5. This work indicates that impaired endosomal transport by GDP-bound Rab5 reduces Wg signaling, whereas enhanced endosomal fusion by GTP-bound Rab5 increases signaling. Because the GDP/GTP-binding state of Rab5 is controlled posttranslationally by GTPase-activating proteins and guanine nucleotide exchange factors, endocytic regulation likely allows more of a rapid adjustment of signaling than regulatory mechanisms requiring transcription and translation. Furthermore, because endocytic rates vary between cell types, this regulation may allow signaling to be adjusted in particular parts of the body or cells of a tissue. Thus, regulated endocytosis allows for precise temporal and spatial control of Wg signaling (Seto, 2006).

Endocytosis is hypothesized to regulate signaling through several mechanisms. For example, lysosomal degradation of internalized active receptor tyrosine kinases serves to attenuate signaling. However, the data suggest that Wg signaling is enhanced by endocytosis. One theory by which intracellular transport facilitates signaling is that the internalization of ligand-receptor complexes promotes interactions with other signaling members recruited to or already present on endosomes. In MAPK signaling, ERK1 receptors form protein complexes with endosomal MP1 and p14, leading to greater activation of signaling. Similarly, TGFβ signaling may be enhanced by receptor internalization to endosomes where the Smad2 anchor protein SARA is enriched. Although this work and that of others suggests that Wg undergoes receptor-mediated internalization in the wing, these data alone cannot explain the enhanced Wg signaling observed. However, not only are Wg and Arrow colocalized in large endosomal accumulations in hrs mutants, but they also colocalize with the cytoplasmic signaling component Dsh. The colocalization of Wg, Arr, and Dsh correlates with the increased expression of Wg readouts. These data suggest that internalization and endosomal transport may promote Wg signaling by facilitating associations between the Wg-receptor complex and downstream signaling components like Dsh. Interestingly, Dsh is reportedly present on intracellular vesicles, and mutations that impair vesicular localization do disrupt canonical Wg signaling (Seto, 2006).

Axin, a protein that inhibits Wg signaling by down-regulating Arm levels, has also been shown to colocalize with Dsh on intracellular vesicles. Upon Wg signaling, Axin relocalizes from intracellular puncta to the plasma membrane. This correlates with Arm stabilization and increased Wg signaling. Because Axin associates with Dsh and the cytoplasmic tail of Arr, it is proposed that internalized Wg forms an endosomal signaling complex that may relocalize Axin, thereby stabilizing Arm and facilitating signaling (Seto, 2006).

A model of intracellular Wg signaling is presented. Based on the data obtained from altering endocytosis, Wg at the cell surface produces only low levels of Wg signaling in the wing. Wg associates with its receptors and is internalized. When endocytic vesicles fuse with the early endosome, the cytoplasmic domains of the Wg receptors Frizzled and Arr are able to associate with downstream signaling components like Dsh, thereby facilitating Wg signaling. Subsequent endosomal sorting into MVB inner vesicles sequesters the Wg-receptor complex from other signaling components, and the activation of signaling transduction is halted (Seto, 2006).

The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly

Cell fate is often determined by the intracellular localization of RNAs and proteins. In Drosophila oocytes, oskar (osk) RNA localization and the subsequent Osk synthesis at the posterior pole direct the assembly of the pole plasm, where factors for the germline and abdomen formation accumulate. osk RNA produces two isoforms, long and short Osk, which have distinct functions in pole plasm assembly. Short Osk recruits downstream components of the pole plasm, whose anchoring to the posterior cortex requires long Osk. The anchoring of pole plasm components also requires actin cytoskeleton, and Osk promotes long F-actin projections in the oocyte posterior cytoplasm. However, the mechanism by which Osk mediates F-actin reorganization remains elusive. Furthermore, although long Osk is known to associate with endosomes under immuno-electron microscopy, it was not known whether this association is functionally significant. This study shows that Rabenosyn-5 (Rbsn-5), a Rab5 effector protein required for the early endocytic pathway, is crucial for pole plasm assembly. rbsn-5- oocytes fail to maintain microtubule polarity, which secondarily disrupts osk RNA localization. Nevertheless, anteriorly misexpressed Osk, particularly long Osk, recruits endosomal proteins, including Rbsn-5, and stimulates endocytosis. In oocytes lacking rbsn-5, the ectopic Osk induces aberrant F-actin aggregates, which diffuse into the cytoplasm along with pole plasm components. It is proposed that Osk stimulates endosomal cycling, which in turn promotes F-actin reorganization to anchor the pole plasm components to the oocyte cortex (Tanaka, 2008).

The polarized targeting and anchoring of specific molecules and organelles to particular subcellular regions are crucial for many cellular processes, including cell-polarity establishment and cell-fate determination. In many animals, germline fate is controlled by maternal factors localized to a specialized cytoplasmic region within the egg, called the germ plasm. Germ plasm contains germ granules, which are electron-dense, and non-membranous structures consisting of maternal RNAs and proteins required for the formation of germ cells. Drosophila germ plasm, also called pole plasm, forms at the posterior pole of the embryo and is inherited by the germline precursors, or pole cells. Because the cytoplasmic transplantation of the pole plasm into recipient embryos causes the ectopic formation of pole cells, the pole plasm contains sufficient factors for germ-cell formation. This observation also highlights the importance of retaining the pole plasm at the posterior cortex of the embryo to ensure the germ cells form at the appropriate location (Tanaka, 2008).

In Drosophila, the pole plasm is assembled during oogenesis, which is divided into 14 morphologically distinct stages of egg chamber development. The egg chamber is composed of a single oocyte and 15 nurse cells, surrounded by a monolayer of somatic follicle cells. During oogenesis, most components of pole plasm are synthesized in the nurse cells and transported into the oocyte via ring canals, which are cytoplasmic bridges interconnecting the oocyte with nurse cells. Within the oocyte, these factors become concentrated at the posterior pole and are assembled into the polar (germ) granules. These factors are transported by a polarized microtubule (MT) array that is initially nucleated at the oocyte posterior and extends into the nurse cells through the ring canals. During stages 6-7, the MT array is reorganized by the transforming growth factor alpha-like Gurken (Grk) signal. In the stage-6 oocyte, posteriorly restricted Grk induces neighboring follicle cells to adopt the posterior fate. These cells send back as-yet unknown signals to the oocyte to trigger the reorganization of the MT cytoskeleton. Consequently, the MT array within the oocyte becomes polarized along the anteroposterior (AP) axis, with the minus ends abundant at the anterior of the oocyte and the plus ends extending toward the posterior. This MT organization promotes the migration of the oocyte nucleus and associated grk RNA to the future anterior-dorsal corner, where Grk signals the follicle cells to define the dorsoventral axis. The polarized MT array also directs the localization of bicoid (bcd) RNA to the anterior and oskar (osk) RNA to the posterior within the oocyte. The anterior accumulation of bcd RNA is required for the proper development of the embryonic head and thoracic structures. The posterior localization of osk RNA is essential for the formation of the germ cells and abdomen (Tanaka, 2008).

osk RNA localization is tightly coupled to translational control: only the posteriorly localized osk message is translated. The localized Osk protein, in turn, recruits downstream components of the pole plasm, such as Vasa (Vas) and Tudor (Tud) proteins, and the nanos, germ cell-less and polar granule component RNAs. Misexpression of Osk at the anterior of the oocyte causes ectopic pole plasm assembly and the formation of germ cells at the new site, indicating that Osk organizes pole plasm assembly (Tanaka, 2008).

Although osk has no known alternatively spliced variants, the osk message produces two protein isoforms, long and short Osk, by translation from in-frame alternative start codons. Short Osk shares its entire sequence with the long isoform. Nevertheless, genetic evidence shows that the two Osk isoforms have distinct functions in the assembly of the pole plasm. Long Osk is required for all the components of the pole plasm, including Osk itself, to be anchored to the posterior cortex, preventing their diffusion into the cytoplasm. However, the mechanism by which long Osk retains pole plasm components at the posterior cortex remains unknown (Tanaka, 2008).

A recent immuno-electron microscopic study revealed that the two Osk isoforms localize to distinct organelles in the oocyte posterior: long Osk associates with endosomes and short Osk is concentrated in the polar granules (Vanzo, 2007). Long Osk also upregulates endocytosis, which occurs preferentially at the oocyte posterior (Vanzo, 2007). Therefore, the endocytic pathway may be involved in pole plasm assembly downstream of long Osk, although data are lacking to show that the association between long Osk and endosomes is functionally significant. Several reports have suggested that vesicular trafficking is involved in pole plasm assembly and germ cell formation. For example, in mutants for Rab11, which encodes a small GTPase involved in the recycling of endosomes, osk RNA fails to be transported to the oocyte posterior, instead forming aggregates close to the posterior. However, the defects in osk RNA localization in Rab11 mutants are thought to be an indirect consequence of the disrupted MT polarization (Tanaka, 2008).

This study shows that Drosophila Rabenosyn-5 (Rbsn-5), a Rab5 effector protein involved in the early endocytic pathway, is required for osk RNA localization and pole plasm assembly. Although the primary defect of the rbsn-5 mutation is, as in the Rab11 mutant, caused by the failure to maintain MT polarity, which secondarily affects osk RNA localization, evidence is provided that the endocytic pathway also functions downstream of Osk to anchor the pole plasm components to the oocyte cortex (Tanaka, 2008).

Vas is a reliable marker for the germline throughout Drosophila development. A GFP-Vas fusion protein enables the direct visualization of the pole plasm and germ cells in the living organism. During oogenesis, GFP-Vas accumulates at the oocyte posterior from stage 9 onward. Using GFP-Vas as a marker, a germline clonal screen was performed targeting chromosome 2L for mutations that disrupted pole plasm assembly. From 5122 lines mutagenized with EMS, 66 mutants were isolated defective in GFP-Vas localization. Twenty-seven of these were alleles of cappuccino, spire or profilin (chickadee), three genes on 2L that are known to be involved in osk RNA localization, which validates the screening strategy (Tanaka, 2008).

Among the other mutants recovered was a recessive lethal mutation, C241, that mapped to 28C2-29E2. Subsequent deficiency mapping and sequencing of the mutant chromosome revealed that the C241 mutation was a single nucleotide substitution in the CG8506 gene (Rabenosyn -- FlyBase), which resulted in a premature stop codon at position 315 of the 505 amino acid open reading frame (ORF). The introduction of a transgene containing a genomic DNA fragment with the CG8506 transcriptional unit rescued the C241 mutant phenotypes (described below). These data show that CG8506 corresponds to the gene that was mutated at the C241 locus. Rabbit and rat polyclonal antisera raised against full-length CG8506 did not detect a truncated form of CG8506 in ovarian extracts from C241 heterozygotes. Furthermore, neither antibody showed immunoreactivity in C241 homozygous clones, suggesting that the truncated protein was not expressed at detectable levels and/or was unstable. Therefore, C241 appeared to be a strong loss-of-function, presumably a protein-null, allele of CG8506 (Tanaka, 2008).

CG8506 (Rabenosyn - FlyBase) encodes a protein homologous to Rabenosyn-5 (Rbsn-5) (Nielsen, 2000). Rbsn-5 interacts with several Rab proteins, including Rab5, which functions in early endosomal transport (de Renzis, 2002; Eathiraj, 2005). Several Rbsn-5 protein domains are conserved across species, including the FYVE domain, which binds phosphatidylinositol-3-phosphate (Nielsen, 2000). However, invertebrate Rbsn-5 homologs lack the C-terminal domain common to the mammalian homologs of this protein. Since the C-terminal domain of mammalian Rbsn-5 is responsible for its interaction with Rab5 (de Renzis, 2002; Eathiraj, 2005), whether CG8506 interacted with Rab5 was examined. Pull-down assays showed that GST-Rab5 efficiently pulled down in-vitro-synthesized CG8506 protein in the presence of a GTP analog, GTP-γS, but inefficiently in the presence of GDP. The interaction between CG8506 and Rab5-GTP was specific, because the interactions of CG8506 with Rab11 and Rab7 were at background levels. Consistent with a physical interaction between CG8506 and Rab5 in vitro, in CG8506C241 GLCs, neither auto-fluorescent granules derived from endocytosed yolk proteins nor the incorporation of a fluorescent marker for endocytosis, FM4-64, were observed in the oocytes, suggesting that CG8506 functions cooperatively with Rab5 in the early endocytic pathway. Thus, CG8506 is the Drosophila ortholog of Rbsn-5 and has an evolutionarily conserved function in the endocytic pathway (Tanaka, 2008).

This study shows that that Osk maintains, but does not establish, the posterior accumulation of endosomal proteins and asymmetric endocytosis, and that Osk can recruit endosomal proteins and stimulate endocytosis even at an ectopic site. It is further shown that the anchoring of the pole plasm components to the oocyte cortex requires the Osk-dependent stimulation of endocytic activity. These data reveal an interdependent relationship between Osk anchoring and localized endocytic activity at the oocyte posterior (Tanaka, 2008).

In rbsn-5- oocytes, the anterior misexpression of Osk induces aberrant F-actin aggregates, which diffuse along with pole plasm components into the cytoplasm. Several lines of evidence suggest that the anchoring of pole plasm components requires the proper organization of F-actin. Since endosomal proteins are recruited by long Osk, the idea is favored that the endocytic pathway functions downstream of long Osk to anchor the pole plasm components at the cortex by regulating F-actin dynamics. Supporting this idea, in addition to its roles in early endosomal sorting, Rab5 acts as a signaling molecule that remodels F-actin networks (Lanzetti, 2004). Rab11, which regulates the recycling of endosomes, is also involved in F-actin organization during cellularization in Drosophila blastoderm embryos (Riggs, 2003). Intriguingly, the recruitment of endosomal proteins by Osk is not sufficient for proper F-actin reorganization to anchor the pole plasm components at the cortex, because their recruitment occurs even in oocytes lacking Rbsn-5, in which cortical anchoring fails. It is therefore proposed that the continuous cycling of endosomes is required for pole plasm components to be anchored to the oocyte cortex. This scenario is compatible with a model in yeasts, which use endocytic cycling coupled with localized exocytosis to maintain their polarity (Valdez-Taubas, 2003), although it is unclear if F-actin reorganization is involved in this process (Tanaka, 2008).

Rbsn-5 is primarily required for the maintenance of MT polarity that directs posterior localization of osk RNA. Rab11 is also required for MT polarization in the oocyte (Jankovics, 2001; Dollar, 2002). However, the accumulation of endosomal proteins and upregulation of endocytosis at the oocyte posterior require the oocyte polarization, which promotes the reorganization of the MT array. Thus, MT polarization and asymmetric activation of the endocytic pathway are probably interdependent as well. Furthermore, maintenance of polarized endocytic activity depends on Osk. Intriguingly, Osk is also thought to maintain MT polarity, as posterior accumulation of Kin-βgal is partially defective in the absence of Osk (Zimyanin, 2007). It is therefore likely that the endocytic pathway and Osk form a positive-feedback loop that maintains oocyte polarity: Osk may maintain MT polarity through recruiting endosomal proteins. Based on these results, a model is proposed in which the endocytic pathway is involved in several distinct steps in pole plasm assembly (Tanaka, 2008).

The localization of bcd RNA to the anterior pole of the oocyte requires the ESCRT-II (endosomal sorting complex required for transport II) complex, which sorts mono-ubiquitinated endosomal transmembrane proteins into multivesicular bodies. Furthermore, Vps36p, a component of the ESCRT-II complex, binds bcd 3' UTR in vitro and co-localizes with bcd RNA at the oocyte anterior, suggesting the direct involvement of ESCRT-II in bcd RNA localization. osk RNA, however, appears to use another mechanism for its posterior localization, since its localization is unaffected in the absence of ESCRT-II function. Several lines of evidence suggest that ER organization and RNA localization are linked. However, it is considered unlikely that the ER directs the posterior localization of osk RNA, because ER components and osk RNP distributed differentially in developing oocytes. Interestingly, the osk RNP and the endosomal proteins are in close proximity during their transport to the oocyte posterior. Although their close association may simply be owing to the dynamic rearrangements of the MT array during stages 7-8, these findings suggest that the endocytic pathway may also play a role in the targeting of osk RNP to the posterior pole of the oocyte. Retroviral genomic RNAs are known to hitchhike on endosomal vesicles to reach the plasma membrane. Therefore, it will be interesting to learn if osk RNA is also transported to the posterior pole of the oocyte along with the endosomes (Tanaka, 2008).

Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration

The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. This study shows that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. It is further demonstrated that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms (Palamidessi, 2008).

Crosstalk among small GTPases is critical in the regulation of numerous cellular functions, and the cell has adopted several strategies to regulate it. In some cases, crosstalk is obtained through simple hierarchical cascades whereby two small GTPases are directly and orderly linked in a positive or negative amplification loop. This is, for instance, the case with Ras-to-Rac signaling or signaling among Rho-like GTPases (Palamidessi, 2008).

In the case described in this study, Rab5 regulates Rac not via a direct biochemical link but, rather, by activating an entire process, endocytosis, that provides the enabling conditions for the activation of Rac and the execution of its function. The data, therefore, add an extra layer to the complex strategies adopted by the cell to use endocytosis as a device to provide spatial and temporal dimensions to signaling. The regulation of Rac activity by endocytosis is reminiscent of the endocytic-dependent regulation of Ras signaling. Ras and/or MAPK signaling has been observed on different endomembranes, and it depends on functional endocytosis. Additionally, H-Ras is dynamically recruited, in both its active form and a Rab5 and Rab11 manner, to recycling endosomes (Palamidessi, 2008).

There are important differences, however, in the finalities of endocytic regulation of Ras and Rac. In the former case, the organelle location of Ras has invariably been proposed to serve as an intracellular platform, conferring signal specificity and diversity possibly by extending and segregating the repertoire of regulatory molecules and/or effectors. In the case of Rac, endocytosis, localization to endosomes, and recycling to the plasma membrane should be viewed primarily as a means to resolve and direct signals in space, thus preventing them from becoming uniformly distributed and, as such, uninformative (Palamidessi, 2008).

Regulation of early endosomal entry by the Drosophila tumor suppressors Rabenosyn and Vps45

The small GTPase Rab5 has emerged as an important regulator of animal development and is essential for endocytic trafficking. However, the mechanisms that link Rab5 activation to cargo entry into early endosomes remain unclear. Rabenosyn (Rbsn) is shown to be a Rab5 effector that bridges an interaction between Rab5 and the Sec1/Munc18-family protein Vps45. The syntaxin Avalanche (Avl) was identified as a target for Vps45 activity. Rbsn and Vps45, like Avl and Rab5, are specifically localized to early endosomes and are required for endocytosis. Ultrastructural analysis of rbsn, Vps45, avl and Rab5 null mutant cells, which show identical defects, demonstrates that all four proteins are required for vesicle fusion to form early endosomes. These defects lead to loss of epithelial polarity in mutant tissues, which overproliferate to form neoplastic tumors. This work represents the first characterization of a Rab5 effector as a tumor suppressor, and provides in vivo evidence for a Rbsn-Vps45 complex on early endosomes that links Rab5 to the SNARE fusion machinery (Morrison, 2008).

The transport of protein cargoes to the numerous compartments within cells requires the budding, movement and fusion of membrane-bound vesicles. The myriad itineraries that vesicles follow require robust regulatory mechanisms to ensure specificity of delivery. One important site of regulation is at the fusion reaction itself. The core machinery that enables vesicle fusion consists of SNARE proteins, which are trans-membrane proteins located on the donor and target membranes that each contribute one of the four α-helices found in an assembled SNARE complex. Formation of a fusion-competent complex requires the incorporation of an α-helix from each of the different subfamilies of SNARE motifs, the Qa-, Qb-, Qc- and R-SNAREs. Individual SNAREs within each subfamily are localized to distinct cellular compartments, suggesting that this distribution along with intrinsic SNARE pairing propensities may contribute to membrane fusion specificity. However, the properties of SNAREs alone appear insufficient to account for the specificity seen in vivo, indicating that other regulators are important to ensure the integrity of intracellular traffic (Morrison, 2008).

Rab proteins play a key regulatory role in SNARE-mediated fusion events. Like SNAREs, these small GTPases show distinct intracellular localization patterns and are required for specific transport steps. Rabs are thought to influence vesicle fusion by serving as molecular switches that, when activated, recruit additional factors -- Rab effectors -- to their site of action. While activated Rabs generally bind to many different proteins, only a subset of these are actually direct effectors of vesicle trafficking. Identification of trafficking effectors requires a demonstration that the Rab and the effector are required for the same transport step. Genetic analyses in yeast have identified such proteins, in which loss-of-function phenotypes mimic those of mutations in specific Rabs and SNAREs. These trafficking effectors are structurally, and apparently functionally, diverse (Morrison, 2008).

Some effectors are thought to act as a physical 'tether' to mediate attachment between an incoming vesicle and its target membrane, bringing them into close proximity prior to vesicle fusion. Other effectors recruit proteins such as the Sec1/Munc-18 family (SM proteins), which bind and regulate the SNARE fusion complex itself; these modes may not be mutually exclusive. Since the mechanisms by which Rab activation controls membrane fusion are varied and unclear, a thorough understanding requires the identification of Rab trafficking effectors and the molecular interactions by which they link the Rabs to the SNARE complexes (Morrison, 2008).

Although yeast genetics has pioneered the determination of Rab effectors that mediate most stages of intracellular transport, an important exception is plasma membrane-to-early endosome traffic. This is a particularly significant step in metazoan organisms, where the internalization of cell surface proteins into the endosomal pathway regulates many critical cell-cell interactions, including signaling and adhesion. Current knowledge of the mechanisms underlying cargo delivery to early endosomes derives from several different approaches in mammalian cells, including biochemical interactions and in vitro reconstitution of endosomal fusion reactions, which have demonstrated the central role of Rab5 in this event. Intriguingly, these studies have also identified two effectors, EEA1 and Rabenosyn-5, which are recruited to endosomes by activated Rab5 and are associated, directly and indirectly respectively, with SNAREs. The indirect association of Rabenosyn-5 with SNAREs is through Vps45, an SM protein that binds various syntaxins in vitro. Despite these interactions, functional studies have not demonstrated that these proteins are required for plasma membrane-to-early endosome transport in vivo; the identity of the Rab5 effector that mediates this trafficking step thus remains unresolved (Morrison, 2008).

Drosophila has emerged as a valuable system to study endocytosis in vivo, in particular for the stage of early endosomal entry. Reverse genetics originally established that, as in mammalian cells, Drosophila Rab5 is required for this trafficking step. Recently, a forward genetic screen identified mutations in a syntaxin, called Avalanche (avl), that cause a similar endocytic phenotype to that of Rab5 mutations. The endocytic defects of Rab5 and avl imaginal disc cells lead to a loss of epithelial architecture, and mutant tissues show dramatic overgrowth to form tumor-like cell masses; this phenotype is termed 'neoplastic'. To identify factors that link Rab5 activation to Avl-mediated vesicle fusion, a screen was performed for new mutations that produced the same tumorous phenotype. This study describes two previously uncharacterized genes, which encode the Drosophila proteins Rabenosyn (Rbsn) and Vps45; both are required for plasma membrane-to-early endosome trafficking. Genetics, ultrastructural analysis and biochemical interactions were used to link Rab5 and Avl activities through Rbsn and Vps45. These data are consistent with a model in which Rbsn, via Vps45 binding, functions as a Rab5 effector and tumor suppressor by mediating early endosomal fusion (Morrison, 2008).

The screen for mutations affecting epithelial polarity and proliferation identified many new complementation groups that control epithelial tissue architecture. This screen used the eyFLP/cell lethal system to generate eye imaginal discs composed predominantly of homozygous mutant cells in an otherwise heterozygous animal (hereafter 'mutant eye discs'). In this assay, eye discs mutant for the MENE(2L)-C complementation group consist of rounded and dramatically disorganized masses of cells. A similar phenotype is seen in the ovarian follicle cells. While wild-type follicle cells form a monolayered epithelium, MENE(2L)-C mutant cells multilayer and often invade the germ cell cluster. Staining for proteins normally localized to apical or lateral plasma membrane domains reveals that these domains are misspecified in MENE(2L)-C mutant cells. The normally apically restricted protein Atypical Protein Kinase C (aPKC) fails to remain distinct from Discs-Large-marked (Dlg) basolateral domains, indicating that apicobasal polarity is disrupted in these mutants. MENE(2L)-C mutant eye discs also show strong upregulation of Matrix Metalloprotease 1 (Mmp1) expression, which correlates with neoplastic transformation. Finally, larvae with MENE(2L)-C mutant eye discs do not pupariate but continue to feed during an extended L3 stage; during this time the eye discs grow to be significantly larger than wild-type eye discs. The polarity, proliferation, and gene expression phenotypes all resemble those seen in tissues mutant for previously characterized neoplastic tumor suppressor genes (nTSGs) including scribble (scrib) and Rab5. However, complementation tests showed that MENE(2L)-C was not allelic to any known tumor suppressor gene. Collectively, these phenotypes therefore indicate that MENE(2L)-C disrupts a novel Drosophila neoplastic tumor suppressor gene (Morrison, 2008).

MENE(2L)-C disrupts a protein related to human Rabenosyn-5 To identify the gene disrupted by MENE(2L)-C alleles, complementation tests were performed with chromosomal deficiency stocks and a small deficiency, Df(2L)Exel7034, was found that failed to complement the two extant MENE(2L)-C alleles. Sequencing of genes located within the genomic region deleted in Df(2L)Exel7034 revealed that each MENE(2L)-C allele carries a lesion in the gene CG8506, which encodes a 505 amino acid protein. MENE(2L)-C40-3 is a missense mutation altering the initiating ATG to ATA; the next in-frame ATG is located at amino acid 116. MENE(2L)-CX17 is a nonsense mutation that introduces a stop codon at amino acid 241. Both alleles show identical phenotypes in imaginal discs as well as in follicle cell epithelia, and animals either homozygous for each allele or hemizygous over Df(2L)Exel7034 die before the second larval instar. In addition, antibodies raised against a GST-CG8506 fusion protein recognize a polypeptide of the expected molecular mass of 56kD in wild-type larval extracts; this polypeptide is absent from extracts of MENE(2L)-C40-3 tissue. These results indicate that MENE(2L)-C40-3 and MENE(2L)-CX17 are null alleles of CG8506 (Morrison, 2008).

Sequence analysis revealed that CG8506 encodes a protein containing a number of conserved domains, including an N-terminal C2H2 zinc finger, a FYVE domain, two repeats of the tripeptide motif NPF, and several coiled-coil regions. BLAST searches indicate that CG8506 has significant homology to the human Rab5-binding protein Rabenosyn-5, which contains each of these domains, although in a different arrangement. CG8506 is shorter than Rabenosyn-5 and lacks a C-terminal helical region, the NPF motifs are N-terminal in CG8506 while they are C-terminal in Rabenosyn-5, and CG8506 contains a single coiled-coil region. Nevertheless, these features are not found together in any other protein encoded by the fly genome. Therefore CG8506 is referred to as rabenosyn (Morrison, 2008).

Mammalian Rabenosyn-5 has been linked to both the endocytic and the recycling pathways in part by virtue of its ability to bind simultaneously to Rab5 and Rab4. To explore whether Drosophila Rbsn might be involved in these trafficking pathways, in vitro binding assays were performed using recombinant Rbsn and Rab GTPases. It was found that Rbsn binds to Rab5 specifically in its GTP-, but not GDP-bound form. By contrast, no significant binding base detected between Rbsn and Rab4 in either GTP or GDP-bound forms. Because Rab11 has been implicated in recycling pathways, tests were performed to see whether Rbsn could bind to Rab11, but no interaction was detected. It is concluded that Rbsn interacts specifically with the endocytic regulator Rab5 at early endosomes but not with Rab proteins that control recycling (Morrison, 2008).

It was also asked whether the results of in vitro binding experiments reflected the protein association in vivo, by examining the subcellular localization pattern of Rabenosyn relative to each of the Rab proteins. In cultured Drosophila S2 cells, Rabenosyn is found in discrete puncta which partially overlap with Avl-positive endocytic compartments. Expression of Rab5-YFP demonstrates Rbsn and Avl colocalization in Rab5-positive puncta, indicating that Rbsn localizes to early endosomes in response to Rab5 activation. By contrast, in cells expressing activated forms of Rab4 or Rab11, Rbsn and Avl colocalize in puncta that are mostly discrete from those marked by Rab4 or Rab11, indicating that Rbsn is not strongly recruited to recycling endosomes, consistent with the in vitro results (Morrison, 2008).

These above data suggest an association between Rbsn and the endocytic pathway, and disruption of several endocytic stages has been previously shown to perturb both cell polarity and cell proliferation control. Therefore directly whether Rbsn was required for endocytosis was directly tested. In wild-type imaginal disc cells, the apically localized transmembrane protein Notch is continuously endocytosed and lysosomally degraded; the endocytic transient population can be visualized as intracellular cytoplasmic puncta. However, in rbsn cells, Notch is present at greater than wild-type levels; a similar elevation is seen with the apical transmembrane protein Crumbs (Crb). To directly analyze cargo internalization, a trafficking assay was performed in living disc tissue. This assay pulse-labels cell surface Notch using an antibody against the Notch extracellular domain; endocytosis is then allowed to occur over varying chase periods. After 10 minutes of chase in WT cells, Notch is internalized and is found in early endosomes, while after 5 hours, no Notch signal remains. In contrast, in rbsn mutant cells no intracellular Notch puncta are seen after 10 minutes; instead Notch remains in the cell periphery and this localization persists even after 5 hours. This pattern strongly resembles that seen in rab5 mutants, but contrasts with that seen with the late-acting ESCRT mutants, in which Notch accumulates in enlarged endocytic compartments; it also contrasts with mutations in 'junctional scaffold' neoplastic tumor suppressor genes such as scrib, where no effect on Notch endocytosis is seen. The activity of a Notch reporter is reduced in rbsn mutant discs as in Rab5 and avl mutants; this is consistent with studies indicating that Notch that does not enter endosomes has reduced signaling function and suggests that Notch accumulation is not involved in the rbsn tumor phenotype. Together, these results establish that rbsn is required for an early step in the endocytic pathway (Morrison, 2008).

The data indicate that rbsn has an endocytic mutant phenotype similar to Rab5 mutants, and Rbsn colocalizes with Rab5 at early endosomes and binds directly to Rab5-GTP. These results suggest that Rbsn might regulate Rab5-dependent fusion events at the early endosome. Interestingly, the Sec-1/Munc-18 (SM) protein Vps45 has been identified as a Rabenosyn-5 interacting protein. While the function of mammalian Vps45 is unknown, the yeast homolog Vps45p has a well documented requirement in biosynthetic Golgi-tolysosome traffic, and interacts with a Rabenosyn-5 like protein Vac1p. To test whether Rbsn might associate with a Vps45-like protein, a single clear Vps45 homolog was identified among the 4 SM proteins in Drosophila, that is encoded by the uncharacterized gene CG8228 (hereafter referred to as Vps45). An MBP-Vps45 fusion protein was expressed in bacteria, and it was found, using in vitro binding assays, that Vps45 strongly binds to Rbsn. These data suggest that Rab5 might regulate traffic through Rbsn-dependent Vps45 recruitment to the early endosome. The localization of Vps45 in animals has not been previously reported. To assess the in vivo localization of Vps45, an epitope-tagged Vps45 construct was expressed in S2 cells. In transfected cells, Vps45 shows a punctate pattern with only partial overlap to that of Rbsn and Avl. Interestingly, upon overexpression of Rab5-YFP, Vps45 relocalizes to the resultant enlarged endosomes; most Vps45 in these cells colocalizes with both Rbsn and Avl. Taken together with the in vitro binding data, this result suggests that Vps45 is recruited to the early endosome in response to Rab5 activation (Morrison, 2008).

Since yeast Vps45p is required for vacuolar but not endocytic traffic (Raymond, 1992; Bryant, 1998), attempts were made to determine whether the Rbsn-Vps45 interaction observed in Drosophila was relevant for function at the early endosome. No mutations in Vps45 have been reported in flies. However, by testing the uncharacterized neoplastic mutants isolated in the screen, it was found that MENE(3R)-B alleles fail to complement deficiencies that remove Vps45. Vps45 was sequenced and lesions were found in the coding region in each of the two MENE(3R)-B alleles. MENE(3R)-BJJ2 causes premature termination of the protein at amino acid 233 of the 574 amino acid coding region, and MENE(3R)-BGG11 converts valine 219 to a glutamine. Larvae homozygous for either allele die before the third instar, and the mutant eye imaginal disc phenotypes are indistinguishable, suggesting that both represent null alleles (Morrison, 2008).

The phenotypes of Vps45 mutant tissues were analyzed. As in rbsn mutants, staining for Notch and Crb showed that these protein levels were elevated in Vps45 mutant discs. To test whether Vps45 mutants might cause neoplastic transformation by blocking endocytosis in a manner similar to rbsn mutants, Notch trafficking was examined. Using live trafficking assays, it was found that Notch was not internalized in Vps45 mutant cells, and accumulated near the cell surface in a manner resembling that of both rbsn and Rab5 mutant cells. Moreover, the cell polarity, proliferation, Mmp1 expression and Notch signaling phenotypes were indistinguishable from those of rbsn mutant discs. These data suggest that in Drosophila, Vps45 function is indeed required for endocytic traffic and in particular for early endosomal stages (Morrison, 2008).

SM proteins are canonically thought to be trafficking regulators, able to bind to either SNARE proteins or complexes and govern fusion between vesicular and target membranes. In Golgi-to-vacuole traffic in yeast, the binding of Vps45p to Tlg2p is necessary for fusion into the vacuole. Physical interactions between the Drosophila homologs of these proteins were tested and a strong interaction was confirmed between Vps45 and Syx16. However, only a small fraction of Drosophila Syx16 localizes to endosomes, and strong expression of a Syx16 RNAi transgene did not generate defects associated with disrupted endocytosis. Since similar expression of a Vps45 RNAi transgene shows strong endocytic defects resembling that seen in null mutant tissue, it was asked whether Vps45 might control endocytosis by associating with early endocytic SNAREs such as Avl. Weak but consistent binding was found between Vps45 and Avl as compared to Syx1 as a negative control; this binding was comparable to that seen with the annotated Drosophila homolog of Syx13, which interacts with Vps45 in humans and C. elegans (Morrison, 2008).

To evaluate whether Vps45 might regulate these SNAREs in vivo, genetic interaction studies were performed. Moderately reducing the protein levels of Vps45 using an RNAi construct expressed in the posterior compartment of the adult wing produces no obvious phenotype. However, removing one copy of the wild-type Vps45 gene to further reduce Vps45 protein levels results in an enhanced phenotype, including aberrant vein formation and ruffling of the posterior margin. Similar defects are also seen when Avl protein levels are reduced by RNAi, suggesting that this phenotype results from impaired endocytosis. The Vps45 RNAi sensitized background was used to test whether other genes might act in the Vps45- regulated endocytic pathway. It was found that removing one copy of Syx13 or Syx16 did not alter the Vps45 RNAi phenotype but removing one copy of avl, as well as Rab5 and to a lesser extent Rbsn, resulted in an enhancement similar to that produced by the removal of one copy of Vps45. Although weak, the interaction between Vps45 and rbsn was consistent; 64% of en>Vps45-IR; rbsn/+ wings show ectopic vein formation across the posterior cross vein, versus only 17% of en>Vps45-IR wings. Analogous results were seen when knocking down avl, further validating the interactions between these genes. Along with the strong phenotypic similarity of the mutants, these results suggest that Vps45 acts together with Rab5, Rbsn and Avl at the early endosome, and point to Avl as a regulatory target for Vps45 (Morrison, 2008).

The genetic and biochemical interactions described above suggest the hypothesis that Rab5, Rbsn, Vps45 and Avl act together to promote a single stage of endocytic traffic, involving fusion of incoming endocytic vesicles into the early endosome. If this hypothesis is correct, then cells lacking any of these proteins should block endocytosis at the same step and show similar disruption of endosomal structures. Since light microscopy does not allow the resolution required to clearly distinguish these structures, transmission and immunoelectron microscopy were used to test this hypothesis. Late-stage oocytes, which are large cells with a defined endocytic pathway required for uptake of yolk proteins, were tested. By making germ-line clones, oocytes mutant for Rab5, rbsn, Vps45, and avl were tested; all four mutants are defective in the formation of yolk granules. In WT oocytes, yolk granules are late endocytic structures that have a characteristic, electron-dense appearance resulting from condensation of internalized yolk proteins. These structures are strikingly absent from mutant oocytes. Because the lack of yolk granules could point to a prior block in the endocytic pathway, endocytic intermediates were analyzed by using an antibody against the yolk proteins, which are produced outside the oocyte, to trace a known endocytic cargo within oocyte vesicular compartments. In wild-type oocytes, yolk proteins are found in numerous endocytic compartments spanning a wide size range. In contrast, in Rab5, avl, rbsn and Vps45 mutant oocytes, yolk proteins are confined to small vesicles with a narrow size distribution; these are primarily found in dense accumulations in close proximity to the plasma membrane. The diameter of the vesicles, approximately 100 nm, is consistent with both the expected size of internalized clathrin-coated vesicles and the size of vesicles present in WT oocytes, and both electron-dense coated and uncoated vesicles are seen. Taken together, these data indicate that endocytic vesicles still form in the absence of Avl, Rab5, Vps45, or Rbsn; these vesicles can uncoat but nevertheless cannot fuse to form later endoyctic structures. The strong phenotypic similarity seen amongst all four mutants using immuno-electron microscopy support a model in which Vps45 and Rabenosyn act with Rab5 and Avl to promote vesicle fusion into the early endosome (Morrison, 2008).

This study has used forward genetics to identify and characterize two essential regulators of plasma membrane-toearly endosome traffic in Drosophila: Rbsn and Vps45. Rbsn and Vps45 are related to proteins implicated in endocytosis in mammalian cells, and their endocytic role in Drosophila has been definitively demonstrated in this study by direct analysis of cargo trafficking in null mutant tissue. Loss of either protein disrupts the flow of information from the activated small GTPase Rab5 to the trans-SNARE complex and blocks the fusion of endocytic vesicles into the endosome. The endocytic defect further causes mispolarization of epithelial cells and consequent overproliferation to form 'neoplastic tumors.' Although it has been shown that Rab5 acts as a Drosophila tumor suppressor, Rab5 has many effectors that regulate cellular processes as diverse as lipid metabolism, cytoskeletal organization, and cargo recycling. The demonstration that Rbsn and Vps45 are effectors of the tumor suppressive-activity of Drosophila Rab5 emphasizes that growth regulation requires endosomal fusion itself. These two proteins therefore extend the list of endocytic regulators that act as tumor suppressors, confirm the critical role of endocytosis in coordinating cell polarity and cell proliferation, and provide insight into the processes controlling entry into the early endosome (Morrison, 2008).

The mechanisms linking Rab-mediated vesicle targeting and SNARE-mediated vesicle fusion are among the least well understood events in cellular trafficking. Drosophila Rbsn is shown to be a Rab5 effector, binding to Rab5-GTP and localizing to early endosomes. Like Rab5, Rbsn is required for early endocytic entry, and rbsn and Rab5 mutants are phenotypically indistinguishable. In particular, this study used high resolution immuno-electron microscopy to identify the site of cargo trapping in cells completely lacking rbsn and Rab5 (as well as Vps45 and avl). These mutants show a striking accumulation of endocytic cargo-containing vesicles of a size consistent with plasma membrane-derived carrier vesicles; the absence of large endosomal compartments suggests that these vesicles fail to undergo fusion to form early endosomes. Together, the genetic, biochemical and in vivo phenotypic data provide strong support for a model in which Rbsn is a Rab5 effector essential for endocytic vesicles to fuse into the early endosome. The severe endocytic block seen in rbsn tissue contrasts with the phenotype of mammalian cells depleted of the related human protein Rabenosyn-5 by RNA knockdown, which allow early endosomal entry but are defective in recycling cargo back to the plasma membrane. The involvement of Rabenosyn-5 in the recycling pathway is supported not only by this phenotype, but also by its ability to bind Rab5 and the recycling regulator Rab4 simultaneously, prompting a model in which Rabenosyn-5 acts to coordinate cargo transfer from the early to recycling endosomes. Drosophila Rbsn, despite its strong association with Rab5-GTP, does not bind to Rab proteins known to regulate recycling, and while Rabenosyn-5 contains separate Rab4 and Rab5 binding domains, these domains show homology to the same single domain in Drosophila Rbsn. It can be speculated that in mammalian Rabenosyn-5, duplication of the Rbsn Rab-binding domain followed by subsequent functional divergence led to its adoption into the recycling pathway, while the mammalian tethering protein and Rab5 effector EEA1 played a greater role in regulating early endosome entry (Christoforidis, 1999a). Although such an evolutionary scenario is possible, the possibility cannot be excluded that Rbsn plays a role in Drosophila recycling, particularly since the strong endocytic defects that were observed in rbsn mutants are upstream of, and thus prevent analysis of, the recycling pathway (Morrison, 2008).

This analysis of rbsn null mutant tissue demonstrates that Rbsn is required for vesicles to fuse into the early endosome. How does Rbsn promote vesicle fusion? The Drosophila SM protein Vps45, which binds to Rbsn, is required for the identical step of endocytosis as Rbsn and Rab5. Vps45 localizes to early endosomes, and this localized is increased by Rab5 overexpression. Recruitment of Vps45 by Rbsn bound to active Rab5 may create a high local concentration of Vps45; once concentrated at the endosome, Vps45 could act on SNARE proteins to enable fusion of incoming carrier vesicles. In contrast to yeast, where Vps45p is required for lysosomal delivery of biosynthetic cargo (Cowles, 1994), this study shows that Drosophila Vps45 is required for trafficking and degradation of surface-derived cargo, thus identifying an SM protein that acts in the endocytic pathway (Morrison, 2008).

Gengyo-Ando has reported that C. elegans oocytes lacking homologs of Vps45 or Rbsn are defective in yolk uptake, but did not distinguish the precise stage of endocytic traffic blocked; moreover, they could not identify any syntaxin required for endocytosis and therefore could not determine a functional target of Vps45 (Gengyo-Ando, 2007. Full text of article). This study provides evidence that the endocytic syntaxin Avl is a key Vps45 target. A clear genetic interaction was found specifically between Vps45 and avl, as well as a weak physical interaction between Vps45 and both Avl and Syx13. While human and C. elegans Syx13 have been shown to bind Vps45 (Nielsen, 2000; Gengyo-Ando, 2007), orthologous relationships with Drosophila syntaxins are ambiguous: both Drosophila Avl and Syx13 are similar to human and C. elegans Syx13 as well as to human Syx7. The data demonstrate that Avl is required for the fusion event required for cargo entry into early endosomes; although RNAi experiments do not reveal a role for Drosophila Syx13 in endocytosis, further experiments will be needed to clarify the function of Syx13 in vesicle trafficking (Morrison, 2008).

The in vitro physical interactions observed between Vps45 and both Avl and Syx13 were notably weaker than that with Syx16. However, the data do not provide evidence for an endocytic role of Syx16. In addition, the significance of SM protein binding to an isolated SNARE remains unclear. While in most cases it correlates with SNARE complex assembly, in some instances this interaction is not necessary for function in vivo, and in others it is associated with inhibition of incorporation into a SNARE complex. Considering these scenarios, phenotypic analysis of mutant tissues completely lacking Vps45 demonstrates common phenotypes to those completely lacking Rab5, Avl, or Rbsn at the tissue, cellular and ultrastructural levels, indicating that Vps45 acts as a positive regulator of early endocytic SNAREs; this is also consistent with the enhancing nature of the genetic interactions. Moreover, these data argue that Avl is a component of the SNARE complex whose activity in vesicle fusion requires Vps45, establishing a functional link between Rab5 and SNAREs essential for early endosomal entry (Morrison, 2008).

Taken together, these genetic, phenotypic, and biochemical analyses provide strong support for a model in which Rbsn, by binding to Vps45 and Rab5, enables incoming cargo vesicles to fuse into the early endosome. This trafficking event is required for the proper control of surface levels of transmembrane proteins and has significant consequences for tissue development. Given that plasma membrane-to-early endosome trafficking is a process by which metazoan animals can control intercellular interactions, Rbsn may be an attractive target for cellular regulation of this event. Indeed, genetic interactions hint at a role for Rbsn in modulating several cell-cell interaction and communication pathways; future work will reveal whether Rbsn activity is modulated in specific contexts to achieve different developmental outcomes (Morrison, 2008).

Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules: Spict is essential for a normal Rab5 compartment at the NMJ

To understand the functions of NIPA1, mutated in the neurodegenerative disease hereditary spastic paraplegia, and of ichthyin, mutated in autosomal recessive congenital ichthyosis, their Drosophila melanogaster ortholog was studied. Spichthyin (Spict) is found on early endosomes. Loss of Spict leads to upregulation of bone morphogenetic protein (BMP) signaling and expansion of the neuromuscular junction. BMP signaling is also necessary for a normal microtubule cytoskeleton and axonal transport; analysis of loss- and gain-of-function phenotypes indicate that Spict may antagonize this function of BMP signaling. Spict interacts with BMP receptors and promotes their internalization from the plasma membrane, implying that it inhibits BMP signaling by regulating BMP receptor traffic. This is the first demonstration of a role for a hereditary spastic paraplegia protein or ichthyin family member in a specific signaling pathway, and implies disease mechanisms for hereditary spastic paraplegia that involve dependence of the microtubule cytoskeleton on BMP signaling (Wang, 2007).

Axonal abnormalities, including impairment of transport, are a hallmark of many neurological and neurodegenerative diseases. These include the hereditary spastic paraplegias (HSPs), a heterogeneous set of diseases characterized by degeneration of corticospinal tract axons and spasticity of the lower extremities. Different forms of the disease are termed either pure or complicated, depending on whether other mainly neurological symptoms are present. The mechanisms of degeneration in HSPs are unknown, but over twenty causative loci (SPG loci) have been mapped and thirteen cloned. Some SPG products are implicated in microtubule function or transport, including the microtubule motor protein kinesin, and the microtubule-severing protein spastin. Since microtubules are the route for fast axonal transport, the most distal portions of axons are likely to be most sensitive to impairments of microtubule function. A second class of SPG products are mitochondrial proteins, but it is not known how mutations in these cause axonal degeneration. A third class of SPG products are apparently associated with endosomes, judged by immunolocalization or the presence of domains such as MIT or FYVE. HSP is also caused by some mutations in the amyotrophic lateral sclerosis gene ALS2, which encodes alsin, a guanine-nucleotide-exchange-factor for the early endosomal GTPase Rab5. However, the mechanism by which impairment of endosomal membrane traffic might cause axonal degeneration is unknown. (Wang, 2007).

One membrane protein encoded by an SPG gene is SPG6, mutations in which cause a dominant pure form of HSP, and which is widely expressed, although enriched in brain tissue. SPG6 is a member of a protein family (Pfam: DUF803) predicted to have between seven and nine transmembrane (TM) domains. Three different amino-acid substitutions are known, one of which is found in ethnically disparate families and another caused by different nucleotide substitutions in the same codon, suggesting a dominant gain-of-function disease mechanism that can be mediated by only a few mutations in the protein. This protein family includes another human disease protein, ichthyin, mutations in which cause autosomal recessive congenital ichthyosis (ARCI), a skin disorder whose cellular basis is not understood. Ichthyin is widely expressed, although with high expression in keratinocytes, and little or no expression in brain, and at least six recessive alleles are known that cause substitutions of mainly conserved amino acids in different parts of the protein. In summary, little is known of the cellular roles of the SPG6 and ichthyin family (Wang, 2007).

To understand the normal role of the SPG6 and ichthyin protein family, and how changes in their function might lead to cellular defects, their Drosophila homolog, spichthyin (Spict) was have studied. Spict shows preferential localization on early endosomes. It regulates growth of the neuromuscular junction (NMJ) presynaptically, by inhibition of BMP (Bone Morphogenic Protein)/TGF-β (Transforming Growth Factor-β) signaling. BMP signaling regulates synaptic growth, function and stabilization at the NMJ. This study shows a novel role for BMP signaling in maintenance of microtubules and axonal transport, and that this function is also inhibited by Spict. These data suggest that Spict inhibits BMP signaling by regulating BMP receptor traffic. These findings provide a cellular role for the Spict family of proteins, and suggest potential mechanisms for the pathology of HSPs and ARCI that include dependence of microtubules on BMP signaling (Wang, 2007).

A BLASTP search using human SPG6 identified one Drosophila homolog, CG12292. A search using CG12292 identified four predicted human proteins that were 40-50% identical to it: SPG6 (NIPA1), NIPA2, ichthyin and NPAL1. Two more distantly related human proteins, NPAL2 and NPAL3 are more closely related to plant and fungal homologs than to CG12292, and probably represent a subfamily lost from the Drosophila lineage. Since Drosophila CG12292 appears orthologous to both SPG6 and ichthyin, it was designated spichthyin (spict).

To generate spict mutant flies, transposase-mediated imprecise excision was used of a P element, EP(2)2202, inserted in the spict 5' untranslated region. One imprecise excision, spictmut, had lost the entire coding region, and was therefore a null allele of spict. Several precise excision events were recovered; one of these was used as a wild-type control in most subsequent experiments, and is referred to as spict+. Homozygous spictmut flies were viable and fertile, and took about a day longer than spict+ flies to reach adulthood (Wang, 2007).

To determine where Spict might act, its expression pattern and subcellular localization was examined. spict mRNA was found ubiquitously during embryogenesis, with elevated expression in some tissues, including CNS and muscles. EGFP-Spict and Spict-EGFP fusions both showed punctate distributions in Drosophila S2 cells, that overlapped substantially with the early endosome compartment detected using anti-Rab5, but showed no striking overlap with the late endosomal/multivesicular body marker Hook, the recycling endosomal marker Rab11, or the late endosomal/lysosomal markers Spinster and LysoTracker. A Spict-mRFP fusion protein also showed a punctate cytoplasmic distribution in wild-type and spictmut third instar larvae, which also overlapped substantially with Rab5, but not with late endosomal/lysosomal markers, in muscles and NMJs. Trypsin digestion of N-terminally and C-terminally tagged Spict, redistibuted to the plasma membrane by blockage of endocytosis, suggested that the N-terminus of Spict is in the endosome lumen, and its C-terminus in the cytosol. This result is consistent with previous suggestions that Spict family members might either have nine transmembrane domains, or be divergent members of the 7-TM superfamily. Attempts to raise an antibody that recognized endogenous Spict in immunomicroscopy were unsuccessful. However, since Spict-EGFP and EGFP-Spict fusions had apparently identical localizations in S2 cells, the Spict-mRFP fusion could rescue a spictmut phenotype and cause the same overexpression phenotypes as wild type Spict, these fusions are likely to have the same localization as endogenous Spict (Wang, 2007).

Since tagged Spict proteins localized with Rab5, tests were performed to see whether Rab5 staining is normal when Spict is lacking. Rab5 staining was less intense in spictmut NMJ boutons compared to wild-type; these phenotypes were rescued by ubiquitous expression of UAS-spict. Rab5 staining was also reduced in muscles but not obviously affected in neuronal cell bodies and axons of spictmut larvae, or in S2 cells treated by spict RNAi. Therefore, Spict is essential for a normal Rab5 compartment at the NMJ, but not in all situations (Wang, 2007).

One of the signaling pathways with the largest effects on synaptic size at the Drosophila NMJ is the BMP pathway, which stimulates synaptic growth. The expanded NMJ phenotype of spictmut is similar to that of spinster (also known as benchwarmer), which also shows defects in endosomal-lysosomal trafficking and requires an active BMP/TGF-β signaling pathway for NMJ expansion. It is also similar to the increase in bouton number of highwire NMJs. Highwire encodes a putative E3 ubiquitin ligase that appears to affect multiple signaling pathways including JNK and BMP. To determine whether the synaptic overgrowth of spictmut larvae requires BMP signaling, key BMP signaling components were genetically removed from spictmut larvae. Mutations affecting the type I receptor subunits Tkv (Thickvein) and Sax (Saxophone), the type II receptor subunit Wit (Wishful Thinking), the type II receptor ligand Gbb (Glass Bottom Boat), or the co-Smad Med (Medea) all suppressed the NMJ overgrowth of spictmut larvae. In all cases, the synaptic undergrowth in larvae that were doubly homozygous for spictmut and BMP pathway mutations was indistinguishable from that of homozygous BMP pathway mutations alone. In addition, all heterozygous BMP pathway mutations tested partly suppressed the NMJ expansion of spictmut larvae, but had no effect on NMJ bouton number in a wild type background. Therefore, BMP signaling is essential for the excessive NMJ growth of spictmut larvae (Wang, 2007).

The contrasting phenotypes of spictmut and loss of BMP signaling, and the genetic interactions between spict and BMP signaling mutants, suggest that Spict antagonizes BMP signaling in the control of NMJ growth. Nevertheless, alternative models are possible: for example, highwire mutations interact with BMP signaling mutations, but Highwire affects synaptic size primarily through a MAPK signaling pathway. However, evidence strongly supports a direct effect of Spict on BMP signaling. During BMP signaling in neurons, the R-Smad protein Mad is phosphorylated by active BMP receptors, and phosphorylated Mad (PMad) is then translocated to the nucleus and acts as a transcription factor. At the NMJ, PMad overlaps mainly with the presynaptic marker cysteine string protein (CSP), but also with the largely postsynaptic marker Discs-large (Dlg). PMad is also found in cell body nuclei in the larval CNS. PMad levels were significantly higher in spictmut than in spict+ larvae, both at the NMJ and in CNS cell bodies, and this phenotype was fully rescued by neuronal expression of UAS-spict. Therefore, BMP signaling is upregulated at spictmut neurons, in contrast to highwire neurons. Next the possibility of upregulation of BMP receptors at spictmut NMJs was tested. HA-tagged Tkv was found mainly in a punctate distribution in the periphery of synaptic boutons, at or close to the plasma membrane, and at higher levels in spictmut than in spict+ boutons. Wit was barely detectable in spict+ boutons, but was present at higher levels in spictmut boutons, also in a punctate pattern mainly at or close to the plasma membrane. The effect of spictmut on Tkv-HA and Wit levels was rescued by neuronal expression of UAS-spict. No effect was found of spictmut on levels of other neuronal membrane proteins (Fasciclin II, Syntaxin), or on the neuronal surface antigen recognized by anti-Horseradish Peroxidase (HRP) at the NMJ. Therefore, Spict action specifically lowers the levels of BMP receptors at the presynaptic NMJ (Wang, 2007).

The opposing effects of Spict and BMP signaling on NMJ and neuronal microtubules suggest that Spict is a novel antagonist of BMP signaling. BMP signaling acts both presynaptically and postsynaptically at the NMJ; rescue experiments show that Spict acts presynaptically to regulate NMJ expansion. The data suggest a direct effect of Spict on the presynaptic BMP signaling machinery. First, elevated levels of PMad and BMP receptors are seen at spictmut NMJs. Second, Spict can be co-immunoprecipitated with Wit. Third, Spict shows partial colocalization with the BMP receptors Tkv-HA or Wit at NMJ boutons. Fourth, Spict promotes relocalization of Wit from the surface of S2 cells to the Rab5 early endosomal compartment. Therefore, the data suggest strongly that Spict antagonizes BMP signaling by regulating its receptor traffic. This is in contrast to Highwire - while synaptic overgrowth in highwire mutants can be suppressed by BMP signaling mutants, the highwire phenotype is more completely suppressed by loss of the Wallenda MAP kinase kinase kinase, and there is no apparent upregulation of PMad in highwire mutants (Wang, 2007).

The posterior crossveinless phenotype in some spictmut adult wings is also typical of reduced BMP signaling in pupal wing discs. At first sight a crossveinless phenotype is inconsistent with Spict being an antagonist of BMP signaling. However, lowered BMP signaling in the posterior crossvein primordium could be due not only to direct downregulation of signaling, but also to upregulation of receptors that reduces diffusion of BMP ligands. No changes were detected in the level of BMP signaling about the time when the posterior crossvein primordium develops, but this could be due to either the partial penetrance of the phenotype, or the robustness of the regulatory and feedback mechanisms that translate smooth gradients of BMP ligands into more sharply defined developmental features (Wang, 2007).

How might an endosomal protein regulate BMP signaling? Membrane trafficking from the plasma membrane to lysosomes regulates many signaling pathways including BMP/TGF-β. For example, mutations that impair endosome to lysosome traffic cause an increase in BMP signaling, in at least some cases accompanied by increased levels of Tkv. However, the predominant localization of Spict on early endosomes, and its ability to internalize Wit to this compartment suggest that Spict functions at some step of plasma membrane to endosome traffic. (1) Rab5 compartments fail to accumulate at spictmut NMJs, rather than enlarge as in Hrs mutants. (2) Spict overexpression in S2 cells redistributes Wit mainly to early endosomes, rather than to late endosomes or lysosomes. (3) There is no obvious degradation of Wit in Spict-overexpressing cells that internalize Wit, suggesting that Spict does not directly target Wit for degradation, at least in S2 cells. While levels of BMP receptors are elevated locally in NMJ boutons that lack Spict, this could be either to altered trafficking or degradation, and BMP signaling in S2 cells can be affected by Spict, without detectable changes in levels of BMP receptors. Therefore, Spict might inhibit BMP signaling by internalizing vacant receptors and thus preventing them from responding to ligand; since clathrin RNAi treatment redistributes Spict to the plasma membrane, Spict probably appears at least transiently at the plasma membrane. However, more complex models are possible. For example, Spict might sequester BMP receptors in a compartment from which they cannot signal; Notch receptors apparently have to reach a specific endosomal compartment before they can signal (Wang, 2007).

By studying Spict, this study has identified a role for BMP signaling in maintenance of axonal microtubules. Notably, local loss of presynaptic microtubules has also been seen in loss of BMP signaling at the NMJ, and apical microtubule arrays are eliminated in tkv mutant clones in wing imaginal discs. Since BMP signaling promotes synaptic growth and synaptic strength at the NMJ, it would be logical for it also to stimulate the additional transport of materials and organelles that a larger more active synapse requires (Wang, 2007).

If human SPG6 alleles are dominant gain-of-function, then the HSP that they cause would resemble the situation of Spict overexpression in Drosophila, and axonal degeneration in HSP could then be caused by inhibition of BMP signaling, loss of axonal microtubules, and impaired axonal transport. Given the effect of BMP signaling on axonal microtubules, other HSP gene products apart from SPG6 may affect BMP signaling and thus maintenance of axonal microtubules. (Wang, 2007).

In contrast to SPG6, ARCI appears to be caused by loss of ichthyin function (Lefevre, 2004). Identification of a role for the ichthyin ortholog Spict in inhibiting BMP signaling suggests upregulation of BMP signaling as a possible disease mechanism in ARCI. Indeed, the BMP-like ligand TGF-β1 has complex roles in maintenance of skin, and its overexpression can cause psoriasis, a condition that bears some resemblance to ichthyosis. Inhibitors of BMP signaling may therefore be candidates for therapeutic purposes in ARCI or similar conditions. (Wang, 2007).

In conclusion, this study has established a cellular role for the SPG6 and ichthyin family of proteins, thus identifying a novel group of players in BMP signaling, and providing a framework for future understanding of diseases caused by mutations that affect these proteins (Wang, 2007).

C. elegans Rab GTPase activating protein TBC-2 promotes cell corpse degradation by regulating the small GTPase RAB-5

During apoptosis, dying cells are quickly internalized by neighboring cells or phagocytes, and are enclosed in phagosomes that undergo a maturation process to generate the phagoslysosome, in which cell corpses are eventually degraded. It is not well understood how apoptotic cell degradation is regulated. This study reports the identification and characterization of the C. elegans tbc-2 gene, which is required for the efficient degradation of cell corpses. tbc-2 encodes a Rab GTPase activating protein (GAP) and its loss of function affects several events of phagosome maturation, including RAB-5 release, phosphatidylinositol 3-phosphate dynamics, phagosomal acidification, RAB-7 recruitment and lysosome incorporation, which leads to many persistent cell corpses at various developmental stages. Intriguingly, the persistent cell corpse phenotype of tbc-2 mutants can be suppressed by reducing gene expression of rab-5, and overexpression of a GTP-locked RAB-5 caused similar defects in phagosome maturation and cell corpse degradation. It is proposed that TBC-2 functions as a GAP to cycle RAB-5 from an active GTP-bound to an inactive GDP-bound state, which is required for maintaining RAB-5 dynamics on phagosomes and serves as a switch for the progression of phagosome maturation (Li, 2009).

Rab5-mediated endocytosis of activin is not required for gene activation or long-range signalling in Xenopus

Morphogen gradients provide positional cues for cell fate specification and tissue patterning during embryonic development. One important aspect of morphogen function, the mechanism by which long-range signalling occurs, is still poorly understood. In Xenopus, members of the TGF-beta family such as the nodal-related proteins and activin act as morphogens to induce mesoderm and endoderm. In an effort to understand the mechanisms and dynamics of morphogen gradient formation, fluorescently labelled activin was used to study ligand distribution and Smad2/Smad4 bimolecular fluorescence complementation (BiFC) to analyse, in a quantitative manner, the cellular response to induction. The results indicate that labelled activin travels exclusively through the extracellular space and that its range is influenced by numbers of type II activin receptors on responding cells. Inhibition of endocytosis, by means of a dominant-negative form of Rab5, blocks internalisation of labelled activin, but does not affect the ability of cells to respond to activin and does not significantly influence signalling range. Together, these data indicate that long-range signalling in the early Xenopus embryo, in contrast to some other developmental systems, occurs through extracellular movement of ligand. Signalling range is not regulated by endocytosis, but is influenced by numbers of cognate receptors on the surfaces of responding cells (Hagemann, 2009).


REFERENCES

Search PubMed for articles about Drosophila rab5

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date revised: 23 August 2017

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