InteractiveFly: GeneBrief

Clathrin heavy chain: Biological Overview | References


Gene name - Clathrin heavy chain

Synonyms -

Cytological map position - 13F5-13F7

Function - principal structural component of vesicles

Keywords - spermatogenesis; Notch pathway; endocytosis; exocytosis; vesicles, tracheal development, eye development, peripheral nervous system, neuromuscular junction, synapses

Symbol - Chc

FlyBase ID: FBgn0000319

Genetic map position - chrX:15,722,019-15,729,507

Classification - Coatomer WD associated region

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Hunter, M.V., Lee, D.M., Harris, T.J. and Fernandez-Gonzalez, R. (2015). Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair. J Cell Biol [Epub ahead of print]. PubMed ID: 26304727
Summary:
Embryonic epithelia have a remarkable ability to rapidly repair wounds. A supracellular actomyosin cable around the wound coordinates cellular movements and promotes wound closure. Actomyosin cable formation is accompanied by junctional rearrangements at the wound margin. This study used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility. Blocking endocytosis with pharmacological or genetic approaches disrupts wound repair. The defect in wound closure is accompanied by impaired removal of E-cadherin from the wound edge and defective actomyosin cable assembly. E-cadherin overexpression also results in reduced actin accumulation around wounds and slower wound closure. Reducing E-cadherin levels in embryos in which endocytosis is blocked rescues actin localization to the wound margin. These results demonstrate a central role for endocytosis in wound healing and indicate that polarized E-cadherin endocytosis is necessary for actomyosin remodeling during embryonic wound repair.



BIOLOGICAL OVERVIEW

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

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

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

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

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

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

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

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

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

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

The results suggest that formation of mucin-containing glue granules and Weibel-Palade bodies might be similar. Weibel-Palade bodies have an unusual cigar-shaped appearance and it was proposed that AP-1 and clathrin might participate in their formation at the TGN by allowing lumenal cargo to properly fold and aggregate or by preventing premature scission (Lui-Roberts, 2005; Metcalf, 2008). Indeed, depletion of AP-1 in endothelial cells results in the formation of small, round von Willebrand factor-containing organelles lacking other Weibel-Palade body markers. The data demonstrate that the requirement for clathrin and AP-1 is not restricted to one specific type of granule. Depletion of clathrin or AP-1 in Drosophila salivary glands resulted in the accumulation of glue protein both at the TGN and in small organelles of aberrant morphology. This finding extends the role of AP-1 and clathrin to the formation of granules containing mucoprotein cargo and suggests a broader requirement for this coat complex in granule production (Burgess, 2011).

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

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

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

On the basis of the small size of mutant cells, AP-1 likely participates in additional trafficking pathways. In mammalian cells, AP-1A is ubiquitously expressed and required for trafficking between TGN and endosomes, whereas AP-1B is present only in polarized epithelial cells and is required for basolateral sorting from recycling endosomes. The sole AP-1 complex in Drosophila might mediate both functions in a single cell type. Interestingly, depletion of AP-1γ in salivary glands after granule formation caused the basolateral protein Discs large to redistribute to the apical surface (Peng, 2009), suggesting that AP-1 is required for basolateral targeting of proteins in this tissue. However, an independent analysis of AP-1μ null cells in the dorsal thorax epithelium failed to reveal a similar polarity defect (Benhra, 2011). This discrepancy might be due to cell type-specific requirements for AP-1 or to differences in RNAi versus mutant clones (Burgess, 2011).

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

In addition to the AP-1 complex, the Drosophila genome encodes two other Golgi-localized clathrin adaptor proteins, EpsinR/LqfR and Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding (GGA) protein (Drosophila has only one GGA) (Hirst, 2009; Lee, 2009; Kametaka, 2010). LqfR partially colocalizes with AP-1 at the TGN in salivary gland cells and lqfR mutants exhibit small salivary glands, suggesting defects in granule biogenesis (Lee, 2009). It will be interesting to determine whether LqfR and GGA participate in glue granule biogenesis, especially since these clathrin adaptors might facilitate sorting of other types of cargo. For example, EpsinR has been shown to bind SNARE proteins and could function to provide vesicle identity to nascent glue-containing granules. SNAP-24 was previously identified as a glue granule-specific SNARE, although whether this SNARE mediates homotypic fusion of granules or functions during exocytosis of granules at the plasma membrane is unclear. Given the apparent similarities between glue granule and Weibel-Palade body biogenesis, as well as the high degree of conservation of TGN sorting machinery in Drosophila, the current findings suggest that Drosophila salivary glands are of great utility to further elucidate the mechanisms of biogenesis of regulated secretory granules (Burgess, 2011).

Expression and localization of clathrin heavy chain in Drosophila melanogaster

Clathrin-coated vesicles mediate cellular endocytosis of nutrients and molecules that are involved in a variety of biological processes. Basic components of the vesicle coat are clathrin heavy chain (Chc) and clathrin light chain molecules. In Drosophila the chc gene function has been analyzed in a number of studies mainly using genetic approaches. However, the chc mRNA and protein expression patterns have not been studied systematically. An antibody as been generated that specifically recognizes Chc, and chc RNA and protein expression patterns have been analyzed throughout embryonic and larval stages. chc mRNA and protein have been found to be highly expressed from early stages of embryogenesis onwards, consistent with genetic studies predicting a maternal contribution of the gene function. During subsequent stages mRNA and protein are co-expressed in all embryonic cells; however an up-regulation was found in specific tissues including the gut, the salivary glands, tracheal system and the epidermis. In addition the central nervous system and the nephrocyte-like garland cells show strong Chc expression at late embryogenesis. In larvae Chc is highly expressed in garland cells, imaginal discs, fat body, salivary glands and the ring gland. Subcellularly, Chc protein was found in a vesicle-like pattern within the cytoplasm and at the plasma membrane. Co-labeling studies show that Chc is partially in contact with the trans-Golgi network and co-localizes with markers for early endocytosis. Together, the antibody may serve as a new tool to study the function of Chc in clathrin-dependent cellular processes, such as endocytosis (Wingen, 2009).

In Drosophila the chc gene is located at the X chromosome (13F5) and contains six putative transcript variants, which differ only in size of the non-coding exon one. All variants encode a single Drosophila Chc protein, which is highly conserved to vertebrate orthologs in sequence (80% identity and 90% similarity to human Chc1) and domain arrangement (Wingen, 2009).

Strong ubiquitous chc mRNA and Chc protein distribution was detected from earliest stages of embryogenesis onwards. These observations are consistent to a predicted maternal chc component that is necessary for early embryo development (Bazinet, 1993). Throughout subsequent embryonic stages chc mRNA and protein were found to be expressed in all cells. However, during second half of embryogenesis chc mRNA and protein levels are up-regulated in specific tissues, including the fore-, mid-, and hind-gut, salivary glands, tracheal system which all form epithelial tubular organs and at the epidermis. At late embryogenesis, strong expression was found in the central nervous system, in the epidermal leading edge cells during dorsal closure and in the nephrocyte-like garland cells, latter are known to have a rapid rate of fluid phase CCV-mediated internalization (Chang, 2002). In summary chc mRNA and protein expression pattern are similar in embryos. The organ specific Chc up-regulation may reflect high endocytosis rates required for organ and tissue development (Wingen, 2009).

In Drosophila larvae clathrin-mediated endocytosis plays an important role in multiple processes. It regulates growth, signaling pathways, autophagy (Hennig, 2006), presynaptic vesicle recycling (González-Gaitán, 1997) and axis formation (Fischer, 2006). In order to analyze Chc activity in third instar larvae, organs were dissected and analyzed for mRNA and immunohistochemical fluorescence antibody stainings. RNA and protein patterns were similar. Chc protein is highly expressed in imaginal discs, including eye and wing discs, in garland cells, fat body, ring gland and salivary glands. The data are in good agreement with the importance of endocytosis required for disc patterning (Fischer, 2006) and uptake of toxic heamolymph material in the nephrocyte-like garland cells (Weavers, 2009). Consistently Chc accumulates predominantly at the cell membrane in discs and garland cells as shown by co-expression with the cell membrane skeleton marker α-Spectrin (Spec). The fat body regulates cell growth and autophagy by endocytosis (Hennig, 2006). On the other hand the fat body, the endocrine ring gland and the salivary glands are secretory active organs involved in metabolism and growth control. In these organs Chc localizes at the membrane as well as in a strong cytoplasmic vesicle-like pattern, which is consistent to observations that clathrin vesicles mediate plasma membrane and intracellular cargo transport processes (Wingen, 2009).

Subcellular Chc distribution was analyzed by a series of co-localization studies on embryonic hindgut cells. Co-expression with Spec reveals localization of Chc at the cell membrane and the cortical cytoplasm in a punctuate pattern. CCVs mediate cargo transport processes from plasma membrane and between trans-Golgi network and endosomes. At the plasma membrane CCV endocytosis uses common core components, such as the adaptor protein AP2 (Brodsky, 2001), while coat formation at the trans-Golgi network requires distinct accessory proteins, e.g., AP1. In both cases CCV budding-in depends on clathrin recruitment, polymerization and membrane curvature. In Drosophila the transmembrane protein Wurst recruits clathrin to the membrane of epithelial cells (Behr, 2007). After inward budding of the forming vesicle, the large GTPase Dynamin mediates vesicle cleavage. Finally clathrin molecules dissociate away and the small GTPase Rab5 is required for vesicle fusion to acceptor compartments such as early endosomes. In order to investigate subcellular Chc localization in Drosophila co-labeling studies were performed with Lava Lamp (Lva) for the Golgi apparatus and Clc, Wurst, Dynamin and Rab5 for endocytotic compartments. For Clc and Rab5 GFP tagged versions were expressed in embryos using the Gal4/upstream activator sequence (UAS) system and the ectodermal driver line 69BGal4. Immune-fluorescence stainings show numerous vesicle-like Chc 'punctae' within the cytoplasm and at the membrane of epithelial hindgut cells. Confocal stacks reveal that only some cytoplasmic Chc vesicles are in close contact to the Golgi apparatus. Most of the identified Chc vesicles co-localize with Clc. Furthermore, Chc shows partial co-localization with Wurst, Dynamin and Rab5 at the membrane as well as in the cortical cytoplasm. The co-localization studies are consistent with published data from vertebrates (Wu, 2003) and suggest that Chc is associated with early endocytotic compartments. Next Chc distribution was analyzed in wurst and dynamin (shibirets1) mutant embryos, which block endocytosis at early stages. In contrast to cytoplasmic and membranous punctate pattern in wildtype, Chc accumulates at the membrane of wurst and dynamin hindgut cells (Wingen, 2009).

During embryogenesis Chc protein is ubiquitously present in all cells. However, individual up-regulation suggests that the rates of Chc activity seem to be organ specific. Subcellularly Chc is involved in clathrin-mediated endocytosis at the plasma membrane and in trafficking between the trans-Golgi network and endosomes. Co-localization studies on hindgut cells confirm that Drosophila Chc is partially in contact with the trans-Golgi network and numerous 'punctae' can be identified as early endocytotic vesicles at and close to the plasma membrane. Consistently, the data show that in larval excretory organs and glands Chc vesicle-like 'punctae; accumulate prominently within the cytoplasm. In contrast, in endocytotic active tissues, as the garland cells, Chc localizes strongly at the plasma membrane (Wingen, 2009).

Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis

Clathrin has been implicated in Drosophila male fertility and spermatid individualization (Fabrizio, 1998). To understand further the role of membrane transport in this process, the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, were analyzed in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm. The reproductive defects of aux males were rescued by male germ cell-specific expression of aux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, this rescue depends on both the clathrin-binding and J domains, suggesting that the ability of Aux to bind clathrin and the Hsc70 ATPase is essential for sperm formation. aux mutant spermatids show a deficit in formation of the plasma membrane during elongation, which probably disrupts the subsequent coordinated migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. By contrast, in aux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its colocalization with AP-1 was disrupted. Based on these results, it is proposed that Golgi-derived clathrin-positive vesicles are normally required for sustaining the plasma membrane increase necessary for spermatid differentiation. These data suggest that Aux participates in forming these Golgi-derived clathrin-positive vesicles and that Aux, therefore, has a role in the secretory pathway (Zhou, 2011).

Defects in phospholipid regulation and vesicle trafficking are known to perturb various aspects of sperm development. For example, mutations in four wheel drive (fwd), the Drosophila homolog of phosphatidylinositol (PI) 4-kinase, affect cytokinesis. Depletion of phosphatidylinositol 4,5-bisphosphate in germ cells causes defects in axoneme biogenesis, cytokinesis and cell polarity, suggesting that phospholipids have multiple distinct functions during spermatogenesis (see Fabian, 2010). Studies on genes encoding Cog5 (Four way stop), a protein required for normal Golgi morphology and localization; Syntaxin 5, a Golgi-associated SNARE protein; and Brunelleschi, a subunit of the Golgi-associated TRAPP-II complex, suggest that Golgi is crucial for both cytokinesis and spermatid elongation. Mutations in Rab11 and Arf6 (Arf51f) GTPases, two of the proteins regulating recycling endosomes, disrupt cytokinesis. Recent evidence shows that Rab11 localization during cytokinesis depends on fwd, providing a functional link between Rab11 and the PI 4-kinase (see Polevoy, 2009). Vps28, a component of the endosomal sorting complex required for transport (ESCRT), participates in individualization. This genetic evidence suggests that secretory and endocytic pathways are utilized during spermatogenesis (Zhou, 2011).

Clathrin has also been implicated in cell morphogenesis during spermatogenesis. In males mutant for Chc4 (a partial loss-of-function mutation), the number of functional sperm is greatly reduced and spermatid individualization is disrupted (Fabrizio, 1998). However, Chc4 flies have poor viability, indicating that other processes besides spermatogenesis are affected. This pleiotropy of clathrin function raises the question of whether disruption of clathrin in germ cells is the direct cause of male sterility in Chc4 mutants. Thus, although the most apparent defect in Chc4 mutant testes is the loss of IC synchrony during individualization (Fabrizio, 1998), the precise role of clathrin in spermatogenesis remains to be determined (Zhou, 2011).

One important regulator of clathrin function is auxilin, first identified in mammals as a cofactor in Hsc70-mediated disassembly of clathrin coats from nascent clathrin-coated vesicles (CCVs) in vitro. The mammalian genome contains two auxilin-related genes, auxilin and cyclin G-associated kinase (GAK). These differ in the presence of a N-terminal Ark (actin-related kinase) family kinase domain and in their respective tissue distributions. GAK contains an Ark domain and is ubiquitously expressed, whereas auxilin lacks the kinase domain and is expressed predominantly in neural tissues. However, expression of auxilin in non-neural HeLa cells has recently been demonstrated. Tissue-specific removal of GAK function in mice reveals that GAK is essential for the development of multiple tissues, whereas auxilin knockout mice show impaired synapse function. Within the cell, auxilin family proteins have been suggested to participate in the disassembly of clathrin coats, recruitment of clathrin and adaptors to membranes, exchange of clathrin during coated-pit formation, constriction of coated-pits, and prevention of precipitous assembly of clathrin cages. GAK has also been implicated in mediating trafficking from the trans-Golgi network (TGN) via its interaction with the clathrin adaptor AP-1 (Zhou, 2011 and references therein).

Drosophila has only one auxilin ortholog, which is more similar to GAK, as Aux contains an Ark domain and is ubiquitously expressed. Like other members of the auxilin protein family, Aux has a PTEN (phosphatase and tensin) homologous region, a clathrin-binding domain (CBD) and a C-terminal J-domain. Mutational analysis suggests that Aux participates in ligand endocytosis during Notch signaling in eye and wing discs. In aux mutant cells, clathrin appears as abnormal aggregates, and formation of these aggregates is thought to deplete the level of functional clathrin in the cytosol, thereby inhibiting Notch ligand internalization. Since localization of the epidermal growth factor (EGF) receptor also appears to be disrupted, Notch ligand is not the sole cargo of aux-dependent transport. Hence, it is likely that Aux has additional roles during fly development (Zhou, 2011 and references therein).

This study employed phenotypic analysis of testes from viable aux mutant males to further elucidate the role of clathrin-dependent transport in Drosophila spermatogenesis. Consistent with the notion that clathrin is crucial for male fertility, aux mutant males contain asynchronous ICs and lack sperm in their seminal vesicles. Using the male germ cell-specific β2-tubulin promoter and fluorescently tagged subcellular markers, evidence is provided that Aux function is required in the germ cells for spermatogenesis, and that Aux participates in the formation of Golgi-derived clathrin-positive vesicles to generate sufficient plasma membrane for spermatid morphogenesis (Zhou, 2011).

Using partial loss-of-function aux mutations, this study showed that auxilin has an important role in Drosophila male fertility and sperm formation. As auxilin is a well-known regulator of clathrin function, the most direct explanation for the observed male sterility is that a clathrin-dependent event crucial for sperm production is disrupted in aux mutant testes. Indeed, the phenotypes of viable aux allele combinations in spermatid individualization are similar to those of Chc4. The finding that the CBD and J-domain are indispensable for rescue of the sterility of aux mutants by exogenously expressed Aux implies that its ability to bind clathrin and Hsc70 is necessary for male germ cell development. The disruption of clathrin distribution in aux mutant germ cells further emphasizes the importance of auxilin in regulating clathrin function. Thus, although GAK has been suggested to function in the nucleus, these data strongly argue that the sterility associated with aux mutant males is caused by a disruption of clathrin in the cytosol (Zhou, 2011).

Using the β2tub promoter, it was shown that male germ cell-specific expression of functional Aux at the primary spermatocyte stage could rescue all aux-associated male reproductive defects (i.e. sterility, absence of sperm in seminal vesicles and asynchronous IC movement). This result demonstrated that male sterility was indeed caused by a disruption of aux function and that Aux is required autonomously in the germ cells for successful spermatogenesis. This is in contrast to the Notch signaling pathway, where requirement for Aux function is non-cell-autonomous. As the β2tub promoter becomes active at the spermatocyte stage, rescue by β2tub-dAux-mRFP also implies that the cause for the sterility in aux males occurs at or after the spermatocyte stage. Consistent with this, processes prior to the spermatocyte stage (e.g. the morphology of the hub, the number of GSCs) appeared unaffected in aux mutant testes. Although Notch has recently been implicated in the maintenance of niche cells in Drosophila testes, no significant reduction was seen in hub cell number in aux mutant testes (Zhou, 2011).

Overexpression of an Aux deletion consisting of just the CBD and the J-domains (dAuxCJ) can rescue the Notch signaling defect and lethality caused by aux (Kandachar, 2008). Similarly, expression of this construct from the β2tub promoter rescues aux-associated male sterility. By contrast, expression of Aux deletions missing either the CBD or the J-domain fail to rescue the sterility and IC defects. These results suggest that the CBD and J-domain are necessary for Aux function in spermatogenesis and that recruitment of Hsc70 to clathrin by Aux is a crucial event for spermatid differentiation. The kinase and PTEN-related regions also have a role in mediating Aux function in spermatogenesis, as the alleles used for generating the viable sterile aux males contain missense mutations in these domains. However, at least in the context of overexpressed rescue constructs, the kinase and PTEN-related domains are dispensable for the functions of auxilin family proteins (Zhou, 2011).

This analysis of aux mutants, in addition to confirming the importance of clathrin function in spermatogenesis, provides a plausible explanation for the observed male sterility. The phenotypes of aux mutant testes suggest that Aux participates in several processes during spermatid differentiation, including cytokinesis, formation of the plasma membrane and individualization. Since the sterility of aux mutant males could be rescued by the expression of dAuxCJ, it is likely that all of these phenotypes are clathrin-related (Zhou, 2011).

In mammals, clathrin is known to mediate vesicular trafficking from the plasma membrane, the TGN and endosomes. In addition to its role in endocytosis, GAK has been implicated in delivering lysosomal proteins from TGN. In Drosophila, although the importance of clathrin-mediated endocytosis in cell-cell signaling and cell morphogenesis is well known, the roles of clathrin-dependent transport from organelles in development are less clear. Using Clc-GFP, this study has shown that in developing spermatids many clathrin-positive vesicular structures are localized in the vicinity of the Golgi. Furthermore, these structures contained AP-1 adaptors, suggesting that they are Golgi-derived CCVs. In aux mutant cells, the distribution of these clathrin structures and their colocalization with AP-1 are disrupted, implying that formation of these Golgi-derived clathrin-positive vesicles requires auxilin. This conclusion is further strengthened by localization of dAuxFL-mRFP at the Golgi. The presence of abnormal clathrin-positive structures, along with the deficit in plasma membrane formation, suggests that Golgi-derived CCVs act as intermediates to provide membrane for the cell surface during spermatid differentiation. In this scenario, the amount of membrane transported from the Golgi to the cell surface is expected to be reduced in aux mutants. Indeed, cytokinesis and spermatid elongation, two processes requiring significant increase in cell surface area, are affected in aux mutants (Zhou, 2011).

The role of auxilin family proteins at the TGN remains unclear. Given the well-established role of auxilin in disassembling clathrin coats, aux mutations might block the transit of Golgi-derived vesicles by inhibiting removal of their clathrin coats. If this scenario is correct, the disrupted colocalization of clathrin and AP-1 in aux mutant cells would imply that disassembly of AP-1 from the newly formed vesicles does not require auxilin. Alternatively, this loss of colocalization of clathrin and AP-1 could suggest that Aux has a role in facilitating the interaction between clathrin and AP-1 at the TGN. In this scenario, its involvement could be direct (e.g., stabilizing binding between clathrin and AP-1), as Aux contains motifs capable of interacting with both clathrin and adaptors. Alternatively, since it was previously shown that clathrin forms aggregates in aux mutant cells (Kandachar, 2008), it is possible that clathrin in these aggregates is incapable of interacting with AP-1. Kametaka (2007) has shown that, in HeLa cells, AP-1 recruits GAK to TGN, and the presence of GAK at the TGN is required for lysosomal trafficking. It is possible that, in Drosophila germ cells, localization of Aux to the Golgi also relies on AP-1 and this recruitment is required for formation of CCVs (Zhou, 2011).

The mechanistic link between clathrin-dependent trafficking and IC migration is less clear. As IC migration excludes cytoplasmic content during individualization, the plasma membrane of spermatids also becomes constricted. It was originally thought that clathrin might mediate this decrease in the cell surface by endocytosis. However, no Clc-GFP was detected within the ICs, where membrane constriction is expected to occur. Furthermore, genetic and pharmacological manipulations have previously shown that endocytosis and exocytosis have no direct role in IC migration during individualization. It is thus speculated that, although the ICs were scattered, aux mutations might not have a direct role in IC organization or migration. Instead, aux mutations might have disrupted an event prior to individualization that would affect IC organization or migration later on. Indeed, it was shown that, in aux mutant germ cells, the plasma membrane is not properly formed, even before IC assembly. It is proposed that this aberrant plasma membrane would impede subsequent IC movement, resulting in IC scattering (Zhou, 2011).

It has long been appreciated that the cell surface of spermatids increases significantly during differentiation. It is proposed that auxilin-dependent membrane trafficking from the Golgi is required to sustain this expansion of the plasma membrane. As clathrin and Lva are also implicated in cellularization during embryogenesis, this mechanism of clathrin-dependent membrane addition during cell separation is probably conserved. Given the large size of Drosophila male germ cells, differentiation of spermatids could be a useful paradigm to dissect genes required for these cell morphogenetic events (Zhou, 2011).

AP-1 controls the trafficking of Notch and Sanpodo toward E-cadherin junctions in sensory organ precursors

In Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. This study shows that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, these data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling (Benhra, 2011).

The dorsal thorax of Drosophila pupae, the notum, is a single-layered neuroepithelium that produces epidermal and sensory organ (SO) cells. Each adult SO is composed of four cell types and is derived from a single cell, the sensory organ precursor (SOP, also called the pI cell). Notch regulates binary cell fate decisions in the SO lineage. Each SOP undergoes asymmetric cell division to generate two distinct daughter cells; Notch is activated in the SOP daughter cell that adopts the pIIa fate and is inhibited in the other cell, which becomes a pIIb cell. The pIIa cell divides to generate the external cells of the SO, the shaft and socket cells. The pIIb cell undergoes two rounds of asymmetric cell division to generate the internal cells of the SO, the neuron, the sheath cell, and a glial cell. Although Notch-mediated binary cell fate decision in the SO lineage is tightly controlled by intracellular trafficking, the exact subcellular location of where Notch ligand and receptor interact to produce a signal is subject to debate (Benhra, 2011).

To identify new regulators of Notch signaling involved in intracellular trafficking, a double-stranded RNA (dsRNA) screen was carried out for genes affecting SO development and the clathrin adaptor AP-1 complex was identified. AP-1 is an evolutionarily conserved heterotetrameric complex. Drosophila AP-1 complex is composed of AP-1γ (CG9113), β-adaptin (CG12532), AP-1μ1 (encoded by AP-47, CG9388), and AP-1σ (CG5864) subunits. Although mammalian AP-1 is involved in lysosome-related organelle (LRO) biogenesis and in polarized sorting of membrane proteins to the basolateral plasma membrane, the function of Drosophila AP-1 remains largely unknown. Each wild-type SO contains only one socket cell. In contrast, tissue-specific gene silencing of any of the three AP-1 specific subunits, AP-47, AP-1γ, or AP-1σ, gives rise to a Notch gain-of-function phenotype that results in a pIIb-to-pIIa cell fate and/or a shaft-to-socket cell transformation, leading to an excess of socket cells. Following knockdown of AP-1 subunits, 4% to 17% of SO show more than one socket cell. To confirm and extend these dsRNA-induced results, classical mutants were analyzed. Two mutations in AP-47, AP-47SHE11, and AP-47SAE10 were previously recovered as genetic modifiers of presenilin hypomorphic mutations. This stud characterized the AP-47SHE11 allele as a genetic null, whereas the second allele, AP-47SAE10, is hypomorphic. AP-47SHE11/Df(3R)Excel 6264 transheterozygotes die at early first-instar larvae stage, indicating that, as in worms, zebrafish, and mice, AP-47 is essential for viability. To assess the AP-47 loss-of-function phenotype in SO, AP-47 mutant mitotic clones were generated and analyzed in the notum. The same two categories of transformed mutant organs were observed as in the dsRNA experiments. Cell fate transformation was seen in 11% of the mutant organs and in 17% following AP-47dsRNA. The difference could be due to protein perdurance in the mutant clones induced during development. The incomplete penetrance suggests that a compensatory mechanism could bypass the requirement for AP-1. In any case, the results suggest a requirement for the AP-1 complex in Notch-dependent binary cell fate acquisition (Benhra, 2011).

Excess Notch signaling can arise from either disruption of epithelial cell polarity or defects in partitioning of cell fate determinants at mitosis. Because cell polarity relies on the proper apicobasal sorting of membrane proteins, a process requiring both clathrin activity in mammals, this study has analyzed the localization of various polarity markers in AP-47 mutant clones. The Notch gain-of-function phenotype observed in the absence of AP-1 activity cannot be explained by a disruption of epithelial cell polarity, nor by a defect in the partitioning of the cell fate determinants Numb and Neuralized (Neur) at mitosis. Thus, AP-1 activity may be required after unequal segregation of cell fate determinants, possibly at the pIIa/pIIb cell stage to control Notch signaling (Benhra, 2011).

Defects in the endolysosomal degradation, such as in vps25 and erupted mutant cells, result in a Notch gain-of-function phenotype that is caused by ligand-independent mechanisms. Because AP-1 is involved in the biogenesis of LROs in mammals, genetic interaction tests were devised to determine whether excess signaling caused by loss of AP-47 requires the activity of the Notch ligands Delta and Serrate (Ser). Loss of Delta and Ser signaling causes Notch loss-of-function phenotypes, a lateral inhibition defect and a pIIa-to-pIIb cell fate transformation that results in generation of extra neurons, the opposite phenotype to what is observed in AP-47 mutant clones. Loss of external sensory cells accompanied by an excess of neurons is observed in AP-47 Delta Ser triple mutant clones, a phenotype indistinguishable from that of Delta Ser double mutant clones. The reversal of pIIb-to-pIIa transformation phenotype of AP-47 in AP-47 Delta Ser triple mutant clones demonstrates that Delta and Ser are epistatic to AP-47. This finding indicates that the AP-47 mutant phenotype is ligand dependent (Benhra, 2011).

The activity of Delta in the SO lineage is controlled by Neur-dependent endocytosis. Following endocytosis, Delta is recycled, and its trafficking toward apical microvilli requires Arp2/3 and WASp. Mutations in WASp prevent Notch signaling, resulting in a pIIa-to-pIIb cell fate transformation. Excess Notch signaling is observed in AP-47 WASp clones, as in AP-47 clones. These data demonstrate that AP-47 is required for SO formation even in the absence of WASp. These findings suggest that AP-1 is unlikely to act by preventing Delta recycling and raise the possibility that AP-1 acts on Notch receptor signaling (Benhra, 2011).

Sanpodo (Spdo) is a four-pass transmembrane protein required for Notch signaling in asymmetrically dividing cells. Because mutations in spdo result in reduced Notch signaling, the opposite phenotype to what was observed in AP-47 mutant clones, it could be that AP-1 normally represses Spdo activity. To test this hypothesis, AP-47 spdo double mutant clones were generated and a phenotype was observed that is indistinguishable from that of spdo mutant clones. The reversal of the pIIb-to-pIIa transformation phenotype of AP-47 in AP-47 spdo double mutant clones indicates that AP-1 requires the activity of Spdo to control Notch signaling and suggests that AP-1 might control Spdo trafficking and/or localization (Benhra, 2011).

To test for a role of AP-1 in Spdo localization, the subcellular distribution of Spdo was compared in wild-type and AP-47 SO lineages. In the wild-type SOP, Spdo is found in intracellular compartments. After division, Spdo-positive vesicles remain localized in the pIIb cell as a consequence of the unequal inheritance of Numb during SOP mitosis, whereas Spdo localizes preferentially at the plasma membrane of the posterior pIIa cell. Spdo is also detected at the apical cortex of SOP and pIIa/pIIb cells, albeit at a low level. In contrast, in AP-47 mutant SO cells, Spdo accumulates apically, as well as at the interface between the AP-47 SOP daughter cells, where DE-Cad is present. It is concluded that loss of AP-1 results in the specific accumulation of Spdo at the apical plasma membrane in SO cells, as well as at the level of adherens junction in SOP daughters. It is suggested that this defect in Spdo trafficking could explain the excess Notch signaling (Benhra, 2011).

Because AP-1 is required for proper localization of Spdo, an anti-AP-1γ antibody was generated to investigate the subcellular distribution of AP-1 relative to Spdo. AP-1γ is closely juxtaposed to the trans Golgi network (TGN) marker GalT::RFP and colocalizes partially with Liquid facet related (LqfR; CG42250), the Drosophila ortholog of Epsin related (Epsin-R), recently reported to localize at the TGN. AP-1γ also partially colocalizes with Rab11-positive recycling endosomes (RE). Thus, in epithelial cells of the notum, AP-1 is found on two membrane-bound compartments, the TGN and RE, as previously reported in tissue culture cells. In SOPs, Spdo was previously shown to partially colocalize with Notch, Hrs, and Rab5. This study reports that Spdo also colocalizes with AP-1γ and Rab11-positive endosomes, suggesting that Spdo traffics within the TGN and RE (Benhra, 2011).

Together with the above genetic data, colocalization of AP-1 with Spdo raises the interesting possibility that AP-1 could control the sorting and transport of Spdo. Furthermore, Spdo contains a conserved N-terminal YTNPAF motif that falls into the Y/FxNPxY/F-consensus sorting signal of the LDL receptor whose localization is regulated by clathrin adaptors. If Spdo is an AP-1 cargo, deletion of the sorting motif of Spdo should prevent its interaction with AP-1. To test this prediction, the localization of AP-47-VenusFP (VFP) was analyzed relative to that of Spdo-mChFP versus Spdo-mChFP deleted of its 18 first amino acids containing the YTNPAF motif (SpdoΔ18-mChFP) in the SOP lineage. On average at the two-cell stage, 69% of the AP-47-VFP-positive vesicles are also positive for Spdo-mChFP, whereas only 14% of AP-47-VFP vesicles are positive for SpdoΔ18-mChFP. Thus, the first 18 amino acids of Spdo may be required for its AP-1-mediated sorting. Nonetheless, SpdoΔ18-mChFP does not accumulate at the apical cortex, suggesting that additional sorting motifs or interacting proteins such as Numb, also interacting with Spdo via the YTNPAF motif, contribute to Spdo apical localization. These data reveal that in addition to AP-2, a second clathrin adaptor complex, AP-1, controls the localization of Spdo and regulates Notch signaling. AP-2 and Numb prevent Spdo accumulation at the plasma membrane, whereas AP-1 prevents Spdo accumulation at the apical plasma membrane. Whether AP-1 binds directly to the YTNPAF motif or indirectly via a yet-to-be-discovered clathrin-associated sorting protein (CLASP) like Numb remains unknown. By analogy to Numb and AP-2, the hypothetical CLASP would function together with AP-1 to sort Spdo at the TGN and/or RE (Benhra, 2011).

Based on its localization at the TGN and the RE, AP-1 may ensure sorting of Spdo from the TGN and/or RE. To test whether AP-1 has a role at RE, a functional Spdo construct was generated in which mChFP is inserted in the second extracellular loop of Spdo (SpdoL2::mChFP) and used in a pulse-chase internalization assay with an anti-RFP that recognizes the extracellularly accessible mChFP tag in epithelial cells of the notum. In the control, following a 45 min chase, the anti-RFP has been efficiently internalized and resides primarily in apically localized endosomes. A small pool of anti-RFP is also detected at the level of adherens junctions labeled with DE-cadherin, suggesting that Spdo can be recycled back to adherens junctions, albeit with low efficiency. In cells depleted of AP-1, anti-RFP internalized from the basolateral membrane is efficiently recycled to the adherens junctions, suggesting that AP-1 acts in RE to limit recycling of Spdo toward adherens junctions. In contrast, when AP-2-dependent endocytosis is prevented, anti-RFP remains mostly localized at the basolateral plasma membrane, even after a chase of 45 min, as predicted for a requirement of AP-2 in the internalization of Spdo. Therefore, the data indicate that AP-1 does not regulate endocytosis of Spdo from the basolateral membrane. To test whether AP-1 could regulate apical endocytosis of SpdoL2::mChFP, a pulse-chase internalization assay was conducted in epithelial cells of the wing imaginal discs, a tissue that, in contrast to the pupal notum, allows for access of anti-RFP at the apical plasma membrane. In cells depleted of AP-47, anti-RFP resides predominantly at the apical side at the level of adherens junction at t = 0 and is internalized with similar kinetics as in the control situation. It is concluded that AP-1 does not regulate SpdoL2::mChFP apical internalization. Altogether, these results indicate that AP-1 acts at the RE to prevent or limit apical recycling of Spdo, giving a rationale for why endogenous Spdo accumulates apically in SO mutant for AP-47 (Benhra, 2011).

Does apical accumulation of Spdo cause the Notch gain-of-function phenotype seen in AP-1 mutant SO? Spdo was previously reported to partially colocalize with Notch in large intracellular structures and at the plasma membrane. In wild-type, Notch localizes at the apical membrane of epidermal cells, SOP cells, and SOP daughter cells. Shortly after SOP division, Notch extracellular domain (NECD) is detected apically together with Spdo at the DE-Cad interface between pIIa and pIIb. This specific localization is transient, because NECD and Spdo are detected at the interface of daughter cells in one-third of the cases and are no longer detectable at the pIIa/pIIb interface when the remodeling of the apical cortex of pIIa/pIIb cells takes place. In AP-47 mutant cells, NECD is stabilized with Spdo at the interface of SOP daughter cells, even at a time when control organs have undergone apical cortex remodeling. Similarly, Notch intracellular domain (NICD) is accumulated at the level of adherens junctions in AP-47 mutant cells, whereas it is detected at the interface of wild-type SOP daughters in only half of the cases. To determine whether the stabilization of Notch at the SOP daughter cell interface is caused specifically by AP-47 loss of function, NECD localization was compared in AP-47 versus spdo or AP-47 spdo double mutant clones. Although NECD is enriched at the apical surface in these three mutant situations compared to control cells, stabilization of NECD at the interface of SOP daughter cells occurs in AP-47 single and AP-47 spdo double clones, but not in spdo single clones. These data indicate that, upon loss of AP-47, Spdo is not required for NECD to accumulate at the junction between SOP daughter cells, which raises the interesting possibility that Notch itself may be an AP-1 cargo. Because Spdo and Notch are transiently detected at the interface of wild-type SOP daughter cells, it is proposed that sustained elevated levels of Spdo and Notch at the interface cause the excess signaling observed in AP-47 mutants. These effects of AP-1 appear to be specific to Spdo and Notch, because Delta is transiently detected in punctuated structures at the level of junctions together with Spdo in a similar manner in both control and AP-47 SOP daughter cells. Furthermore, endocytosis of Delta is unaffected by the loss of AP-1. It is thus concluded that AP-1 regulates the amount of Notch and Spdo at this junctional domain, which could serve as a launching pad from which endocytosed Notch ligand is trafficked for signaling (Benhra, 2011).

These data have uncover a novel function for AP-1 complex during development. The observations suggest that AP-1 participates in the polarized sorting of Spdo and Notch from the TGN and/or RE toward the plasma membrane. The correlation between the Notch gain-of-function phenotype and the stabilization of Notch and Spdo at the junctions suggests that adherens junctions may be particularly important for Notch activation. Because the effect of loss of AP-1 on Spdo and Notch localization is completely penetrant, it is proposed that a threshold of Spdo and Notch localized at the junctional domain has to be reached in order to cause the cell fate transformation, explaining why only 10% to 20% exhibit the Notch gain-of-function phenotype (Benhra, 2011).

Previous reports have suggested that trafficking of endocytosed Delta to the apical membrane in the pIIb cell is required for its ability to activate Notch that localizes at the apical side in the pIIa cell. Recently, it was reported that most endocytosed vesicles containing the ligand Delta traffic to a prominent apical actin-rich structure (ARS) formed in the SOP daughter cells. Based on phalloidin staining, the ARS appears to be unaffected by the loss of AP-47. Notch and Spdo are stabilized at the junctional domain that is included within the ARS and are therefore poised to receive the Delta signal. This would place this domain of the ARS as an essential site for Delta-Notch interaction, leading to productive ligand-dependent Notch signaling (Benhra, 2011).

Could this novel function for AP-1 be conserved in mammals? Spdo is specifically expressed in Dipterans, and no functional ortholog has been described so far, raising the question of the role of AP-1 in Notch signaling in mammals. Nonetheless, Notch is also mislocalized in AP-1 mutant cells even when Spdo activity is missing. Notch also contains evolutionarily conserved tyrosine-based sorting signals, and it cannot be excluded at present that Notch is itself an AP-1 cargo. Finally, the facts that Notch controls several early steps of T cell development and that mice heterozygous for γ-adaptin exhibit impaired T cell development raise the interesting possibility that Notch-dependent decisions in mammals also required AP-1 function (Benhra, 2011).

Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. By generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, tests were performed of each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand-expressing cells. First, it was discovered that deletion or mutation of both Ubiquitin interaction motifs (UIM) destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the Epsin-N-terminal homology (ENTH domain) was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, it was found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand-expressing cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. It is proposed that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin (Xie, 2012).

Epsin is a complex multi-modular protein that functions differently in different contexts. Each Lqf isoform has two UIMs, two Clathrin binding motifs (CBMs), seven DPW motifs that bind the AP-2 endocytic adapter complex, and two NPF motifs that bind EH-domain-containing endocytic factors such as Eps15. In C. elegans, Drosophila, and mice, Epsin is needed specifically in Notch ligand cells. The structure/function analysis of Epsin performed in this study shows that modules of Epsin associate with the internalization step of endocytosis - the lipid binding function of the ENTH domain and the C-terminal modules that bind proteins present in Clathrin-coated vesicles - are required for Epsin's function in Notch ligand cells. In addition, it was shown that a UIM is necessary (Xie, 2012).

The dispensability of the Cdc42 GAP binding function of the ENTH domain suggests that in ligand cells the primary role of Drosophila Epsin, unlike yeast Ent1, is not regulation of actin dynamics. The other known function of the ENTH domain is the endocytic function, and the results suggest that the ability of the ENTH domain to interact with PIP2 explains why it is needed for maximal Epsin function in Notch ligand cells. These observations are consistent with the lack of typical Notch signaling defects in Drosophila cdc42 mutants. In contrast, flies with mutations in genes for either of two actin regulators, the Arp2/3 complex and WASp, do have notal bristle defects indicative of Notch signaling failure. The notal bristle phenotype described in this study is not due to failure of the Epsin-dependent endocytosis of ligand that activates Notch in all cell types, but instead to failure of ligand transcytosis required in only some cell types to relocalize ligand prior to signaling. The absence of the Arp2/3 complex or WASp in mutants inhibits signaling by blocking traffic of endocytosed Delta to apical microvilli of sensory organ precursors. Whether or not Delta transcytosis in sensory organ precursors also depends on Epsin is unknown. If Epsin is involved, it may be interesting to use the Epsin variant transgenes generated in this study to determine whether or not the Cdc42 GAP interaction function of the ENTH domain is required (Xie, 2012).

There are two types of UIMs: single-sided UIMs that bind one Ubiquitin, and double-sided UIMs that bind two Ubiquitins simultaneously. As the affinity between a UIM and Ubiquitin is low, successful interaction between a mono-ubiquitinated protein and a UIM-containing protein is thought to require either one double-sided UIM, or two single-sided UIMs. Epsins have single-sided UIMs, and so the observation that only one single-sided UIM is required for Drosophila Epsin function in Notch signaling is unexpected. The simplest explanation is that Notch ligands use multiple mono-Ubiquitins or Ubiquitin chains as a signal for Epsin-mediated internalization (Xie, 2012).

Two distinct Lysine residues in the intracellular domains of both Delta and Serrate have been implicated as important for the function of each ligand. In the case of Serrate, simultaneous mutation of both of these Lysines results in a Serrate ligand that can neither activate Notch nor be endocytosed in wing discs. These observations identify two particular Lysines as candidates for the critical Ub attachments, but do not distinguish whether one or both Lysines are required. In the case of Delta, single mutation of either of two specific Lysines results in accumulation of Delta at the cell surface of eye discs and failure to signal. Although Delta is thought to be mono-ubiquitinated, these results suggest the possibility that Delta is multiply mono-ubiquitinated. An alternative explanation for Epsin's ability to promote ligand endocytosis with a single UIM is that mono-ubiquitinated ligands cluster to generate an environment where multiple Ubiquitins attract Epsin to ligand at the plasma membrane (Xie, 2012).

There is compelling evidence that in somatic cells, Notch ligand endocytosis associated with signaling is Clathrin-dependent. First, there are exceedingly strong genetic interactions between the Clathrin heavy chain (Chc) gene and lqf, the gene for Epsin. Flies with only one Chc+ gene copy are wild-type, but this condition is lethal in homozygotes for a normally viable hypomorphic allele of lqf. Second, the Clathrin-coated vesicle uncoating protein Auxilin is, like Epsin, required specifically for Notch signaling in Drosophila and in ligand cells. Given the clear involvement of Clathrin and the lack of strong genetic interaction between α-Adaptin (the gene for an AP-2 subunit) and lqf, the simplest model for Epsin function in Notch signaling was as an adapter protein that links Clathrin and the plasma membrane, independent of AP-2. This model predicted that direct interaction between Epsin and Clathrin would be necessary, and thus the most surprising result of this work is that deletion of the CBMs had no detectable effect on Epsin activity. The dispensability of the CBMs rules out models where Epsin acts as a monomeric Clathrin adapter that links ligand to Clathrin cages (Xie, 2012).

In the Drosophila female germline, Notch signaling requires Epsin but neither Clathrin nor Auxilin. Although this is surprising, Epsin has been shown to function in Clathrin-independent internalization of ubiquitinated transmembrane cargos in vertebrate cell culture. Epsin must therefore function differently in Notch signaling in the female germline than in somatic cells. It is speculated that the ENTH domain and UIMs may be required in germline cells to guide the ubiquitinated proteins into Q6 an endocytic vesicle. However, it is not clear how any of the characterized modules within Epsin's C-terminus might be involved in Clathrin-independent endocytosis. It would be of interest to use the transgenes that were generated in this study to determine which motifs are required in the female germline. Additional experiments could potentially identify unknown C-terminal interaction motifs used in Clathrin-independent endocytosis (Xie, 2012).

Does Epsin function in the same way in the embryo, eye, and wing? The experiments began with the assumption that Epsin functions through the same mechanism in all signaling contexts, and thus it was expected the same Epsin modules would be required for Epsin function in all contexts. Epsin appears to be required in every Notch signaling event and thus could be regarded as a core component of Notch signaling. It therefore seems reasonable to expect that Epsin would function in the same manner in all tissues. The female germline is apparently an exception. Nevertheless, in the three assays used for Epsin activity - rescue of lethality and eye morphology defects due to lqf mutations and rescue of the ability of lqf null cells to activate Cut expression in cells at the D/V boundary in the wing disc - only subtle differences were detected between the eye and the wing in the activity of two Epsin variants, δENTH and δUIM. (The only major difference was with the highly artificial Epsin variant, 4XNPF.) Despite these differences, it is thought that Epsin likely functions the same way in the eye and wing, as well as during embryogenesis. For one, the differences in activity that were observed be explained easily without invoking different mechanisms for Epsin in the eye and wing. Importantly, no even one case was observed where modules were essential in one context (embryogenesis, eye, or wing development) and dispensable in another one. In fact, it is possible to observe all-or-none differences in requirements for Epsin modules. Epsin was found to function outside of Notch ligand cells and modules were found that were dispensable completely in this context yet absolutely essential for Epsin's function in ligand cells (Xie, 2012).

Notch ligands require ubiquitination and (usually) Clathrin-dependent endocytosis, and formation of Clathrin-coated vesicles requires adapter proteins that link the plasma membrane with Clathrin. The absolute necessity of at least one UIM and the observation that the lipid-binding function of the ENTH domain plays a role in ligand cells suggests that Epsin indeed binds ubiquitinated Notch ligands at the plasma membrane.However, as an Epsin derivative lacking CBMs functions as well as wild-type Epsin in ligand cells, the essential role of Epsin in Notch signaling cannot be as a monomeric Clathrin adapter that links Clathrin directly to ligand at the plasma membrane. As any pair of the three types of modules is sufficient for Epsin function (CBMs+DPWs, CBMs+NPFs, or DPWs+NPFs), Epsin must be able to support Notch activation by linking ligand to Clathrin in a variety of different ways. It is speculated that Eps15, the second Drosophila Epsin, is involved because of the three EH-domain proteins in Drosophila (Eps15, Dap160, Past1), none have Clathrin binding motifs, and Eps15 is the only one with motifs for a known Clathrin-binding protein (AP-2). From analysis of mutant phenotypes and genetic interaction studies, there is no evidence for the involvement of Eps15 nor AP-2 in Notch signaling (Xie, 2012). The results presented in this study suggest that Eps15 and AP-2 may play redundant roles in the presence of intact Epsin and this idea could be tested with additional genetic experiments. In light of the evidence indicating a requirement for Clathrin in ligand cells (outside of the germline), the results suggest that Epsin is required absolutely for Notch signaling not because it generates a special endocytic environment, but simply because it is the only UIM-containing endocytic protein with the appropriate complement of interaction modules to target ubiquitinated cargo to Clathrin-coated vesicles (Xie, 2012).

The functions of auxilin and Rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. This study used Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, Rab11 was found to be dispensable for several Notch signaling events in the eye disc. Second, Drosophila female germline cells, the one cell type known to signal without clathrin, was also found not to require auxilin to signal. Third, much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling (Banks, 2011).

Rab11 is not required for several Notch signaling events in the developing Drosophila eye that require epsin and auxilin. Thus, as in the female germline cells, ligand recycling, at least via a Rab11-dependent pathway, is not necessary for Notch signaling in the eye disc. The one Notch signaling event presently known to be clathrin-independent is also auxilin-independent. This result reinforces the idea that rather than performing some obscure function, the role of auxilin in Notch signaling cells is to regulate clathrin dynamics. Overexpression of both clathrin heavy chain and epsin rescues to nearly normal the severely malformed eyes and semi-lethality of aux hypomorphs. Presumably, vesicles uncoated of clathrin fuse with the sorting endosome, and so it seems reasonable to assume that uncoating clathrin-coated vesicles containing ligand is preprequisite for trafficking ligand through endosomal pathways. Thus, if ligand endocytosis is prerequisite to recycling, efficient production of uncoated vesicles would be required. In aux mutants with severe Notch-like mutant phenotypes, clathrin vesicle uncoating is inefficient. It is presumed that this remains so even when clathrin and epsin are overexpressed, yet the eye defects and lethality are nearly absent. Thus, it is reasoned that auxilin is required not for efficient production of uncoated vesicles per se, but for the other product of auxilin activity - free clathrin (and possibly also free epsin). Taken together, these results argue strongly that at least in some cell types, the fundamental role of Notch ligand endocytosis is not ligand recycling (Banks, 2011).

Is it possible that the fundamental mechanism of Notch signaling is so completely distinct in different cell types, that ligand endocytosis serves only to activate ligand via recycling in some cellular contexts, and only for exerting mechanical force on the Notch receptor in others? While formally possible, this is not parsimonious. Thus, a model is favored where the fundamental role of ligand endocytosis is to exert mechanical force on the Notch receptor. In addition, some cell types will also require ligand recycling. As no altered, activated form of ligand has yet been identified, while ligand transcytosis has been well-documented , the most likely role of recycling is to relocalize ligand on the plasma membrane prior to Notch receptor binding (Banks, 2011).

Clathrin is required for Scar/Wave-mediated lamellipodium formation

The Scar/Wave complex (SWC) generates lamellipodia through Arp2/3-dependent polymerisation of branched actin networks. In order to identify new SWC regulators, a screen was conducted in Drosophila cells combining proteomics with functional genomics. This screen identified Clathrin heavy chain (CHC) as a protein that binds to the SWC and whose depletion affects lamellipodium formation. This role of CHC in lamellipodium formation can be uncoupled from its role in membrane trafficking by several experimental approaches. Furthermore, CHC is detected in lamellipodia in the absence of the adaptor and accessory proteins of endocytosis. It was found that CHC overexpression decreased membrane recruitment of the SWC, resulting in reduced velocity of protrusions and reduced cell migration. By contrast, when CHC was targeted to the membrane by fusion to a myristoylation sequence, an increase was observed in membrane recruitment of the SWC, protrusion velocity and cell migration. Together these data suggest that, in addition to its classical role in membrane trafficking, CHC brings the SWC to the plasma membrane, thereby controlling lamellipodium formation (Gautier, 2011).

The dual screen using Drosophila cells was aimed at identifying novel regulators of the SWC involved in lamellipodium formation. Surprisingly, this screen identified CHC, a major coat protein involved in membrane trafficking. A molecular interaction between the Sra1 subunit of the SWC and CHC was recently reported by Anitei (2010). However, that study found that this interaction played a role in the generation of tubular carriers derived from the TGN, a classical function for clathrin. By contrast, the atypical function of CHC in lamellipodium formation reported in this study seems to be independent from its well-established role in membrane trafficking. The evidence against an indirect effect on lamellipodium formation through defective membrane trafficking is threefold. (1) It was possible to uncouple the two functions of CHC, using adaptor depletions or BFA. These experiments impaired trafficking, but not lamellipodium formation. (2) Conversely, overexpression of CHC impaired lamellipodium formation, but not trafficking. (3) CHC was detected in lamellipodia without the adaptor and accessory proteins mediating endocytosis. The two functions associated with the complex formed by CHC and SWC, generation of carriers from the TGN and lamellipodium formation, thus appear distinct, even though they share components. These shared components provide a simple explanation of why AP1-depleted cells spread slightly more than control cells in response to Rac, because the SWC, which is no longer recruited to the TGN in AP1-depleted cells, can then perform its function at lamellipodia (Gautier, 2011).

The novel function of clathrin in controlling lamellipodia adds to the case for unconventional roles of clathrin. Indeed, clathrin was previously shown to stabilise the mitotic spindle and to mediate p53-dependent transcription. This latter role is also independent of the light chains, as is the role of CHC in lamellipodium formation described in this study. CHC promotes lamellipodium formation through membrane recruitment of SWC. Indeed, experiments using Myr-CHC suggest that CHC is able to bring the SWC to the membrane. In line with this idea, CHC depletion, or overexpression, greatly decreases the amount of SWC in the membrane pool. This role of clathrin in the recruitment of the SWC to the plasma membrane might explain the recent finding that clathrin is required for actin polymerisation at the immunological synapse (Calabia-Linares, 2011), a structure that also depends on SWC activity (Gautier, 2011).

The recent success of reconstituting the activation of purified SWC in vitro using prenylated Rac and liposomes containing PtdIns(3,4,5)P3 suggest that clathrin is not absolutely required to induce and maintain the active conformation of the SWC (Lebensohn, 2009). The situation is analogous to the one described for BAR-domain-containing molecules of the IRSp53 family, which are crucial in vivo to deform the plasma membrane, to recruit and activate the SWC, but which are similarly dispensable in in vitro assays. These results suggest that, even though actin dynamics have been beautifully reconstituted in vitro, the complexity of a lamellipodium, especially its membrane dynamics, has not yet been fully understood and recapitulated in vitro (Gautier, 2011).

It is striking that a major component of the endocytic machinery such as CHC should also be involved in SWC activation and in the formation of lamellipodia. Protrusion of the plasma membrane through actin polymerisation and membrane retrieval through endocytosis are antagonistic. Indeed, biophysical experiments have revealed that either one of these events, but never an unproductive combination of the two, is triggered by the same stimulus, a decrease in membrane tension. The interaction between CHC and SWC might thus be involved in this coordination by locally shutting down endocytosis in membrane protrusions (Gautier, 2011).

LRRK2 localizes to endosomes and interacts with clathrin-light chains to limit Rac1 activation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of dominant-inherited Parkinson's disease (PD), and yet the physiological functions of LRRK2 are not fully understood. Various components of the clathrin machinery have been recently found mutated in familial forms of PD. This study provides molecular insight into the association of LRRK2 with the clathrin machinery. Through its GTPase domain, LRRK2 binds directly to clathrin-light chains (CLCs). Using genome-edited HA-LRRK2 cells, LRRK2 was localized to endosomes on the degradative pathway, where it partially co-localizes with CLCs. Knockdown of CLCs and/or LRRK2 enhances the activation of the small GTPase Rac1, leading to alterations in cell morphology, including the disruption of neuronal dendritic spines. In Drosophila, a minimal rough eye phenotype caused by overexpression of Rac1, is dramatically enhanced by loss of function of CLC and LRRK2 homologues, confirming the importance of this pathway in vivo. These data identify a new pathway in which CLCs function with LRRK2 to control Rac1 activation on endosomes, providing a new link between the clathrin machinery, the cytoskeleton and PD (Schreij, 2014).

Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster

Targeting of proteins to their final destination is a prerequisite for living cells to maintain their homeostasis. Clathrin functions as a coat that forms transport carriers called clathrin-coated vesicles (CCVs) at the plasma membrane and post-Golgi compartments. This study established an experimental system using Schneider S2 cells derived from Drosophila as a model system to study the physiological roles of clathrin adaptors, and to dissect the processes of CCV formation. It was found that a clathrin adaptor Drosophila GGA (Golgi-localizedγ-adaptin ear containing, ARF binding protein or dGGA), a homolog of mammalian GGA proteins, localizes to the trans-Golgi network (TGN) and is capable of recruiting clathrin from the cytosol onto TGN membranes, through direct protein interaction. dGGA itself is recruited from the cytosol to the TGN in an ARF1 small GTPase (dARF79F)-dependent manner. dGGA recognizes the cytoplasmic acidic-cluster-dileucine (ACLL) sorting signal of Lerp (lysosomal enzyme receptor protein), a homolog of mammalian mannose 6-phosphate receptors. Moreover, both dGGA and another type of TGN-localized clathrin adaptor, AP-1 (adaptor protein-1 complex), are shown to be involved in the trafficking of Lerp from the TGN to endosomes and/or lysosomes. Taken together, these findings indicate that the protein-sorting machinery in fly cells is well conserved relative to that in mammals, enabling the use of fly cells to dissect CCV biogenesis and clathrin-dependent protein trafficking at the TGN of higher eukaryotes (Kametaka, 2010).

The trans-Golgi localization of dGGA suggested that this protein plays a role in the formation of clathrin-coated transport intermediates that transport integral membrane cargo proteins from the TGN to endosomes. To assess the involvement of clathrin in this process, an antibody to dCHC was raised. To examine the contribution of dGGA to the recruitment of dCHC onto the trans-Golgi, in vitro pull-down assays were performed to see whether dGGA interacts with dCHC. A series of dGGA recombinant proteins was prepared as GST-fusion proteins. The full-length dGGA, Hinge and GAE region, and Hinge region efficiently pulled-down dCHC from a cytosolic extract from S2 cells, whereas VHS-GAT pulled-down dCHC at levels close to the control. Thus, the binding site maps to the hinge region (amino acid position 292-590 in dGGA). Mutations were introduced in two putative clathrin binding sequences, D331LL to DAA and/or E327LL to EAA , and in a putative ACLL motif, D496VPLL to DVPAA. That mutations in the two putative clathrin-binding boxes were observed to strong affect the interaction with dCHC, whereas the putative ACLL motif was dispensable for this interaction. These results indicate that the D331LL and E327LL sequences in the hinge region of dGGA have crucial roles in interaction with dCHC. Golgi-association of clathrin heavy chain (Kametaka, 2010).

The intracellular localization of dCHC was also examined in S2 cells. The antibody preferentially labeled large perinuclear puncta and smaller dots beneath the cell surface. The dCHC-positive, perinuclear large structures were juxtaposed to p120-positive structures and colocalized with the signal for HA-dGGA, indicating that they correspond to the trans-Golgi compartments. By contrast, the small peripheral dots colocalized with AP50, a mu2 subunit of AP-2, indicating that they represent the plasma-membrane-associated clathrin-coated pits. These results indicated that a significant amount of cellular clathrin localizes to the trans-Golgi, together with dGGA (Kametaka, 2010).

In mammals, expression of the VHS-GAT domains of mammalian GGAs has been previously shown to exert a dominant-effect on clathrin localization and MPR sorting at the TGN (Puertollano, 2001). Thus, to further assess the role of dGGA vis-à-vis clathrin in vivo, the effect of overexpression of dGGA-VHS-GAT on clathrin localization was examined in S2 cells. Overexpression of the truncated dGGA reduced dCHC association with the Golgi compartment. Together with the above GST-pull down experiments, these results strongly suggest that dGGA has a role in clathrin recruitment at the TGN (Kametaka, 2010).

Genome sequencing projects have revealed the conservation of clathrin adaptor genes throughout the eukaryotic kingdom, including well-established model organisms such as Drosophila and C. elegans. Drosophila is a particularly appealing organism for analysis of GGA function because of the existence of a single dGGA, the ease of RNAi approaches and the conservation of Golgi function. Although the morphology of the Drosophila Golgi is different from that of mammals, the basic features of the organelle (e.g., polarity, number of cisternae per stack and function in the secretory pathway) are quite similar (Kondylis, 2003; Kondylis, 2005). The molecular machineries responsible for protein sorting from the Golgi complex to the endosomal system, however, have not been characterized in Drosophila. Dennes (2005) showed that Lerp, a Drosophila homolog of the mammalian CI-MPR, could rescue the defects in the lysosomal cathepsin sorting in MPR-deficient fibroblasts. However, the properties and functions of Lerp and its putative adaptor dGGA in Drosophila cells have remained uncharacterized. In this study Drosophila S2 cells were used to assess the role of dGGA and its regulators in the trafficking of Lerp (Kametaka, 2010).

The results indicate that dGGA meets the requirements to be a clathrin adaptor for membrane cargo molecules such as Lerp. Its ability to interact with GTP-ARF1, the ACLL motif of Lerp and the dCHC all imply that dGGA functions in CCV formation for trafficking of Lerp between the TGN and endosomal compartments. In addition to the molecular interaction between dGGA and Lerp in vitro, it was also observed that mCherry-tagged Lerp and EGFP-dGGA depart together from the Golgi in vesicular structures in living cells. Moreover, a dominant-negative form of dGGA caused redistribution of clathrin from the Golgi complex and also inhibited recruitment of clathrin in vitro. Furthermore, dGGA knockdown caused a decrease in the level of Lerp processing at endosomal and/or lysosomal compartments in vivo. Thus, dGGA is likely to function at the exit step of Lerp from the TGN. These assays, however, do not allow precise determination of dGGA function at a molecular level. More extensive biochemical and in vivo functional analyses will be required (Kametaka, 2010).

Despite the overlapping localization and common biochemical features of GGAs and AP-1, they are thought to function differently in mammalian cells. It has been shown that AP-1 and cargo molecules, but not GGAs, are concentrated in purified CCV fractions (Hirst, 2000). Moreover, knockdown of GGAs or AP-1 in mammalian cells causes only slight missorting of pro-cathepsin D to the extracellular space without detectable perturbation in the localization of its sorting receptors, MPRs. On the basis of these findings, it has been presumed that GGAs and AP-1 function cooperatively, but not at exactly the same step in cargo trafficking. Indeed, Kornfeld's group proposed a 'hand-off' model in which GGAs first prime the cargo molecule and AP-1 is subsequently recruited through direct interaction with GGAs (Bai, 2004). GGAs are thus replaced by AP-1 to execute clathrin-coated carrier formation (Ghosh, 2003). In this model, GGA is displaced by AP-1 through a phosphorylation state-dependent structural change. Internal ACLL motifs in the hinge region of human GGA1 and GGA3 are thought to mediate this structural change (Doray, 2002). Through the characterization of dGGA and Drosophila AP-1 (dAP-1) in the current study, it was found that many structural and functional features of these adaptors were strikingly conserved from mammals to fly. Like mammalian AP-1, dAP-1 was shown to localize to the TGN in S2 cells and the Golgi localization was sensitive to BFA. Moreover, knockdown of AP47, which encodes a mu1 subunit of dAP-1, accentuated the defect in processing of Lerp seen in dGGA-depleted cells, even though knockdown of AP47 itself had little effect. These observations support the notion that dGGA and dAP-1 function closely together in Lerp trafficking in S2 cells (Kametaka, 2010).

It was also noticed that dGGA has one putative internal ACLL sequence in the hinge region. Interestingly, this ACLL motif is preceded by a Ser493 residue that could be a target of casein kinase II (CKII). CKII is known to be involved in the phospho-regulation of CI-MPR trafficking in mammals through phosphorylation of the cytoplasmic tail of CI-MPR, human GGA1 and GGA3, and PACS-1. Although this idea needs further assessment in the future, the dGGA signal was often observed as a doublet on immunoblotting, so dGGA might be under phospho-regulation control like the mammalian GGAs. Taken together, these findings strengthen the idea that Drosophila possesses similar mechanisms of CCV formation at the TGN and of regulation of clathrin adaptors (Kametaka, 2010).

This is the first report that the putative lysosomal enzyme receptor Lerp and its sorting proteins dGGA and dCHC localize to trans-Golgi compartments in Drosophila cells, where Lerp is believed to be sorted and packaged into CCV destined for endosomal compartments. It was also found that the entire molecular system responsible for post-Golgi protein trafficking in Drosophila is highly conserved relative to that in mammals. This conservation should enable genome-wide screens for novel factors involved in the complex processes of CCV formation and regulation of protein sorting at the TGN. Recently, it has been shown with isolated CCVs that double knockdown of dGGA and dAP-1 causes significant reduction of Lerp incorporation into the CCVs in Dmel2, one of another Drosophila cell lines (Hirst, 2009). Although the molecular relationship of these clathrin adaptors needs to be assessed more carefully, these results support the current results showing the physiological consequence of dGGA in cargo sorting at the trans-Golgi. Thus, this approach using the insect systems will lead to a better understanding of how those clathrin adaptors are important in the development of multicellular organisms and in the molecular basis for lysosomal diseases in higher organisms (Kametaka, 2010).

The Disabled protein functions in Clathrin-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Members of the Disabled (Dab) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric Clathrin-associated sorting proteins that recruit cargo proteins into endocytic vesicles. This study reports a Drosophila disabled mutant revealing a novel role for Dab proteins in chemical synaptic transmission. This mutant exhibits impaired synaptic function, including a rapid activity-dependent reduction in neurotransmitter release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila Dab and Clathrin are highly colocalized within two distinct classes of puncta, including relatively dim puncta that are located at active zones and may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins, including Dynamin, supported a general role for Clathrin-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool (Kawasaki, 2011).

These results reveal a function for the Disabled family of Clathrin-associated sorting proteins (CLASPs) in synaptic vesicle endocytosis and further define the molecular basis for a rapid role of endocytic mechanisms in sustaining neurotransmitter release during synaptic activity (Kawasaki, 2011).

By revealing a function for Dab proteins in synaptic vesicle endocytosis, the present study has implicated a novel molecular component as well as an established set of Dab protein interactions in this process. dDab function in synaptic vesicle endocytosis appears to involve interactions with Clathrin, and possibly AP-2, which are likely to be mediated by conserved binding motifs. In addition, the PTB/DH domain of Dab proteins binds phosphoinositides, which are known to play an important role in synaptic vesicle trafficking. Finally, and importantly, the CLASP function of Dab proteins involves PTB/DH domain binding to a sorting motif (NPxY) in the cytosolic domain of cargo proteins (Yun, 2003). An initial survey of Drosophila synaptic vesicle proteins revealed an NPxY motif in the cytoplasmic domain of Drosophila Synaptotagmin 1 (dSyt1). Syt1 proteins play an important role in synaptic transmission by serving as a calcium sensor for neurotransmitter release and also function in synaptic vesicle endocytosis. The dSyt1 NPxY motif (residues 387-390; NPYY), which is identical in mammalian Syt1 proteins and conserved in other Syts, is located within the C2B domain near a basic region previously implicated in SYT1-AP-2 interactions. This motif is of great potential interest because no classic endocytosis signals have been identified previously in Syts. Finally, it is of interest to consider the roles of co-active-zone and non-active-zone populations of dDab (and Clathrin). The rapid-onset synaptic phenotypes observed are likely to reflect localization and function of dDab and Clathrin at the active zone. Further study is required to address whether non-AZ domains may mark an endosomal compartment from which Clathrin-mediated vesicle formation may occur. Thus far, immunocytochemistry with markers for several endosomal membrane compartments did not show strong colocalization with dDab and Clathrin (Kawasaki, 2011).

The preceding considerations raise interesting questions about how the loss of specific dDab molecular interactions may contribute to the resulting synaptic phenotype. It seems unlikely that the dab synaptic phenotype reflects simple mis-sorting of a synaptic vesicle protein, given the similar phenotypes associated with loss of function for several different endocytic proteins. Rather, as described in the following section, it appears that common features of these phenotypes, including a rapid activity-dependent reduction in neurotransmitter release, slowed recovery in paired-pulse depression (PPD), enlarged membrane cisternae, and persistence or accumulation of AZ-associated and docked synaptic vesicles, reflect a general loss of endocytic function. Consistent with this interpretation, dSyt1 exhibited a WT distribution at dab mutant synapses. Thus, either dDab does not participate in dSyt1 sorting or sufficient redundancy is provided by interactions of Syt1 with at least two other Clathrin-associated adaptor proteins, AP-2 and Stonins (Dittman, 2009; Maritzen, 2010). Furthermore, the distributions of other synaptic vesicle proteins at dab mutant synapses, including neuronal Synaptobrevin and the vesicular glutamate transporter, were similar to those of WT. Thus, synaptic vesicle composition appears to be preserved in dab. These findings are consistent with the WT excitatory postsynaptic currents observed in response to the first stimulus and complete recovery in PPD, as expected for the presence and recovery of a fully functional release-ready vesicle pool (Kawasaki, 2011).

Finally, dab mutant synapses exhibit strong depression during prolonged stimulation but sustain a reduced steady-state level of neurotransmitter release. This is in contrast to the Dynamin mutant, shiTS1, in which EPSC amplitudes progressively decline to zero. In light of the severe nature of the molecular lesion in dabEC1, these findings suggest persistence of residual synaptic vesicle endocytosis in the absence of dDab. This likely reflects redundancy in the mechanisms of synaptic vesicle endocytosis, as shown for several other endocytic proteins (Kawasaki, 2011).

A previous analysis in the shibire (Dynamin) mutant demonstrated a rapid activity-dependent reduction in neurotransmitter release that could not be explained simply by the classic role of Dynamin in recycling synaptic vesicles (Kawasaki, 2000). Rather, it was suggested that accumulation of endocytic intermediates at release sites may occlude fast refilling of the release-ready vesicle pool and that their rapid clearance contributes to maintenance of neurotransmitter release during synaptic activity. Recent studies at the Calyx of Held have further established a rapid role for Dynamin and AP-2 in maintaining neurotransmitter release, which preceded formation of endocytic vesicles, and directly demonstrated its requirement for fast refilling of the release-ready vesicle pool. The present study provides several unique insights into the mechanisms by which endocytic processes regulate exocytosis. First, localization of both dDab and Clathrin at the AZ suggests a local role for endocytic mechanisms near release sites. Second, persistence or accumulation of the docked synaptic vesicle pool at AZs indicates a postdocking role for endocytic mechanisms in synaptic vesicle fusion. Third, the similar electrophysiological and ultrastructural phenotypes observed following loss of Dynamin, dDab, or Clathrin function strongly support a general role for Clathrin-mediated endocytic mechanisms in this process. Finally, comparing and combining endocytic loss of function with a SNAP-25 TS mutant suggests that rapid endocytic mechanisms are required for t-SNARE-mediated synaptic vesicle priming (Kawasaki, 2011).

Together with previous work, the results reported here support a working model in which components of the Clathrin-mediated endocytic machinery first interact at the AZ to clear neurotransmitter release sites and subsequently mediate vesicle formation in the PAZ. Key features of this model are discussed in the following text, including the spatial distribution of endocytic proteins and Clathrin-coated vesicle intermediates with respect to neurotransmitter release sites (Kawasaki, 2011).

The observation of dDab, Clathrin, and AP-2 localization to the AZ was greatly facilitated by the ability to examine isolated AZs at DLM neuromuscular synapses and by the restricted spatial distributions of these proteins. Note that these features are distinct from those of larval neuromuscular synapses and that previous studies suggest no rapid role for Dynamin in exoendocytic coupling in this preparation (Wu, 2005). At adult neuromuscular synapses, the rapid functional roles of Dynamin and DAP160 suggest these proteins are also present at the AZ and participate in early stages of Clathrin-mediated endocytosis. However, their broader distribution within boutons makes it more difficult to confirm localization to the AZ. With respect to Dynamin, which is known to complete vesicle formation through membrane fission (Mettlen, 2009), previous studies have shown it is also present and functionally important at early stages of Clathrin-mediated endocytosis (Evergren, 2004; Loerke, 2009). AZ localization of endocytic proteins might suggest a mechanism of release site clearance involving rapid formation of Clathrin-coated vesicles directly from the AZ; however, this mechanism is not favored, primarily because ultrastructural studies indicate that Clathrin-coated vesicles form at PAZ, rather than AZ, regions of the plasma membrane. Although elongated membrane invaginations occur at the AZ in the shibire (Dynamin) mutant during recovery from massive synaptic vesicle depletion, these do not appear to have Clathrin coats. In the present study, smaller membrane invaginations (Ω-structures) observed at the AZ were not Clathrin-coated but often exhibited filamentous connections with the presynaptic dense body (t-bar). These structures occurred at low frequencies that were not significantly different among all genotypes. It remains unclear whether or not they are related to rapid synaptic vesicle endocytosis and how they may contribute to neurotransmitter release (Kawasaki, 2011).

Regarding the spatial distribution of endocytic intermediates relative to AZs, remarkable ultrastructural analysis has defined a sequence of events in synaptic vesicle exocytosis and endocytosis with high time resolution. Vesicle fusion was observed within several milliseconds after a stimulus and appeared to deposit clusters of large particles into the AZ region of the plasma membrane. Particle clusters were maximally abundant at 20 ms after stimulation and disappeared rapidly over the following 200 ms, consistent with a role for rapid clearance of release sites in synaptic vesicle priming and short-term depression. Particle clusters were thought to dissipate after vesicle fusion, and further analysis suggested they may reassemble within Clathrin-coated pits at the PAZ. In contrast, recent superresolution light microscopy studies showed that native synaptic vesicle proteins deposited in the plasma membrane from different vesicles do not mix before endocytosis but, instead, remain in distinct clusters and are retrieved separately. Moreover, plasma membrane-resident synaptic vesicle proteins may tend to be excluded from the AZ, as observed in conventional confocal imaging of the native synaptic vesicle protein Synaptobrevin. In this current working model, endocytic mechanisms facilitate rapid clearance of synaptic vesicle proteins from release sites either within larger assemblies corresponding to single vesicles or within smaller protein complexes that subsequently assemble in the PAZ as Clathrin-coated pits. A parallel process was described in recent work at DLM neuromuscular synapses demonstrating activity-dependent redistribution of t-SNARE proteins from AZ to PAZ regions, which likely reflects their participation in plasma membrane cis-SNARE complexes (Kawasaki, 2009). In comparing these studies, it is of interest to consider the relative contributions of release site clearance and t-SNARE availability to synaptic vesicle priming and whether AZs maintain their distinctive protein composition, as well as functionality of neurotransmitter release sites, through sorting mechanisms that distinguish among different functional or biochemical states of a protein (Kawasaki, 2011).

Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis

E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. This study shows that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis results in cell intercalation defects. A pathway is delineated that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, evidence is provided that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin (Levayer, 2011).

Epithelial tissues have a robust architecture that is essential for their barrier function. This barrier function depends on their ability to build adhesive contacts at adherens junctions through the recruitment and stabilization of E-cadherin (E-cad), β-catenin (β-cat) and α-catenin (α-cat) by actin filaments (F-actin). During development, epithelia are also extensively reshaped by remodelling of cell contacts. This plasticity is essential for morphogenesis during embryogenesis and organogenesis. Work in the past decade showed that this requires force generation by actomyosin networks and their anchoring at cell junctions by E-cad/β-cat/α-cat complexes. Thus, E-cad plays a pivotal role in junction robustness and plasticity by mediating both adhesion (cohesion) and tension transmission (remodelling). Understanding what controls the distribution and dynamics of E-cad/β-cat/α-cat complexes is therefore key to understanding cell packing and the mechanics of tissue morphogenesis. Disruption of this balance marks key steps in the progression of solid tumours. The loss of epithelial organization during the epithelial to mesenchymal transition is an extreme example in which E-cad endocytosis causes the loss of adhesion and tension transmission at the cell cortex (Levayer, 2011).

The early development of the Drosophila embryo is a powerful system to study epithelial morphogenesis. Spatial regulation of force generation by actomyosin networks and force transmission to adhesion by E-cad both contribute to apical cell constriction in the invaginating mesoderm1, and cell intercalation in the elongating ectoderm called the germ band. Germ-band extension (GBE) is driven by cell intercalation in the ventrolateral region, whereby cells exchange neighbours through planar polarized junction remodelling, namely shrinkage of 'vertical' junctions (that is, junctions oriented along the dorsoventral axis). Intercalation is powered by non-muscle Myosin-II (Myo-II): anisotropic actomyosin contractile flows from the medial apical region to 'vertical' junctions drive junction shrinkage. The shortening of vertical junctions is stabilized by Myo-II at the cortex. Actomyosin contractility is transmitted at the cortex by E-cad complexes through β-cat. Interestingly, E-cad/β-cat/α-cat complexes also exhibit a planar polarized distribution complementary to that of Myo-II: E-cad is less abundant in shrinking 'vertical' junctions. This E-cad polarity is also required to orient actomyosin flows to 'vertical' junctions. It is unknown what controls the planar polarized distribution of E-cad. This may depend on Rho kinase (ROCK), which is required for the polarized distribution of Par3 (Levayer, 2011).

This study shows that the planar polarized distribution of E-cad is also controlled by an upregulation of clathrin- and dynamin-mediated endocytosis at adherens junctions, in particular in 'vertical' junctions of intercalating cells. Blocking endocytosis causes the loss of E-cad planar polarization and a block of intercalation. This led to an investigation of the mechanisms that control planar polarized upregulation of clathrin-mediated endocytosis (CME) of E-cad at adherens junctions. Activation of WASP (Wiscott-Aldrich Syndrome Protein) and the Arp2/3 (Actin-Related Protein 2/3) complex by Cdc42 controls the branched actin polymerization that is required for vesicular scission. This study identified an additional pathway controlling the initiation of E-cad endocytosis through the recruitment of the AP2 (Adaptor Protein 2) complex and clathrin. This recruitment is driven by lateral clustering of E-cad that relies on unbranched actin polymerization induced by Dia, and the presence of Myo-II. Dia and Myo-II are both activated by the guanine exchange factor RhoGEF2 (Levayer, 2011).

This study has delineated two distinct roles for actin in E-cad endocytosis. Dia and Myo-II control the initiation of E-cad endocytosis by enrichment of clathrin and AP2 in an E-cad-dependent manner. This is tightly spatially regulated in the ventrolateral region and in 'vertical' junctions during cell intercalation by cortical RhoGEF2 localization, an activator of Dia and Myo-II in Drosophila embryos. This is distinct from the role of branched actin polymerization by Arp2/3, which promotes vesicular scission similarly to dynamin. At later stages of development, this depends on WASP and is controlled by Cdc42, aPKC (atypical protein kinase C) and Cip4 (Cdc42-interacting protein 4. In early embryos, as WASP is inhibited by the JAK/STAT pathway (Janus kinase/signal transducer and activator of transcription), Scar instead plays a critical role in vesicular scission. Inhibition of Arp2/3 in scar mutants and its constitutive activation (artificially induced by myrWASP) did not affect clathrin and AP2 concentration at adherens junctions, unlike Dia. The different tiers of regulation of E-cad endocytosis by Arp2/3 and Dia may reflect different roles for actin in constitutive versus regulated E-cad endocytosis. Certain situations require a rapid change in the rate of endocytosis, and may do so by tuning the rate of initiation by clathrin and AP2. It will be interesting to see whether rapid collapse of adherens junctions during epithelial to mesenchymal transition relies on a similar process (Levayer, 2011).

Crosslinking E-cad with an IgG is sufficient to promote dorsal endocytosis of E-cad by upregulating the concentration of clathrin, similarly to Dia activation, even following inhibition of Dia, Myo-II or RhoGEF2. Considering the highly correlated localizations of E-cad complexes with AP2 and RhoGEF2, it is proposed that Dia and Myo-II control the initiation of E-cad endocytosis by inducing lateral clustering of E-cad, similar to Fc receptor clustering during phagocytosis or nanoclusters of GPI (glycosylphosphatidylinositol)-anchored proteins. This may have been co-opted by the pathogen Listeria, whose entry into epithelial cells requires E-cad endocytosis. This mechanism may also require specific 'priming' of E-cad, by ubiquitylation as in mammals, although these tyrosines are not conserved in flies. Importantly, the mechanism of AP2 recruitment by E-cad remains unknown in all systems (Levayer, 2011).

Inhibition of E-cad endocytosis increased E-cad levels and disrupted its planar polarized distribution. Myo-II also accumulated in the medial apical region of cells. The GBE defects in shi-ts mutants or following clathrin inhibition are the result of the altered distribution of actomyosin tensile forces. E-cad/β-cat/α-cat complexes affect the lateral flow of medial actomyosin pulses and Myo-II polarized junctional accumulation, presumably through the regulation of tension transmission within the medial network and/or at the junctions. The medial accumulation of Myo-II when E-cad endocytosis is inhibited may thus reflect an inhibition of actomyosin flow towards the cortex. These results emphasize the interplay between actomyosin contractile dynamics and E-cad adhesive complexes during epithelial morphogenesis (Levayer, 2011).


Notch down-regulation by endocytosis is essential for pigment cell determination and survival in the Drosophila retina

The clathrin heavy chain is a fundamental element in endocytosis and therefore, in the internalization of several cell-surface receptors through which cells interact with their environment. The only non-lethal mutant allele of the clathrin heavy chain identified to date in metazoans, the Drosophila Chc4, involves the substitution of a residue at the knee region of the molecule that impairs clathrin-dependent endocytosis. This study investigated the consequences of this endocytic defect in Drosophila retinal development and found that it produces an inhibition of programmed cell death in the retinal lattice, followed by widespread death of interommatidial pigment cells once retinal development has been completed. Through genetic interactions and transgenic analyses, Chc4 phenotypes were shown to be cauesed by a Notch receptor gain-of-function, providing a dramatic example of the importance of Notch down-regulation by endocytosis. An increase in Notch signaling is also observed in Drosophila wings in response to the mutant clathrin, suggesting that Notch levels are controlled by clathrin-dependent endocytosis. The implications of these findings are discussed for current models on eye-development and for the role of endocytosis in Notch signaling (Peralta, 2009).

The clathrin heavy chain (CHC) is a highly conserved polypeptide comprised of five functionally distinct domains: a globular N-terminal domain; the proximal leg; the knee domain; the distal leg; and a C-terminal domain (Schmid, 1997). The functional unit of CHC is the triskelion, that is formed by three CHC molecules bound at their C-terminal ends, and by three clathrin light chains (CLC) associated to their proximal legs. The clathrin coat is formed through interactions between the proximal and distal legs of different triskelions, while the N-terminal domain is responsible for binding clathrin-interacting proteins that contain a clathrin-box related sequence (Ybe, 1999). The knee is the only domain of CHC that is not known to participate in any protein-protein interactions (Peralta, 2009 and references therein).

The dominant nature and temperature sensitivity of the hypomorphic Chc4 allele, make it a particularly useful tool to study the biology of the many processes in which clathrin-dependent endocytosis participates. The change of a highly conserved alanine at position 1082 to threonine is responsible for the Chc4 mutation. This alanine lies within a region of the CHC known as the knee that separates the proximal from the distal leg. This region is believed not to interact with other proteins, and is thought to be responsible for much of the flexibility required by CHC to adapt to the bending of the clathrin lattice. Nevertheless, it still remains to be determined if the change in size or charge introduced by the mutant threonine residue in the knee region affects the flexibility of Clathrin legs, and whether this may translate into changes in the rate of assembly or disassembly of the coat that could explain the phenotypes of the Chc4 mutants (Peralta, 2009).

The Chc4 mutation halves the endocytic capacity of Garland cells, a type of nephrocyte with a high rate of endocytosis). The assembly of clathrin in forming the coat can explain the semi-dominant nature of this hypomorphic mutation (Bazinet, 1993), particularly since the mutant and wild-type CHC molecules are mixed in the triskelion of a heterozygous individual, and triskelions with different compositions are mixed in the coat. This might also explain the variable penetrance displayed by the mutation and by the different transgenic lines expressing wild-type or mutant forms of CHC. This variability may reflect the sensitivity to changes in the proportion and/or amount of mutant and wild-type forms of Clathrin (Peralta, 2009).

The reduced endocytic capacity of the mutant becomes lethal at higher temperatures (28°C), although it is sufficient to allow a small percentage of escapers to complete development at lower temperatures. In these escapers, the retina, the sperm production (Fabrizio, 1998), and the digestive system appear to be particularly affected (Peralta, 2009).

Although endocytosis is required at many steps during larval retinal development, the first phenotype in the eye that can be detected in the Chc4 mutant at 25°C is the partial inhibition of interommatidial PCD during pupal retinal development. The correct spacing of the ommatidia depends on the elimination of roughly one third of the interommatidial cells by PCD, leaving nine interommatidial pigment cells (IOPCs) to isolate each ommatidium. The mechanisms that direct interommatidial PCD are not completely understood, but while Notch and the roughest-irregular chiasma C receptor are required for apoptosis, the EGFR-Ras pathway provides a survival signal for IOPCs. Contrary to certain aspects of this model, yet in support of an increase in Notch signaling explaining the Chc4 phenotype, the expression of Notch (NFL) in IOPCs inhibits PCD in the pupal retina, rather than augmenting apoptosis. The same inhibition of PCD was obtained when a constitutively active form of Notch is expressed, or its downstream target, Suppressor of Hairless, as indicated previously. Moreover, an increase in PCD is seen when a dominant-negative form of Notch is expressed . In the pupal retina, Notch is mainly expressed by IOPCs and to a lesser extent by primary pigment cells (PPCs), and the Notch receptor is endocytosed and directed toward the Hrs degradation pathway. Indeed in a mutant in which endocytosis is intensified Notch loss of function phenotypes are enhanced in the Drosophila wing, constituting additional evidence that endocytosis negatively influences Notch signaling. It remains to be explained why the absence and the excess of Notch signals in interommatidial cells, produce a similar phenotype, (i.e., inhibition of PCD and excess of IOPCs). It can only be speculated that the response of the cells to the absence of such signaling might be mediated by a mechanism other than that which responds to changes in the strength of its signal (Peralta, 2009).

Once PCD is completed and the final number and identity of all the retinal cells have been established, all the IOPCs in the Chc4 mutant retina start to die, such that the retina is totally devoid of IOPCs upon completion of this process. The resulting loss of the ommatidial arrangement probably leads to a severe reduction in the capacity to form images, which relies on the precise orientation of the optical elements in the Drosophila neural superposition eye, a type of compound eye where the image is formed in the brain through parallel processing of the signals from multiple ommatidia. The mutant clathrin is responsible for this pigment cell death because it could be rescued by specifically expressing a wild-type copy of CHC in them. The induction of pigment cell death by disrupting endocytosis with a dominant-negative form of dynamin further confirms that this process is dependent on endocytosis (Peralta, 2009).

As in the case of the PCD phenotype, the IOPC death phenotype displayed by the mutant can also be reproduced by the sole expression of Notch (NFL) in wild-type pigment cells. Indeed, NFL enhances the mutant IOPC phenotype when expressed in Chc4 mutant cells, resulting in a more rapid disappearance of IOPCs. Conversely, expression of a dominant-negative form of Notch (NECN), provokes the suppression of the mutant phenotype. These results suggest that although the defect in Chc4 affects endocytosis, the ultimate cause of the IOPC death phenotype was the excess of Notch signaling that ensued. Genetic analysis supports this notion, as two Notch alleles, Nfa-g62 that causes specific loss of Notch function in the pupal retina, and the temperature-sensitive allele Nts1, both completely suppress the demise of IOPCs caused by the Chc4 mutation (Peralta, 2009).

Once the correct number of IOPCs have been specified, Notch lacks an obvious role in late pupal development, as demonstrated by the absence of retinal defects when Notch activity is lost in late pupas after PCD. At this time, the survival signal mediated by EGFR that protects IOPCs during PCD also disappears, consistent with the failure of overexpression of the EGFR negative regulator Argos to produce an effect, similar to the activated form of armadillo from whose deleterious effects on retinal cells are protected by EGFR activity until about P40%. However, the presence of Notch is required for the IOPC death induced by the Chc4 mutation. If Chc4 mutants fail to down-regulate Notch at this time, an increase in the receptor would be expected at the membrane of IOPCs and PPCs. Indeed, Notch is more prominent at the apical membranes between IOPCs and PPCs, and even between PPCs and cone cells (CCs) in Chc4 mutants. At present there is no evidence that the levels or localization of Delta are altered in the Chc4 mutant retina, raising the possibility that membrane-bound Notch receptor could to be the main target for the Chc4 reduced endocytosis (Peralta, 2009).

It has been shown that there is a strict requirement for Delta ligand endocytosis in the signal-sending cell, while an equivalent requirement for Notch receptor endocytosis in the signal-receiving cell has not been demonstrated yet, although a requirement for dynamin in both cells raises that possibility. Surprisingly, Notch signaling continues to take place in Chc4 during the unaffected larval retinal development, and as required for PPC specification in pupal development. It is believed that is due to Chc4 hypomorphic nature that retains a significant part of its endocytic capacity. If clathrin is responsible for the endocytosis of the empty receptor, it would provide a mechanism for Notch down-regulation consistent with current observations. A lesser decrease in endocytosis would first increase the amount of empty receptor at the membrane, increasing sensitivity and therefore signaling, when ligand is available. A greater reduction in endocytosis would eventually reduce Notch signaling by interference with ligand endocytosis, while a block in endocytosis would prevent Notch from signaling (Peralta, 2009).

One would expect the regulation of Notch receptor levels through clathrin-dependent endocytosis to be a general phenomenon, even if the retina constitutes a particularly sensitive system to such alterations. Indeed, it was found that Notch signaling in the wing is also affected by the Chc4 mutation, although it was necessary to sensitize the system to detect these defects. This sensitization was achieved by either halving normal Notch levels using a null mutation in heterozygosis, or by expressing an antimorphic mutated form of the receptor. The reproduction of the Notch gain-of-function phenotype of Chc4 in the wing suggests that endocytic down-regulation of the Notch receptor is a general mechanism by which cells can control Notch responsiveness (Peralta, 2009).

Although the results shed light over the role of clathrin-dependent endocytosis in Notch down-regulation, the effect of Chc4 on pupal retinal PCD indicates that Notch induces survival among interommatidial cells. This finding apparently contradicts two well established facts about retinal PCD: that lack of Notch reduces cell death, and that the EGFR-Ras pathway is responsible for the survival of interommatidial cells in the last instance. However, all these results are not necessarily incompatible. Throughout retinal development one of the main roles of Notch signaling is to maintain cells in an undifferentiated state, while the EGFR-Ras pathway is responsible for differentiating all the cell types in the retina in successive waves of signaling. At the end of cell specification all extra cells must be lost through apoptosis. If, as the results suggest, Notch promotes the maintenance of the undifferentiated state and opposes the apoptotic pathway, cells with a stronger Delta-Notch signal will avoid being culled by apoptosis, and eventually the Ras/Notch signal ratio will be sufficiently favorable to specify them as IOPCs. In this model, stronger Notch signaling (as in Chc4 or by expressing NFL, NICD, or Su(H)) will increase the number of IOPCs by reducing the number of cells that die. Conversely less Notch signaling (as following NECN expression), will reduce the number of IOPCs by increasing the number of cells that die. However, the absence of Notch in this model forces the cells with a low EGFR signal towards the IOPC fate, where activation of the Ras pathway inhibits apoptosis through a different mechanism, the activation of caspase inhibitors. This model allows inhibition of PCD both by an increase in Notch and by the lack of Notch, although through a different pathway. A prediction of this model is that in the absence of both Notch and EGFR, all the cells should be killed by the apoptotic pathway. Indeed this result should be indistinguishable from eliminating just the Ras pathway, as this prevents the interommatidial cells from differentiating as IOPCs, leaving apoptosis as the only possible outcome. These experiments have already been performed indicating that EGFR/Notch double mutants show extensive cell death that is no different from the EGFR mutants alone, therefore placing EGFR downstream of Notch, as in the current model (Peralta, 2009).

One intriguing aspect of these results is that Notch appears to work in opposite directions in both Chc4 retinal phenotypes. During PCD Chc4-induced excess Notch signaling causes an increase in cell survival, while afterwards it appears to provoke cell death. Currently no explanation is available for this disparity, since the final cause for the IOPC death is not known. It is possible that since, Notch is neither required nor appears to have a function in IOPCs after PCD, its abnormal activation by Chc4 might provoke some aberrant situation that causes the cells to die. Indeed evidence is available to suggest that this could be the case. While investigating the death of the IOPCs, the baculovirus caspase inhibitor P35 was expressed in the interommatidial cells using the GAL454 driver. While PCD was completely inhibited, P35 failed to significantly impair IOPC death in Chc4 retinas, suggesting that the cells did not die through apoptosis. However surprisingly, expression of P35 in control pigment cells also caused the IOPCs to die, despite inhibiting PCD, a retinal phenotype identical to Chc4 and excess of Notch, that also inhibit PCD. P35-induced IOPC death phenotype can be appreciated at its early stages in the original work reporting P35 expression in the Drosophila retina with a different driver. Since Notch appears to inhibit the death pathway, and P35 does precisely that, it is speculated that the underlying cause of IOPC death is the inhibition in them of the apoptotic machinery. Some aspect of pigment granules synthesis might require non-apoptotic caspase activity, as it has been reported for sperm production. This explanation would be consistent with Chc4 other major phenotype: male sterility due to defects in sperm production (Peralta, 2009).

A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis

Clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) are two predominant forms of synaptic vesicle (SV) endocytosis, elicited by moderate and strong stimuli, respectively. They are tightly coupled with exocytosis for sustained neurotransmission. However, the underlying mechanisms are ill defined. Previous work has shown that the Flower (Fwe) Ca2+ channel present in SVs is incorporated into the periactive zone upon SV fusion, where it triggers CME, thus coupling exocytosis to CME. This study shows that Fwe also promotes ADBE. Intriguingly, the effects of Fwe on CME and ADBE depend on the strength of the stimulus. Upon mild stimulation, Fwe controls CME independently of Ca2+ channeling. However, upon strong stimulation, Fwe triggers a Ca2+ influx that initiates ADBE. Moreover, knockout of rodent fwe in cultured rat hippocampal neurons impairs but does not completely abolish CME, similar to the loss of Drosophila fwe at the neuromuscular junction, suggesting that Fwe plays a regulatory role in regulating CME across species. In addition, the function of Fwe in ADBE is conserved at mammalian central synapses. Hence, Fwe exerts different effects in response to different stimulus strengths to control two major modes of endocytosis (Yao, 2017).

A tight coupling of exocytosis and endocytosis is critical for supporting continuous exocytosis of neurotransmitters. CME and ADBE are well-characterized forms of SV endocytosis triggered by moderate and strong nerve stimuli, respectively. However, how they are coupled with exocytosis under distinct stimulation paradigms remains less explored. A model is proposed based on the present data. When presynaptic terminals are mildly stimulated, SV release leads to neurotransmitter release and the transfer of Fwe channel from SVs to the periactive zone where CME and ADBE occur actively. The data suggest that this channel does not supply Ca2+ for CME to proceed. However, intense activity promotes Fwe to elevate presynaptic Ca2+ levels near endocytic zones where ADBE is subsequently triggered. Thus, Fwe exerts different activities and properties in response to different stimuli to couple exocytosis to different modes of endocytosis (Yao, 2017).

It has been previously concluded that Fwe-dependent Ca2+ influx triggers CME (Yao, 2009). However, the current results suggest alternative explanations. First, the presynaptic Ca2+ concentrations elicited by moderate activity conditions, i.e., 1-min 90 mM K+/0.5 mM Ca2+ or 20-s 10-20 Hz electric stimulation, are not dependent on Fwe. Second, expression of 4% FweE79Q, a condition that abolishes Ca2+ influx via Fwe, rescues the CME defects associated with fwe mutants, including decreased FM1-43 dye uptake, a reduced number of SVs, and enlarged SVs. Third, raising the presynaptic Ca2+ level has no beneficial impact on the reduced number of SVs observed in fwe mutants. These data are consistent with the observations that a Ca2+ influx dependent on VGCCs triggers CME at a mammalian synapse. Hence, Fwe acts in parallel with or downstream to VGCC-mediated Ca2+ influx during CME (Yao, 2017).

ADBE is triggered by intracellular Ca2+ elevation, which has been assumed to be driven by VGCCs that are located at the active zones. However, the data strongly support a role for Fwe as an important Ca2+ channel for ADBE. First, following exocytosis, Fwe is enriched at the periactive zone where ADBE predominates. Second, Fwe selectively supplies Ca2+ to the presynaptic compartment during intense activity stimulation, which is highly correlated with the rapid formation of ADBE upon stimulation. Third, 4% FweE79Q expression, which induces very subtle or no Ca2+ upon strong stimulation, fails to rescue the ADBE defect associated with loss of fwe. Fourth, treatment with a low concentration of La3+ solution that specifically blocks the Ca2+ conductance of Fwe significantly abolishes ADBE. Lastly, the role of Fwe-derived Ca2+ influx in the initiation of ADBE mimics the effect of Ca2+ on ADBE at the rat Calyx of Held. As loss of fwe does not completely eliminate ADBE, the results do not exclude the possibility that VGCC may function in parallel with Fwe to promote ADBE following intense stimulation (Yao, 2017).

Interestingly, Ca2+ influx via Fwe does not control SV exocytosis during mild and intense stimulations. How do VGCC and Fwe selectively regulate SV exocytosis and ADBE, respectively? One potential mechanism is that VGCC triggers a high, transient Ca2+ influx around the active zone that elicits SV exocytosis. In contrast, Fwe is activated at the periactive zone to create a spatially and temporally distinct Ca2+ microdomain. A selective failure to increase the presynaptic Ca2+ level during strong stimulation is evident upon loss of fwe. This pinpoints to an activity-dependent gating of the Fwe channel. Consistent with this finding, an increase in the level of Fwe in the plasma membrane does not lead to presynaptic Ca2+ elevation at the Calyx of Held when the presynaptic terminals are at rest or subject to mild stimulation. However, previous studies showed that, in shits terminals, blocking CME results in the accumulation of the Fwe channel in the plasma membrane, elevating Ca2+ levels. It is possible that Dynamin is also involved in regulating the channel activity of Fwe or that the effects other than Fwe accumulation associated with shits mutants may affect intracellular Ca2+ handling. Further investigation of how neuronal activity gates the channel function of Fwe should advance knowledge on the activity-dependent exo-endo coupling (Yao, 2017).

Although a proteomic analysis did not identify ratFwe2 in SVs purified from rat brain, biochemical analyses show that ratFwe2 is indeed associated with the membrane of SVs. The data show that 4% of the total endogenous Fwe channels efficiently promotes CME and ADBE at the Drosophila NMJ. If a single SV needs at least one functional Fwe channel complex during exo-endo coupling, and one functional Fwe complex comprises at least four monomers, similar to VGCCs, transient receptor potential cation channel subfamily V members (TRPV) 5 and 6, and calcium release-activated channel (CRAC)/Orai1, then it is anticipated that each SV contains ~100 Fwe proteins (4 monomers x 25). This suggests that Fwe is highly abundant on the SVs. It is unlikely that many SVs do not have the Fwe, as a 25-fold reduction of the protein is enough to ensure functional integrity during repetitive neurotransmission. Finally, the results for the SypHy and dextran uptake assays at mammalian central synapses indicate the functional conservation of the Fwe channel in promoting different modes of SV retrieval. In summary, the Fwe-mediated exo-endo coupling seems to be of broad importance for sustained synaptic transmission across species. (Yao, 2017).

Bicaudal-D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

Cargo transport by microtubule-based motors is essential for cell organisation and function. The Bicaudal-D (BicD) protein participates in the transport of a subset of cargoes by the minus-end-directed motor dynein, although the full extent of its functions is unclear. This study reports that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co-precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin-mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high-frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. These results implicate BicD as a new player in clathrin-associated trafficking processes and show a novel requirement for microtubule-based motor transport in the synaptic vesicle cycle (Li, 2010).

The genetic requirement of BicD at the organismal level had previously only been investigated in detail during maternal stages in Drosophila. This study describes the unexpected finding that zygotic BicD function is obligatory only in the nervous system, despite its widespread expression during larval stages. This function seems to be independent of BicD's well-known role in Egl-dependent mRNA transport (Li, 2010).

Chc was identified as the major BicD-associated protein in head extracts, and a direct interaction was mapped between the C-terminal third of BicD and the Chc ankle domain. This region of BicD provides a link between cellular cargoes and the dynein motor. Consistent with clathrin acting as a cargo for BicD/dynein, the directed transport of Chc::eGFP in association with microtubules is strongly reduced in BicD mutant presynaptic boutons leading to aberrant accumulation of the protein internally and a partial reduction in clathrin levels at the plasma membrane. Changes in microtubule integrity were not observed in BicD mutant boutons. In addition, a statistically significant portion of Chc signals overlapped with BicD signals within boutons and highly motile structures containing both Chc and BicD were observed in egg chambers, which are suited to sensitive time-lapse imaging. Collectively, these data build a strong case for disruption of Chc motility in boutons being a consequence of a direct requirement for BicD in microtubule-based transport complexes (Li, 2010).

In the case of embryonic transport of mRNA and lipid droplets in Drosophila, BicD does not seem to be obligatory for linkage of cargoes to the bidirectional motor complex, but leads to efficient transport by augmenting the persistence of motor movement. The presence of some residual directed motion of Chc::eGFP in mutant boutons raises the possibility that BicD serves an analogous, stimulatory function in the transport of clathrin-associated cargoes (Li, 2010).

It should also be pointed out that although the readily discernible phenotypic requirement for Drosophila BicD is restricted to neuronal tissue, this does not rule out a role for BicD in modulating the kinetics of clathrin-mediated mechanisms in non-neuronal cells, especially in other species. Highly motile clathrin-labelled structures within cultured mammalian cells are translocated by dynein on microtubules , raising the possibility of a conserved involvement of the BicD-Chc interaction (Li, 2010).

Functional assays show a novel requirement for BicD and dynein in the maintenance of normal levels of neurotransmission during high-frequency electrical stimulation. The function of these factors is not, however, limiting during low-frequency stimulation. BicD and dynein therefore join a group of other clathrin-associated proteins in Drosophila, such as Dap160, AP180, EndophilinA and Synaptojanin, in being required to maintain normal levels of neurotransmission specifically during periods of intense stimulation (Li, 2010).

Experiments assaying FM1-43 dye uptake and release showed that BicD and dynein are required for efficient membrane uptake specifically during intense electrical stimulation. This defect is associated, at least in part, with a requirement for augmenting the rate of release of pre-internalised vesicles. However, because rates of membrane uptake are intimately linked to rates of membrane release, the experiments performed could not directly measure endocytic rates in isolation. Ultrastructural analysis did not show dramatic changes in the organisation or morphology of the membrane trafficking system in resting or stimulated BicD mutant synapses. Collectively, these data indicate that BicD has a kinetic function in stimulating the rate of recycling of pre-internalised vesicles, as opposed to an obligatory role in any one step. This requirement for BicD is almost certainly associated with its well-characterised role in stimulating cargo transport by microtubule-based motors, as inhibition of dynein heavy chain and dynactin resulted in very similar effects on vesicle recycling to those seen in BicD mutants (Li, 2010).

To date the actin cytoskeleton has been heavily implicated in synaptic membrane recycling. However, the absence of chronic problems in synaptic morphology and membrane organisation in BicD mutant boutons is consistent with microtubule-based transport also participating directly in the synaptic vesicle cycle. In support of this notion, microtubules are prominent in boutons and acute, efficient interference with their integrity inhibits neurotransmission (Li, 2010).

BicD is likely to have a role in the transport of a subset of cargoes by dynein. However, the previously known BicD interaction partners do not seem to contribute significantly to the synaptic vesicle recycling phenotype in the BicD mutants; Egl function appears to be strictly maternal and Rab6 is not detectable in presynaptic boutons and its distribution in axons of motor neurons is not sensitive to the absence of BicD (Li, 2010).

It is possible that other, potentially unidentified, BicD cargoes contributing to the synaptic vesicle recycling phenotype in the mutants -- in fact, it is quite plausible. However, several lines of evidence point towards an important involvement of the interaction of BicD with Chc: (1) Chc is by far the most abundant protein stably associated with BicD in head extracts; (2) presynaptic overexpression of EndoA, which is rate-limiting for clathrin-mediated endocytosis and/or uncoating, is sufficient to completely suppress the defects in synaptic vesicle recycling of BicD mutants; (3) the requirement for BicD and dynein in synaptic vesicle recycling mirrors the requirement for high rates of clathrin-mediated membrane retrieval; (4) synaptic vesicle diameter and synaptic bouton number are increased in BicD mutants, reminiscent of when components of the machinery for clathrin-mediated endocytosis are mutated; and (5) despite extensive efforts, no factor other than Chc was found that was mislocalised in BicD mutant synapses; the factors tested include markers of membrane compartments (Hrs, Rabs 5, 6, 7 and 11), active zones (nc82) and synaptic vesicles (Vglut, Csp and Synaptotagmin (Syt) (Li, 2010).

How might a BicD-Chc interaction contribute to efficient synaptic membrane recycling? It seems unlikely that BicD has a direct role in stimulating clathrin-mediated internalisation of the plasma membrane because BicD, unlike several components of the clathrin-mediated endocytic machinery, is not enriched within periactive zones. BicD accumulates predominantly beneath these zones and the overlap of Chc and BicD puncta can occur even more internally within the bouton. These observations also suggest that BicD does not have a critical role in uncoating of clathrin from synaptic vesicles, which occurs very shortly after scission from the plasma membrane. Consistent with this notion, an accumulation of densely coated vesicles, indicative of uncoating defects in NMJ synapses of other mutants, was not detectable in electron micrographs of either resting or stimulated BicD mutant synapses (Li, 2010).

One possibility is that BicD augments dynein-based transport of clathrin that has disassociated from internalised vesicles back to the plasma membrane. This could account for two observations in the BicD mutants: the partially reduced concentration of Chc at periactive zones and the lack of co-localisation of internally accumulated Chc with clathrin adaptor proteins and markers of membrane compartments tested (AP180, α-adaptin/AP2, Hrs and Rab5). Reduced levels of available clathrin can compromise the ability to sustain high rates of membrane uptake. Thus, rates of Chc recycling to the plasma membrane in BicD and Dhc mutants may not be limiting during low-frequency stimulation, but may be unable to maintain sufficient levels of plasma membrane clathrin during bouts of intense stimulation. Recent studies in Drosophila synapses show that the membrane internalised in the absence of clathrin function is greatly increased in size and is not competent for recycling (Heerssen, 2008; Kasprowicz, 2008). Thus, a partial decrease in clathrin availability at the plasma membrane could conceivably contribute to the subtle increase in vesicle diameter and the inefficient recycling of pre-internalised membrane in BicD mutants. Alternatively, the internally mislocalised Chc in BicD mutants may interfere with normal sorting or maturation of synaptic vesicles by acting at ectopic sites or by sequestering important, as-of-yet unidentified, co-factors. Sequestration of clathrin co-factors could also account for changes in the diameter of nascent clathrin-coated vesicles in the mutants (Li, 2010).

Another possibility, which is by no means mutually exclusive, is that the ability of BicD to stimulate Chc transport on microtubules could directly augment translocation of recycling membrane during the time it is associated with clathrin (note that recent EM tomography studies in non-neuronal cells suggest that an incomplete uncoating reaction leads to the retention of some clathrin on vesicles until the exocytic event. Once again, such a kinetic requirement for BicD may only be limiting when there is a demand for a rapid rate of membrane recycling. BicD-Chc could potentially have a role in a process analogous to the microtubule-based pre-endosomal sorting process operating shortly after internalisation in other cell types. Alternatively, BicD might recognise Chc associated with endosomal structures. Indeed, dynein has a key role in non-neuronal cells in the sorting and subsequent transport of specific subsets of endosomal vesicles. Long-term experiments in neuronal and non-neuronal cells will be needed to resolve to what extent BicD participates in motor complexes that directly transport different post-endocytic intermediates, and the involvement of the interaction of BicD with Chc in these events. Future studies will also test directly the contribution of the interaction with Chc to other BicD-dependent processes, including sculpting of NMJ morphology (Li, 2010).

Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake

Synaptic vesicle reformation depends on clathrin, an abundant protein that polymerizes around newly forming vesicles. However, how clathrin is involved in synaptic recycling in vivo remains unresolved. Clathrin function during synaptic endocytosis was tested using clathrin heavy chain (chc) mutants combined with chc photoinactivation to circumvent early embryonic lethality associated with chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads to substantial membrane internalization visualized by live dye uptake and electron microscopy. However, chc-inactivated membrane cannot recycle and participate in vesicle release, resulting in a dramatic defect in neurotransmission maintenance during intense synaptic activity. Furthermore, inactivation of chc in the context of other endocytic mutations results in membrane uptake. The data not only indicate that chc is critical for synaptic vesicle recycling but they also show that in the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization (Kasprowicz, 2008).

Inhibition of chc function through a variety of approaches has been achieved in cell culture, resulting in defects in clathrin-dependent receptor-mediated endocytosis. However, most of these studies did not probe into the function of chc during vesicle formation, nor did they address the role of chc during synaptic vesicle endocytosis. In this work, chc was inactivated using three independent approaches, chc hypomorphic mutants, pharmacological inhibition of chc, and FlAsH-FALI of natively expressed chc, and the effects on synaptic vesicle recycling were studied (Kasprowicz, 2008).

This work expands on understanding of chc function in endocytosis of synaptic vesicles in two ways. First, the data indicate the critical role of chc in synaptic recycling. Synapses that lack chc function show a progressive decline in synaptic transmission during intense activity and membrane that is internalized during neuronal stimulation cannot be released in a second round of stimulation. Ultrastructural data indicates that small vesicles fail to be formed in synapses lacking functional chc, indicating a role for chc to resolve synaptic membrane into functional vesicles. Second, the data also suggest that in the absence of chc, another form of membrane internalization, not observed in controls, takes over. Indeed, loss of chc function does not block membrane uptake as gauged by fluorescent FM 1-43 dye uptake. In addition, ultrastructural studies show massive membrane folds and cisternae in synapses that underwent FlAsH-FALI of chc. Collectively, these data suggest a role for chc in maintaining synaptic membrane integrity during stimulation, preventing massive bulk membrane retrieval. Interestingly, reminiscent of the large vacuoles and cisternae, loss-of-function synapses were observed in chc. Similar structures can also be seen in strongly stimulated synapses of different organisms or in D. melanogaster temperature-sensitive shi mutants that are shifted back to low temperature after endocytic blockade at high temperature. It is surmised that under such conditions, vesicle fusion rate exceeds endocytic capacity and clathrin demand may be higher than supply, resulting in bulk membrane uptake. This work not only highlights the central role of chc in vesicle recycling and in the creation of small-diameter fusion-competent vesicles but also suggests that in the absence of chc function, a form of bulk endocytosis mediates the retrieval of synaptic membrane (Kasprowicz, 2008).

The observation that bulk retrieval takes over in the absence of chc function is consistent with loss-of-function studies of clc and α-adaptin, two other components of the endocytic coat. In nonneuronal TRVb cells, cross-linking most of the clc proteins did not inhibit membrane uptake, and recent data on inactivation of clc in neurons indicates internalization of membranous structures upon stimulation. Similarly, in embryonic lethal α-adaptin mutants, where chc polymers fail to be efficiently linked to the synaptic membrane, a dramatic depletion in vesicle number and large membrane invaginations can be observed. Thus, the data suggest that in the absence of functional clathrin coats, membrane internalizes by bulk retrieval; however, the formation of small synaptic vesicles from these endocytic structures appears inhibited during a time period that would normally be sufficiently long to repopulate the entire vesicle pool at wild-type D. melanogaster NMJs. In this context, it is interesting to note that stimulated dynamin 1 knockout neurons also show large endocytic membranes, which is consistent with the presence of bulk retrieval in these mutants. Interestingly, bulk membrane retrieval was observed in other endocytic mutants linked to dynamin function (synj) or controlling dynamin function (dap160, eps15, and shi) upon inactivation of chc. The bulk uptake in these double mutant animals is reminiscent of that observed in mouse dynamin 1 knockouts and that in fly clathrin mutant synapses and suggests an intriguing possibility where clathrin may coordinate with dynamin to form synaptic vesicles. In the absence of this function, bulk endocytosis appears to then mediate membrane retrieval (Kasprowicz, 2008).

Although it is well established that synaptic vesicles recycle by clathrin-mediated endocytosis, recovery by alternative routes, including direct closure of the fusion pore, remains controversial. At the D. melanogaster NMJ, endophilin (endo) knockouts dramatically impair clathrin-mediated endocytosis; however, some neurotransmission endures during intense stimulation. These data are consistent with either the presence of an alternative mode of vesicle recycling or with the persistence of low levels of clathrin-mediated endocytosis in endo mutants. Interestingly, removal of an additional component involved in clathrin-mediated endocytosis, synj, does not exacerbate the endo recycling defect. Thus, these data suggest that endo mutants block most clathrin-mediated recycling and indicate that an Endo- and Synj-independent recycling mechanism can maintain the neurotransmitter release observed in these mutants (Kasprowicz, 2008).

This work on chc, as well as recent data on clc, now allows the further scrutiny of the mechanisms of vesicle recycling at the D. melanogaster NMJ (Heerssen, 2008). FlAsH-FALI of 4C-clc leads to a complete block in synaptic transmission during high frequency stimulation, indicating that this condition blocks all vesicle recycling. However, the data indicates that photoinactivation of endogenously expressed 4C-chc only blocks transmission in 50% of the recordings, suggesting that some synapses may retain low levels of clathrin-independent recycling and that clc and chc may have partially divergent functions. Although the possibility cannot be excluded that in these studies some functional chc remains at the synapse after FlAsH-FALI, very similar inactivation conditions were used to those that lead to complete inactivation of clc (Heerssen, 2008). Furthermore, FlAsH-FALI of chc and chemical inhibition of chc, or both together, does not show a quantitative difference in membrane uptake during stimulation, indicating that the current protocols lead to severe, if not complete, inhibition of chc function. In addition, 4C-chc but was not overexpressed, but was expressed under native control in chc1-null mutants, ideally controlling protein levels and avoiding overexpression artifacts. Finally, EM of stimulated synapses where chc was acutely inactivated consistently show persistent active zone-associated synaptic vesicles. Some of these vesicles are similar in size to those observed in controls, and these vesicles are well positioned to participate in alternative recycling mechanisms. Hence, it is believed that although the data clearly support a critical role for clathrin-mediated endocytosis in the recycling of synaptic vesicles in D. melanogaster, this work does not exclude the possibility that alternative synaptic vesicle recycling routes operate at the larval NMJ (Kasprowicz, 2008).

Clathrin dependence of synaptic vesicle formation at the Drosophila neuromuscular junction

Among the most prominent molecular constituents of a recycling synaptic vesicle is the clathrin triskelion, composed of clathrin light chain (Clc) and clathrin heavy chain (Chc). Remarkably, it remains unknown whether clathrin is strictly necessary for the stimulus-dependent re-formation of a synaptic vesicle and, conversely, whether clathrin-independent vesicle endocytosis exists at the neuronal synapse. FlAsH-FALI-mediated protein photoinactivation was used to rapidly (3 min) and specifically disrupt Clc function at the Drosophila neuromuscular junction. Clc photoinactivation was demonstrated to not impair synaptic-vesicle fusion. Electrophysiological and ultrastructural evidence is provided that synaptic vesicles, once fused with the plasma membrane, cannot be re-formed after Clc photoinactivation. Finally, it was demonstrated that stimulus-dependent membrane internalization occurs after Clc photoinactivation. However, newly internalized membrane fails to resolve into synaptic vesicles. Rather, newly internalized membrane forms large and extensive internal-membrane compartments that are never observed at a wild-type synapse. Three major conclusions are drawn. (1) FlAsH-FALI-mediated protein photoinactivation rapidly and specifically disrupts Clc function with no effect on synaptic-vesicle fusion. (2) Synaptic-vesicle re-formation does not occur after Clc photoinactivation. By extension, clathrin-independent 'kiss-and-run' endocytosis does not sustain synaptic transmission during a stimulus train at this synapse. (3) Stimulus-dependent, clathrin-independent membrane internalization exists at this synapse, but it is unable to generate fusion-competent, small-diameter synaptic vesicles (Heerssen, 2008).

This study provides data that advances understanding of clathrin function at neuronal synapses in several fundamental ways. First, evidence is provided that stimulus-dependent synaptic vesicle reformation is blocked following Clc photoinactivation. Then membrane recycling was examined at the ultrastructural level. It was shown that clathrin-independent mechanisms of membrane internalization exist at the Drosophila NMJ, but these mechanisms are unable to generate fusion competent, small-diameter synaptic vesicles. Thus, vesicle re-formation requires clathrin and, by extension, clathrin-independent vesicle re-formation does not occur at the Drosophila NMJ (Heerssen, 2008).

Several observations indicate that Clc4C photoinactivation (in which the amino terminus of the Drosophila clathrin light chain gene is tagged with a 6-amino acid tetracysteine motif) does not cause distributed, non-specific damage to other proteins in the presynaptic nerve terminal. First, photoinactivation of Clc4C specifically disrupts synaptic vesicle recycling, as predicted, without altering baseline vesicle release in response to single action potentials delivered at low frequency. By contrast, prior work demonstrated that photoinactivation of Syt1 (Syt14C) substantially disrupts evoked transmitter release in seconds. Thus, the photoinactivation phenotypes that were observe are specific to the targeted protein, arguing against non-specific, widespread damage in the presynaptic nerve terminal. Second, when Clc4C was completely photoinactivate and a short stimulus train was delivered, EPSP amplitudes were observed to recover toward baseline amplitudes. This is consistent with normal vesicle mobilization from the reserve pool to re-populate the readily releasable pool of synaptic vesicles at the Drosophila NMJ. Thus, the mechanisms of vesicle mobilization remain intact despite Clc photoinactivation. Finally, the general architecture of the NMJ remains normal at the light level following complete photoinactivation and depletion of the vesicle pool. In summary, although collateral protein damage cannot be ruled out altogether, the data are consistent with prior studies suggesting that Lumio-FALI mediated protein photoinactivation can disrupt the function of a 4C tagged protein with remarkable specificity, and with the spatial and temporal precision of light (Heerssen, 2008).

It has been proposed that synaptic vesicles can transiently fuse with the plasma membrane, release their contents into the synaptic cleft, and re-internalize in a dynamin-dependent but clathrin-independent manner. This mechanism of vesicle recovery, termed 'kiss-and-run' is proposed to account for a significant fraction of internalized synaptic vesicles at mammalian central synapses. However, recent work examining vesicle recycling at mammalian hippocampal synapses depleted of clathrin using gene specific RNAi demonstrates that the reacidification of pH-sensitive synaptophluorin GFP is blocked following a short train of action potentials (4 stimuli), a result inconsistent with the 'kiss and run' hypothesis (Heerssen, 2008).

A second line of experimentation has been used to argue that 'kiss-and-run' can sustain synaptic transmission even in the background of an endophilinA mutation. At the Drosophila NMJ, two independent studies demonstrate that 80-90% of the synaptic vesicle pool is depleted during prolonged synaptic stimulation in endophilinA mutant animals. Both studies demonstrate that residual vesicle release can persist for prolonged periods of time (> 10 minutes). However, these studies ultimately arrive at different conclusions, based in part upon differing results from FM-dye uptake experiments. The current study concludes that kiss-and-run endocytosis sustains low levels of vesicle recycling in the endophilinA mutant while a previous study concludes that persistent release is achieved by crippled clathrin-dependent endocytosis. This study demonstrates that the synaptic vesicle pool at the Drosophila NMJ can be completely depleted during a stimulus train following Clc photoinactivation. Furthermore, the rate of vesicle depletion is identical to that observed in a dynamin mutant background. When considered in combination with ultrastructural data, it is concluded that synaptic vesicles cannot be reformed in the absence of functional Clc. Therefore, if 'kiss-and-run' is defined as a clathrin-independent form of synaptic vesicle recycling, then this study found no evidence for the existence of 'kiss-and-run' at the Drosophila NMJ (Heerssen, 2008).

It has been proposed that additional mechanisms of endocytosis exist at neuronal synapses that can be engaged during periods of prolonged nerve stimulation. This study demonstrated the stimulus-dependent formation of large internal membrane compartments within synapses following Clc photoinactivation. Since the distribution of synaptic antigens such as anti-Brp and anti-HRP is grossly unaffected at the light level and the decay of EPSP amplitudes during a stimulus train is quantitatively similar to that observed following nerve stimulation in dynamin mutant animals, it is proposed that these internal membrane structures represent re-internalized membrane rather than the aberrant fusion of synaptic vesicles within the nerve terminal. Interestingly, in the dynamin knockout, interconnected tubules of recycling membrane are capped by clathrin-coated buds of relatively uniform dimension (though not as uniform as the normal vesicle population). By contrast, the compartments observed following Clc photoinactivation lack any tubule-like restriction. These data suggest that clathrin normally participates in establishing the dimensions of internalized membrane, perhaps through the early recruitment of essential adaptor proteins. Remarkably, combined electrophysiological and ultrastructural data demonstrate that the large internal membrane compartments that form following Clc4C photoinactivation and nerve stimulation are unable to resolve into functional synaptic vesicles within 6 minutes, a time frame that is normally sufficient to re-populate a significant fraction of the synaptic vesicle pool at wild-type synapses. Thus, the resolution of these internal membrane compartments into synaptic vesicles requires clathrin. It is possible that these unusual membrane structures represent dramatically enlarged compartments that are normally limited by clathrin function. However, because membrane topology can be quite unusual at the EM level following Clc4C photoinactivation and stimulation, it is hypothesized that clathrin normally imposes some form of regulation on bulk membrane endocytosis, though not being strictly necessary for this process (Heerssen, 2008).

The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism

The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc1, Chc2 or Chc3 alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc4 germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc4 males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis (Bazinet, 2003; Full text of article).


Functions of Clathrin heavy chain orthologs in other species

Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos

Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). This paper reports that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. A similar role was established for the cytokinesis/apoptosis/RNA-binding protein CAR-1, and it was uncovered that CAR-1 is needed to maintain proper levels of CHC-1. CHC-1 was shown to be necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, it was established that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1RNAi embryos. A 2D computational model was developed to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, these findings lead to a proposal that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension (Spiro, 2014).

Regulation of clathrin-mediated endocytosis by dynamic ubiquitination and deubiquitination

Clathrin-mediated endocytosis in budding yeast requires the regulated recruitment and disassociation of more than 60 proteins at discrete plasma membrane punctae. Posttranslational modifications such as ubiquitination may play important regulatory roles in this highly processive and ordered process. However, although ubiquitination plays an important role in cargo selection, functions for ubiquitination of the endocytic machinery are not known. This study identified the deubiquitinase (DUB) Ubp7 as a late-arriving endocytic protein. Deletion of the DUBs Ubp2 and Ubp7 resulted in elongation of endocytic coat protein lifetimes at the plasma membrane and recruitment of endocytic proteins to internal membranes. These phenotypes could be replicated by expressing a permanently ubiquitinated version of Ede1, the yeast Eps15 homolog, which is implicated in endocytic site initiation, whereas EDE1 deletion partially suppresses the DUB deletion phenotype. Both DUBs are capable of deubiquitinating Ede1 in vitro. It is concluded that deubiquitination regulates formation of endocytic sites and stability of the endocytic coat. This regulation appears to occur through Ede1, because permanently ubiquitinated Ede1 phenocopies deletion of UBP2 and UBP7. Moreover, incomplete suppression of the ubp2Δ ubp7Δ phenotype by ede1Δ indicates that ubiquitination and deubiquitination are likely to regulate additional components of the endocytic machinery (Weinberg, 2014).


REFERENCES

Search PubMed for articles about Drosophila Clathrin heavy chain

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Biological Overview

date revised: 15 March 2015

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