InteractiveFly: GeneBrief

auxilin: Biological Overview | References

BIOLOGICAL OVERVIEW

Mutations were isolated in the Drosophila homologue of auxilin, a J-domain-containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, these data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-receiving cells needs to proceed past coat disassembly to activate Notch (Hagedorn, 2006).

Endocytosis, a process characterized by the internalization of extracellular materials and membrane proteins via vesicular intermediates, plays many roles in regulating cell-cell signaling pathways. In addition to the well-established role of attenuating signaling activity by clearing active receptor molecules from the cell surface, endocytosis has been proposed to facilitate signaling by transporting active receptor molecules to sites where downstream effectors are localized. A novel role of endocytosis has recently been proposed for the Notch signaling cascade, in which the internalization of the ligand facilitates activation of the receptor, although the exact mechanism of this critical event remains elusive (Hagedorn, 2006).

The Notch pathway is a signaling module that is highly conserved in all metazoans and has been implicated in a variety of developmental processes. How Notch transduces signals from the plasma membrane and affects gene regulation has been extensively analyzed in Drosophila, as well as several other model systems. It is now apparent that proteolytic processing of the Notch receptor is tightly associated with its ability to transduce signals. Notch is first cleaved during its transit through the biosynthetic pathway, thereby reaching the cell surface as a heterodimer of Notch extracellular domain (NECD) and a membrane-tethered intracellular domain. The binding of Notch to its ligand induces two additional cleavage events, releasing a signaling-competent Notch intracellular domain fragment from the plasma membrane. Notch intracellular domain then translocates into the nucleus and regulates gene expression by acting as a transcriptional coactivator (Hagedorn, 2006 and references therein).

Endocytosis appears to play a key role in regulating the activity of the Notch pathway. The importance of vesicular trafficking in Notch signaling was first noticed when mutations in Drosophila dynamin, a GTPase required for the detachment of vesicles from plasma membrane, was found to produce a Notch-like phenotype (Poodry, 1990). Clonal analysis suggested that in Notch signaling, dynamin function is required in both signal-sending and signal-receiving cells (Seugnet, 1997), suggesting that endocytosis impinges on the pathway at two independent steps. Although the role of endocytosis in signal-receiving cells is less clear, the internalization of ligand for the Notch receptor in the signal-sending cells appears to be a key event in activating the Notch cascade (Hagedorn, 2006 and references therein).

In Drosophila, there are two Notch ligands, Delta (Dl) and Serrate (Ser), members of the Dl, Ser, and Caenorhabditis elegans Lag-2 protein family (DSL). Both Dl and Ser appear to use an ubiquitin-mediated endocytic pathway to activate Notch receptors (Lai, 2005; Le Borgne, 2005b; Pitsouli, 2005; Wang, 2005). The covalent addition of ubiquitin to polypeptides, besides being a tag for proteasome-mediated protein degradation, can serve as a sorting signal for membrane protein internalization. The ubiquitination of Dl and Ser for subsequent internalization is mediated by Neuralized (Neur) and Mind bomb (Mib1), which encode two structurally unrelated E3 ubiquitin ligases. Although Neur and dMib regulate distinct Notch-dependent processes, they appear to be interchangeable in mediating the ubiquitination and internalization of the DSL ligand. Another critical component of this process is Liquid facets (Lqf), the D. melanogaster homologue of epsin. Lqf contains an ubiquitin-interacting motif, as well as motifs that bind to clathrin and other classes of adaptors . Thus, it is thought that Lqf functions as a cargo-specific clathrin adaptor, capable of recognizing and sequestering monoubiquitinated DSL ligand into clathrin-coated vesicles (CCVs), although an alternative function for epsin in nonclathrin endocytosis has been proposed (Hagedorn, 2006 and references therein).

Although a requirement of ligand endocytosis for Notch activation seems clear, the mechanism of how the internalization of the DSL ligand in the signal-sending cells promotes the proteolytic processing of Notch in the neighboring signal-receiving cells remains poorly understood. One set of models proposed that the internalization of Notch bound DSL ligand could either clear NECD from the extracellular space or generate physical force to dissociate NECD from the membrane-tethered intracellular domain, allowing the subsequent cleavage processing to occur (Parks, 2000). Alternatively, it has been suggested that endocytosis is required to transport DSL ligand to subcellular compartments, where the ligand is rendered signaling competent before being recycled back to the cell surface (Wang, 2004; Emery, 2005). Because, at present, the analysis of the roles of DSL endocytosis in Notch signaling relies on those mutations disrupting the assembly of cargo-containing CCVs, it is difficult to distinguish whether it is the internalization by itself or the transit of Dl through specific endocytic compartments that is critical for Notch activation. To better understand the mechanism of this critical process, the effects of additional endocytic mutations in Notch signaling need to be assessed (Hagedorn, 2006 and references therein).

The clathrin coats of newly formed CCVs need to be dissociated so the vesicles can fuse with target organelles and the released clathrin triskelions can be reutilized for subsequent rounds of endocytosis. Drosophila Hsc70, a constitutively expressed member of the Hsp70 chaperone family, has been implicated in promoting the release of clathrin triskelions and other coat proteins from CCVs in vitro (Schlossman, 1984; Chappell, 1986; Ungewickell, 1995; Hagedorn, 2006 and references therein).

In addition to Hsc70, another important factor in the clathrin uncoating reaction is thought to be auxilin, which contains clathrin binding domains, as well as a J-domain (Ungewickell, 1995; Umeda, 2000). The J-domain, a conserved motif shared by members of the DnaJ protein family, can bind to Hsp70 family proteins and stimulate their low intrinsic ATPase activity (Ungewickell, 1995). Thus, auxilin is thought to function as a cofactor in the uncoating reaction by recruiting ATP bound Hsc70 proteins to CCVs (Ungewickell, 1995; Holstein, 1996). In support of this, inhibition of auxilin function in vivo using yeast mutants, RNAi, or injection of interfering peptides can disrupt clathrin function (Gall, 2000; Pishvaee, 2000; Greener, 2001). Recent biochemical analysis suggests that auxilin participates in other steps of the CCV cycle, in addition to clathrin coat disassembly (Newmyer, 2003). Still, it is unclear what the relevant endocytic cargo of auxilin may be under physiological conditions or whether auxilin has any role in regulating cell-cell signaling in metazoan systems (Hagedorn, 2006 and references therein).

To further understand the roles of endocytosis in cell signaling during animal development, loss-of-function mutations were generated in auxilin from an F2 complementation screen in D. melanogaster. From this screen, six loss-of-function mutations in were isolated auxilin. In support of previous biochemical data, it was found that auxilin interacts genetically with Hsc70 and clathrin. In addition, the location of the genetic lesion in one of the alleles suggests that the putative lipid binding tensin domain plays a role in regulating clathrin function. The auxilin mutations also interact specifically with Notch and disrupt several Notch-mediated processes, suggesting that auxilin participates in an endocytic event critical for regulating the Notch cascade. Indeed, this analysis suggests that D. melanogaster auxilin is required for internalization of the Dl proteins that are critical for activating the Notch receptor (Hagedorn, 2006).

This study isolated and characterized mutants in Drosophila auxilin. In support of its well-known biochemical role in Hsc70-mediated disassembly of CCVs, this dAux^I670K mutation was shown to interact genetically with Hsc70-4 and the Clc. The in vivo link between auxilin and Hsc70 is further strengthened by the observation that a nonsense mutation (dAux^W1150X) near the very COOH terminus, where the J-domain is located, can strongly disrupt dAux function. These genetic observations are in agreement with in vivo analyses of auxilin function from other systems, which showed that clathrin function was disrupted in auxilin-deficient cells (Gall, 2000; Greener, 2001; Morgan, 2001). In addition, genetic data of dAux^I670K suggest a relevance of the tensin-related domain, a putative lipid binding domain, in clathrin-mediated endocytosis, despite the fact that it does not appear to be required for catalyzing the dissociation of clathrin triskelions from CCVs in vitro (Holstein, 1996; Newmyer, 2003; Hagedorn, 2006).

It has been suggested that, in addition to disassembling clathrin coats, auxilin participates in the dynamin-mediated constriction during CCV formation (Newmyer, 2003). However, subcellular localization analysis did not reveal dAux proteins colocalizing with clathrin at the cell periphery. Instead, most auxilin proteins appear to be associated with intracellular structures, in regions devoid of clathrin staining. This lack of overlap between dAux and Clc seems more consistent with the notion that auxilin is required for the dissociation of clathrin coats from CCVs under physiological conditions (Hagedorn, 2006).

Analysis of dAux clearly suggests that auxilin plays an important role in the Notch cascade in multiple Notch-dependent processes. Supportive evidence comes from the strong genetic interactions between dAux and Notch and the phenotypic similarities ranging from eye and wing development to neural development during embryogenesis. Moreover, the in vivo function of auxilin in the Notch signaling cascade seems specific, since dAux^I670K has no dominant effect on the phenotype caused by the overexpression of EGFR. Together, these observations argue that dAux acts specifically as a general component in the Notch cascade (Hagedorn, 2006).

Analysis from several groups has suggested that ligand internalization is a key event for Notch activation. The neurogenic phenotypes exhibited by dAux^I670K tissues and other genetic data further support this notion. The distribution of phenotypically mutant clusters in a genotypically mutant clone suggests that dAux acts noncell autonomously. In addition, the epistasis analysis places dAux function upstream of an activated form of Notch. Based on the phenotypic resemblance of dAux^I670K to those reported for neur (Lai, 2001; Pavlopoulos, 2001) and lqf (Overstreet, 2003), it is suspected that dAux functions along with neur and lqf in the ubiquitin-dependent endocytic pathway in the signal-sending cells (Hagedorn, 2006).

The identification of dAux as a critical factor in Notch ligand endocytosis has strong implications on the mechanism of Notch activation. Unlike Neur and Lqf, which are postulated to tag and sequester cargos into vesicles, auxilin is thought be involved in disassembly of clathrin coats. Thus, the revelation of dAux as another component in this pathway suggests that Dl-containing endocytic vesicles need to proceed past the clathrin uncoating step to activate Notch. One possible mechanism is that recycling of Dl is a prerequisite to form signaling-competent Dl-containing exosomes (Mishra-Gorur, 2002), although the presence of these structures under physiological conditions remains to be demonstrated. Alternatively, it may be that, as previously proposed, the DSL ligand is not signaling competent before endocytosis but is 'activated' during transit through recycling compartments. Indeed, the transit through Rab11-positive recycling endosomes has been suggested as a critical step for Dl activity (Emery, 2005). However, although Dl appears to colocalize extensively with coalesced perinuclear Rab11-positive structures in the sensory organ precursor cells (Emery, 2005), the current analysis found little spatial overlap between Rab11 and Dl in cells near the furrow. One possible explanation for this apparent difference is that the transit of Dl through Rab11-positive structures in the eye disc cells occurs more transiently, therefore evading detection by immunostaining at a steady state (Hagedorn, 2006).

Another explanation for the relevance of ligand endocytosis hypothesizes that Dl internalization causes a mechanical stress on the Notch receptors, which then induces subsequent cleavages. A variation of this model proposes that the objective of Dl internalization is to remove the NECD fragment from the intercellular space so proteolytic processing can occur. If auxilin is solely involved in clathrin-coat disassembly, it will be difficult to reconcile the current data with these two models because the internalization of Dl into CCVs, the presumed force-generating event, should have already been completed in dAux mutants (Hagedorn, 2006).

Auxilin is essential for Delta signaling

Endocytosis regulates Notch signaling in both signaling and receiving cells. A puzzling observation is that endocytosis of transmembrane ligand by the signaling cells is required for Notch activation in adjacent receiving cells. A key to understanding why signaling depends on ligand endocytosis lies in identifying and understanding the functions of crucial endocytic proteins. One such protein is Epsin (Drosophila Liquid facets), an endocytic factor first identified in vertebrate cells. This study shows in Drosophila that Auxilin, an endocytic factor that regulates Clathrin dynamics, is also essential for Notch signaling. Auxilin, a co-factor for the ATPase Hsc70, brings Hsc70 to Clathrin cages. Hsc70/Auxilin functions in vesicle scission and also in uncoating Clathrin-coated vesicles. Like Epsin, Auxilin is required in Notch signaling cells for ligand internalization and signaling. Results of several experiments suggest that the crucial role of Auxilin in signaling is, at least in part, the generation of free Clathrin. These observations in the light of current models for the role of Epsin in ligand endocytosis and the role of ligand endocytosis in Notch signaling (Eun, 2008).

A role for Clathrin in Notch signaling cells was originally inferred from the observation that Chc mutants are strong dominant enhancers of lqf hypomorphs (Cadavid, 2000). Since Epsin has both Ubiquitin- and Clathrin-binding motifs, and also binds the plasma membrane, the simplest scenario imaginable for Clathrin and Epsin function in Delta internalization is for Epsin to act as a Clathrin adapter that recognizes ubiquitinated Delta, and brings Clathrin to the membrane for CCV formation (Wendland, 2002). However, in light of evidence that Epsin-dependent endocytosis of ubiquitinated transmembrane proteins such as Delta may not occur through formation of CCVs, it has become unclear how to interpret the Chc/lqf genetic interaction. The results presented in this study point to a crucial role for Clathrin in Notch signaling cells. One intriguing possibility is that Delta internalization depends on Clathrin not because Delta is endocytosed in CCVs, but because Clathrin is a positive regulator of Epsin function. More experiments are required to test this idea (Eun, 2008).

Why do tissues that lack Epsin or Auxilin display Delta-like phenotypes, rather than phenotypes indicating failure of many signaling pathways or even cell death? One possibility is that the apparent specificity of both Epsin and Auxilin might simply reflect the usual redundancy of endocytic protein functions, and an unusual dependence of Notch signaling on efficient endocytosis. Alternatively, a special function of Epsin might be crucial to Notch signaling cells. Two kinds of models have been proposed to explain why Notch signaling requires ligand endocytosis by the signaling cells (Le Borgne, 2005a; Le Borgne, 2006; Chitnis, 2006; Nichols, 2007a). One idea (the 'pulling model') is that after receptor binding, ligand endocytosis generates mechanical forces that result in cleavage of the Notch intracellular domain (Notch activation), either by exposing the proteolytic cleavage site on the Notch extracellular domain, or by causing the heterodimeric Notch receptor to dissociate (Parks, 2000; Nichols, 2007b). Alternatively, ligand internalization prior to receptor binding might be required to process the ligand endosomally, and recycle it back to the plasma membrane in an activated form (the 'recycling model'). Epsin might generate an environment particularly conducive to either pulling or recycling, and Auxilin might be required specifically by Notch signaling cells because it activates Epsin, perhaps by providing free Clathrin. Alternatively, Auxilin might be needed to provide free Clathrin because Delta is internalized through CCVs. In this case, if Auxilin is required in Notch signaling solely to provide free Clathrin, the implication would be that efficient CCV uncoating is not important for generating uncoated Delta-containing vesicles per se, which are prerequisite for travel through an endosomal recycling pathway. Further understanding of the role of Auxilin in Notch signaling cells might be key to understanding the role of ligand endocytosis (Eun, 2008).

The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood. To understand the role of auxilin in Notch ligand endocytosis, several point mutations affecting specific domains of dAux were analyzed. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures. These data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, these observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization (Kandachar, 2008).

From a F₂ non-complementation screen, several new dAux alleles were isolated, some of which contain point mutations disrupting specific domains. Consistent with previous analysis of a viable dAux allele, strong dAux mutations affect several Notch-mediated processes, including photoreceptor specification in the eye and DV boundary formation in the wing. These phenotypes are consistent with the genetic interactions exhibited between dAux and Notch and between dAux and lqf (Eun, 2007). Taken together, these genetic observations strengthen the notion that endocytosis plays a critical role in Notch signaling, and suggest that dAux functions in multiple Notch-dependent events (Kandachar, 2008).

Since the functional importance of endocytosis has been suggested for both the signaling and receiving cells during Notch signaling, it is critical to determine in which cell is dAux function required. Although it has been previously concluded that dAux is needed in the signaling cells, the evidence, obtained from mitotic clones of a weak dAux allele, was less than convincing (Hagedorn, 2006). To adequately address this critical issue, the expression of E(spl), a Notch target gene, was examined in clones mutant for strong dAux alleles. Using these reagents, it is clear that dAux mutant cells at the clone border can still activate Notch (a similar result was seen with Cut and Ato staining), suggesting dAux acts non-cell autonomously. These genetic data imply that the relevant cargo is likely to be the Notch ligand. Indeed, as shown by the surface labeling experiment, Dl internalization is disrupted in dAux mutant cells (Kandachar, 2008).

Inhibition of auxilin function by mutations (Eun, 2007; Hagedorn, 2006; Gall, 2000; Pishvaee, 2000), RNAi (Zhang, 2005; Greener, 2001; Zhang, 2004; Lee, 2005), or injection of inhibitory peptides (Morgan, 2001) is known to interfere with the endocytosis of many molecules. In mammalian cells, inhibition of GAK function causes a decrease in the internalization of EGFR and transferrin (Eisenberg, 2007; Zhang, 2004). The current observations suggests that, similar to the mammalian cells, dAux participates in the endocytosis of EGFR, although a genetic interaction between DER and dAux was not previously (Hagedorn, 2006). It is possible that this lack of interaction between dAux and DER reflects the low sensitivity of the genetic assay. Alternatively, it may be that a defect in DER internalization does not significantly impact its signaling during eye development. Consistent with this, no drastic increase was observed in the phosphorylation of MAP kinase, a downstream event of DER activation, in dAux^F956* mutant clones. Nevertheless, the data show that, although the developmental defects of dAux resemble those of Notch, Notch ligand is not the sole cargo of auxilin-mediated endocytosis. This apparent specificity of dAux's Notch-like phenotypes suggests that the Notch pathway, compared to other signaling cascades, may be more sensitive to disruptions in the clathrin-mediated endocytosis (Kandachar, 2008).

Sequencing analysis of the dAux alleles revealed that disruptions in the kinase, the PTEN-related region, and the J-domain could all result in abnormal Notch signaling. Noticeably, the screen did not isolate any point mutation in the clathrin-binding motif (CBM), although the deletion analysis suggests that the CBM is critical for dAux function. This apparent discrepancy is likely due to the fact that the CBM domain contains multiple redundant clathrin-binding motifs, thereby obscuring the effect of eliminating one single motif by a point mutation. Interestingly, the removal of the CBM from the yeast auxilin (swa-2) does not completely eliminate its function in vivo. The reason for this difference is unclear but it is possible that swa-2 contains other protein domains capable of substituting for the CBM. Similar to a study of the mammalian GAK , a deletion analysis confirmed the importance of the J-domain, because over-expression of the dAux^deltaJ construct fails to restore the extra photoreceptor cell defect. The CBM and J domains are thought to facilitate the recruitment of Hsc70 to CCVs, and a fragment consisting of CBM and J domain alone has been shown to support clathrin uncoating in vitro. In support of this notion that the recruitment of Hsc70 to CCVs is likely to be a critical step, over-expression of the CBM and J domain alone could restore the supernumerary Elav-positive cell phenotype (Kandachar, 2008).

Conversely, these observation also implies that the loss of the kinase and PTEN-related region could be compensated by the over-expression of the CBM and J-domain. The PTEN-related region is thought to participate in the membrane recruitment of auxilin during CME (Morgan, 2000; Xiao, 2006). Thus it is imaginable that a defect in the subcellular localization is less deleterious when the fragment consisting of CBM and J-domain is over-expressed. It is unclear how the requirement of kinase domain can be compensated by the over-expression of the CBM and J-domain, as the relevant substrate for dAux kinase domain during Notch signaling is not known. It should be mentioned that elevated expression of dAux^CJ rescued the extra Elav-positive cell phenotype in both dAux^F956* and dAux^L78H (point mutations disrupting the J-domain and the kinase domain respectively), arguing against a scenario in which the kinase domain of endogenous dAux^F956* mutant proteins could complement the over-expressed dAux^CJ in trans. It is possible that some functional redundancy exists between dAux and Numb-associated kinase (NAK, the Drosophila homolog of adaptin-associated kinase) (Chien, 1998), since the kinase domains from both factors are known to phosphorylate adaptor complexes. However, although mutations in subunits of Drosophila AP1 and AP2 complexes have been implicated in other Notch-dependent processes, it is not clear if these adaptor complexes have a role in the Notch processes that were examined. Homozygous α -adaptin mutants do not appear to exhibit a neurogenic phenotype. Furthermore, the removal of one copy of AP2 mu subunit (by a deletion) has no effect on the dAux^I670K rough eye phenotype. In any case, it should be stressed that the kinase and the PTEN-related region do play a role in Notch signaling, since point mutations disrupting these domains cause Notch-like defects, albeit to a weaker extent. Taken together, these results suggest the role of the kinase and the PTEN-related region during Notch ligand endocytosis is less than obligatory (Kandachar, 2008).

What is the role of ligand endocytosis in Notch signaling? It has been suggested that, after receptor-ligand binding, ligand endocytosis may provide a mechanical stress or other types of micro-environment (clustered ligand and receptor, etc.) to facilitate Notch cleavage or NECD shedding. Alternatively, before binding to Notch, the ligands may have to enter a particular recycling pathway to render them active. The linking of dAux to Notch was initially viewed as evidence favoring the latter model because it suggests that ligand endocytosis needs to proceed past clathrin uncoating. However, as an increased level of the Dl appeared to be trapped at the mutant cell surface, not inside CCVs, the linking of dAux to Notch certainly does not exclude the model that ligand internalization per se is critical for Notch signaling. Biochemical analysis has suggested several additional functions for auxilin during the CCV cycle besides uncoating (Eisenberg, 2007 ). Although abnormal clathrin distribution was observed in dAux cells, given the resolution of the analysis, it is unclear which particular step(s) were affected. It is possible that mutations in dAux directly inhibit Notch ligand endocytosis by disrupting one or more of these early steps during CCV formation. Alternatively, dAux mutations may indirectly inhibit Notch ligand internalization by causing an excessive formation of non-functional clathrin-dependent structures, thereby decreasing the cytosolic clathrin pool. Indeed, in dAux mutant cells, those large clathrin-positive structures did not appear to contain an elevated level of Dl. Consistent with this, it was recently shown (Eun, 2008) that over-expression of Chc could restore the dAux-associated defects (Kandachar, 2008).

This genetic analysis of strong dAux alleles clearly strengthens the notion that ligand endocytosis plays a critical role in Notch signaling. Furthermore, the deletion analysis suggests that the recruitment of Hsc70 to clathrin is a key event for dAux to facilitate Notch signaling. More importantly, this study showed that Dl accumulates at the cell surface in dAux mutant cells. This suggests that the linking of dAux to the Notch pathway does not exclude the model in which ligand endocytosis activates Notch by physically dissociating the receptor (Kandachar, 2008).

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 use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, it was found that Rab11 is 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 found to not require auxilin to signal. Third, it was fond that 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).

There are three major results of this work. First, it was found that 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. Second, 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. Finally, overexpression of both clathrin heavy chain and epsin were found to rescue 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).

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. To understand further the role of membrane transport in this process, the phenotypes were analyzed of mutations in auxilin (aux), a regulator of clathrin function, 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 (IC) during individualization. In wild-type germ cells, GFP-tagged clathrin localizes to clusters of vesicular structures near the Golgi. These structures also contain the Golgi-associated clathrin adaptor AP-1, suggesting that they are Golgi-derived. By contrast, in aux mutant cells, clathrin localizes to abnormal patches surrounding the Golgi and its colocalization with AP-1 is 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. The 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).

Using partial loss-of-function aux mutations, this study has shown that auxilin has an important role in Drosophila male fertility and sperm formation. Since 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 cyclin G-associated kinase (GAK), a mammalian Auxilin related gene, has been suggested to function in the nucleus (Sato, 2009), 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, this study has 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, this study did not observe significant reduction in hub cell number in aux mutant testes (Zhou, 2011).

Previous studies have shown that overexpression of an Aux deletion consisting of just the CBD and the J-domains (dAux^CJ) could rescue the Notch signaling defect and lethality caused by aux. Similarly, expression of this construct from the β2tub promoter rescued aux-associated male sterility. By contrast, expression of Aux deletions missing either the CBD or the J-domain failed 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, since 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 dAux^CJ, 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 were localized in the vicinity of the Golgi. Furthermore, these structures contained AP-1 adaptors, suggesting that they were 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 dAux^FL-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 previous studies have shown that clathrin forms aggregates in aux mutant cells, it is possible that clathrin in these aggregates is incapable of interacting with AP-1. 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 was 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).

Search PubMed for articles about Drosophila Auxilin

Banks, S. M., et al. (2011). The functions of auxilin and rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling. PLoS One. 6(3): e18259. PubMed ID: 21448287

Cadavid, A. L. M., Ginzel, A. and Fischer, J. A. (2000). The function of the Drosophila Fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis. Development 127: 1727-1736. PubMed ID: 10725248

Chappell, T. G., et al. (1986). Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell 45: 3-13. PubMed ID: 2937542

Chien, C. T., Wang, S., Rothenberg, M., Jan, L. Y. and Jan, Y. N. (1998). Numb-associated kinase interacts with the phosphotyrosine binding domain of Numb and antagonizes the function of Numb in vivo. Mol. Cell. Biol. 18: 598-607. PubMed ID: 9418906

Chitnis, A. (2006). Why is Delta endocytosis required for effective activation of Notch? Dev. Dyn. 235: 886-894. PubMed ID: 16425217

Eisenberg, E. and Greene, L. E. (2007). Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis. Traffic 8: 640-646. PubMed ID: 17488288

Emery, G., et al. (2005). Asymmetric rab11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system. Cell 122: 763-773. PubMed ID: 16137758

Eun, S. H., et al. (2006). Identification of genes that interact with Drosophila liquid facets. Genetics 175(3): 1163-74. PubMed ID: 17179082

Eun, S. H., Banks, S. M. and Fischer, J. A. (2008). Auxilin is essential for Delta signaling. Development 135: 1089-95. PubMed ID: 18256200

Gall, W. E., et al. (2000). The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast. Curr. Biol. 10: 1349-1358. PubMed ID: 11084334

Greener, T., et al.(2001). Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo. Nat. Cell Biol. 3: 215-219. PubMed ID: 11175756

Hagedorn, E. J., Bayraktar, J. L., Kandachar, V. R., Bai, T., Englert, D. M. and Chang, H. C. (2006). Drosophila melanogaster auxilin regulates the internalization of Delta to control activity of the Notch signaling pathway. J. Cell Biol. 173(3): 443-52. PubMed ID: 16682530

Holstein, S. E., Ungewickell, H. and Ungewickell, E. (1996). Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin. J. Cell Biol. 135: 925-937. PubMed ID: 8922377

Kandachar, V., Bai, T. and Chang, H. C. (2008). The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis. BMC Dev. Biol. 8: 50. PubMed ID: 18466624

Lai, E.C., et al. (2005). The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta. Development. 132: 2319-2332. PubMed ID: 15829515

Le Borgne, R., Bardin, A. and Schweisguth, F. (2005a). The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 132: 1751-1762. PubMed ID: 15790962

Le Borgne, R., Remaud, S., Hamel, S. and Schweisguth, F. (2005b). Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila. PLoS Biol. 3(4): e96. 15760269

Le Borgne, R. (2006). Regulation of Notch signalling by endocytosis and endosomal sorting. Curr. Opin. Cell Biol. 18: 213-222. PubMed ID: 16488590

Lee, D. W., Zhao, X., Zhang, F., Eisenberg, E. and Greene, L. E. (2005). Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors. J. Cell Sci. 118: 4311-4321. PubMed ID: 16155256

Mishra-Gorur, K., et al. (2002). Down-regulation of Delta by proteolytic processing. J. Cell Biol. 159: 313-324. PubMed ID: 12403816

Morgan, J. R., Prasad, K., Hao, W., Augustine, G. J. and Lafer, E. M. (2000). A conserved clathrin assembly motif essential for synaptic vesicle endocytosis. J. Neurosci. 20: 8667-8676. PubMed ID: 11102472

Morgan, J. R., Prasad, K., Jin, S., Augustine, G. J. and Lafer, E. M. (2001). Uncoating of clathrin-coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin. Neuron 32: 289-300. PubMed ID: 11683998

Newmyer, S. L., Christensen, A. and Sever, S. (2003). Auxilin-dynamin interactions link the uncoating ATPase chaperone machinery with vesicle formation. Dev. Cell. 4: 929-940. PubMed ID: 12791276

Nichols, J. T., Miyamoto, A. and Weinmaster, G. (2007a). Notch signaling-constantly on the move. Traffic 8: 959-969. PubMed ID: 17547700

Nichols, J. T., Miyamoto, A., Olsen, S. L., D'Souza, B., Yao, C. and Weinmaster, G. (2007b). DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur. J. Cell Biol. 176: 445-458. PubMed ID: 17296795

Overstreet, E., Chen, X., Wendland, B. and Fischer, J. A. (2003). Either part of a Drosophila epsin protein, divided after the ENTH domain, functions in endocytosis of delta in the developing eye. Curr. Biol. 13(10): 854-60. PubMed ID: 12747835

Parks, A. L., et al. (2000). Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127: 1373-1385. PubMed ID:

Pavlopoulos, E., et al. (2001). neuralized encodes a peripheral membrane protein involved in Delta signaling and endocytosis. Dev. Cell. 1: 807-816. PubMed ID: 11740942

Pishvaee, B., et al. (2000). A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo. Nat. Cell Biol. 2: 958-963. PubMed ID: 11146663

Pitsouli, C. and Delidakis, C. (2005). The interplay between DSL proteins and ubiquitin ligases in Notch signaling. Development. 132: 4041-4050. PubMed ID: 16093323

Poodry, C. A. (1990). shibire, a neurogenic mutant of Drosophila. Dev. Biol. 138: 464-472. PubMed ID: 2108067

Sato, J., et al. (2009). GAK, a regulator of clathrin-mediated membrane trafficking, localizes not only in the cytoplasm but also in the nucleus. Genes Cells 14: 627-641. PubMed ID: 19371378

Schlossman, D. M., et al. (1984). An enzyme that removes clathrin coats: purification of an uncoating ATPase. J. Cell Biol. 99: 723-733. PubMed ID: 6146630

Seugnet, L., Simpson, P. and Haenlin, M. (1997). Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev. Biol. 192: 585-598. PubMed ID: 9441691

Umeda, A., Meyerholz, A. and Ungewickell. E. (2000). Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation. Eur. J. Cell Biol. 79: 336-342. PubMed ID: 10887964

Ungewickell, E., et al. (1995). Role of auxilin in uncoating clathrin-coated vesicles. Nature. 378: 632-635. PubMed ID: 8524399

Wang, W. and Struhl, G. (2004). Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch. Development 131: 5367-5380. PubMed ID: 15469974

Wang, W., and G. Struhl. 2005. Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytosis and signaling in Drosophila. Development. 132: 2883-2894. PubMed ID: 15930117

Wendland, B. (2002). Epsins: adaptors in endocytosis? Nat. Rev. Mol. Cell Biol. 3: 971-977. PubMed ID: 12461563

Xiao, J., Kim, L. S. and Graham, T. R. (2006). Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo. Mol. Biol. Cell. 17: 3281-3290. PubMed ID: 16687570

Zhang, L., Gjoerup, O. and Roberts, T. M. (2004). The serine/threonine kinase cyclin G-associated kinase regulates epidermal growth factor receptor signaling. Proc. Natl. Acad. Sci. 101: 10296-10301. PubMed ID: 15240878

Zhang, C. X., et al. (2005). Multiple roles for cyclin G-associated kinase in clathrin-mediated sorting events. Traffic 6: 1103-1113. PubMed ID: 16262722

Zhou, X., et al. (2011). Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis. Development 138(6): 1111-20. PubMed ID: 21343365

date revised: 1 March 2024

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