BIOLOGICAL OVERVIEW

The Arp2/3 complex (see Drosophila Arp2/3 component Suppressor of profilin 2) and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. The Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. Thus SCAR is a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrates that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation (Zallen, 2002).

Biochemical studies have provided detailed information about the molecules that influence actin dynamics. Of particular significance is the Arp2/3 complex that stimulates microfilament nucleation, the rate-limiting step in actin polymerization. The Arp2/3 complex consists of seven protein subunits, including the actin-related Arp2 and Arp3, and is conserved among eukaryotes. Members of the evolutionarily conserved Wiskott-Aldrich Syndrome protein and Scar/WAVE family function as strong potentiators of Arp2/3 complex activity. Distinct WASp and Scar/WAVE branches of this family have been recognized in diverse organisms, including Dictyostelium, Caenorhabditis elegans, Drosophila, and mammals. WASp and Scar/WAVE proteins share a common domain structure that mediates activation of the Arp2/3 complex in response to multiple signaling pathways. All members of the WASp-Scar/WAVE family possess a common COOH-terminal (WA) domain that stimulates actin polymerization through association with monomeric actin and the Arp2/3 complex, whereas their NH₂-terminal domains are structurally distinct and serve as signal-responsive regulatory regions. The molecular mechanisms controlling WASp function are well characterized, whereas regulatory aspects of Scar function are only now beginning to emerge (Takenawa, 2001; Zallen, 2002 and references therein).

The single WASp/Scar protein in budding yeast is required for processes that have been shown to be Arp2/3 dependent (Li, 1997; Naqvi, 1998), indicating a functional connection in vivo as well as in vitro. In what cellular contexts does this system operate during development of multicellular organisms? Are the distinct WASp and Scar homologs present in such organisms involved in common or separate Arp2/3-dependent processes? The first mutant alleles of the single Drosophila Scar/WAVE homolog, SCAR, have now been studied and SCAR functions have been compared to those Drosophila of WASp (Ben-Yaacov, 2001). SCAR and WASp appear to represent the only homologs of their respective subfamilies in the Drosophila genome, providing an opportunity to compare the functional requirements for these two major branches of the WASp/Scar protein family. Furthermore, the in vivo relevance of WASp and Scar to the Arp2/3 complex functions, which occur during development of a multicellular organism, can be assessed using mutant alleles in components of the Drosophila Arp2/3 complex. The results suggest that WASp and SCAR mediate distinct subsets of Arp2/3-dependent processes during Drosophila development. Although WASp is required specifically for proper execution of asymmetric cell divisions in neural lineages, SCAR plays a major role in the Arp2/3 complex-dependent regulation of cell morphology (Zallen, 2002).

Highly similar requirements for Drosophila SCAR and Arp2/3 complex components in regulating cytoplasmic organization in the blastoderm and cell morphology in CNS neurons, egg chambers, and adult eyes are demonstrated. These results suggest that SCAR and Arp2/3 complex components function in a common pathway in vivo, consistent with their well-established regulatory interaction in vitro. These roles of SCAR and the Arp2/3 complex are largely independent of WASp function, suggesting that SCAR is the primary regulator of Arp2/3-dependent morphological processes in Drosophila. In contrast, WASp is specifically required for the Arp2/3-dependent regulation of asymmetric cell divisions (Ben-Yaacov, 2001; Tal, 2002) a process that is independent of SCAR. These results demonstrate that SCAR and WASp perform generally nonoverlapping functions during Drosophila development and that the Arp2/3 complex can participate in distinct cell biological events in response to different regulators. Although SCAR and WASp can account for all characterized Arp2/3 complex functions in Drosophila, recent studies have described Arp2/3 complex regulators outside of the Scar/Wasp family (Jeng, 2001). Therefore, homologs of such elements (such as Cortactin and Eps15/Pan1p) may also play a role in Arp2/3-dependent processes during Drosophila development (Zallen, 2002).

A requirement for SCAR in the regulation of axon morphology in the Drosophila CNS has been demonstrated. The striking enrichment of SCAR protein in axons is consistent with a direct role for SCAR in axon development. In particular, the breaks in longitudinal and commissural axon bundles in SCAR mutant embryos may indicate a defect in axon growth. However, these phenotypes could also reflect defects in other aspects of nervous system formation, such as axon guidance, axon initiation, or neuronal differentiation. Morphological characterization of SCAR mutants at single neuron resolution will provide greater insight into the processes that require SCAR function (Zallen, 2002).

The CNS axon defects in SCAR mutant embryos resemble defects caused by simultaneous zygotic disruption of the Abl tyrosine kinase and a diverse set of elements including the Fasciclin I transmembrane protein, Armadillo/ß-catenin, Chickadee/profilin, and the Trio Rac/Rho guanine nucleotide exchange factor. Interestingly, Scar/WAVE-1 has been shown to associate with the SH3 domain of the Abl tyrosine kinase, suggesting that they may directly interact in vivo (Westphal, 2000). The observation that multiple zygotic mutations are required to replicate the SCAR phenotype is consistent with a model where SCAR functions downstream of multiple signaling pathways that converge on regulation of the actin cytoskeleton (Zallen, 2002).

The defects in axon morphology caused by reduction of maternal and zygotic SCAR are similar to those produced by zygotic disruption of Arp3 or simultaneous zygotic disruption of Arp3 and Arpc1 or SCAR and Wasp. These results suggest that SCAR, Wasp, and the Arp2/3 complex may affect a common process in neuronal development involving actin regulation. The contribution of both SCAR and Wasp to axon morphology could be explained by several possible mechanisms. In one model, SCAR and Wasp might regulate a common activity of the Arp2/3 complex, such as in the context of a specific actin structure or in contribution to bulk actin levels. Their functional differences in vivo could be achieved through differences in expression, activation, or subcellular localization. Alternatively, SCAR and Wasp could regulate distinct activities of the Arp2/3 complex, producing different actin structures that participate in diverse cell biological processes such as cell morphology (SCAR) and asymmetric cell division (Wasp). It will be interesting to examine how SCAR and Wasp intersect with regulators and effectors to achieve the specific organization of actin structures in different contexts (Zallen, 2002).

SCAR and the Arp2/3 complex regulate actin polymerization and organization in the blastoderm embryo. The dramatic reduction in actin levels of Drosophila Arpc1 mutants indicates that the Arp2/3 complex is an essential source of filamentous actin in the blastoderm embryo. This is consistent with experiments in other systems, where the Arp2/3 complex is required for actin polymerization in yeast actin patches (Pelham and Chang, 2001) and cell extracts in response to the Cdc42 GTPase (Ma, 1998; Mullins and Pollard, 1999) or the Listeria pathogen (Welch, 1997). These results also suggest that the SCAR regulator mediates this Arp2/3-dependent actin polymerization in the blastoderm. A similar reduction in filamentous actin is observed in Dictyostelium Scar mutants (Bear, 1998), and budding yeast Bee1 is required for actin polymerization at actin patch structures in a permeabilized cell assay (Lechler and Li, 1997). Together, these results demonstrate a conserved role for the Arp2/3 complex and WASp/Scar proteins in promoting actin polymerization in vivo as well as in vitro (Zallen, 2002).

In budding and fission yeast, inducible disruption of Arp2/3 complex function first leads to a cessation of actin patch movement followed by eventual actin patch dissolution (Winter, 1997; Pelham, 2001). Therefore, the Arp2/3 complex is required for the motility of actin structures and their formation. Similarly, Dictyostelium Scar mutants exhibit a selective disruption of specific actin structures that cannot easily be explained by an overall reduction of actin. Actin correctly localizes to the cell cortex and extends pseudopods as in wild type; however, leading edge actin fails to coalesce in response to chemoattractant, often leading to the aberrant formation of multiple pseudopods (Bear, 1998). These results suggest that Scar is involved in the dynamic organization of actin structures as well as their generation (Zallen, 2002).

An exciting possibility is that Scar and the Arp2/3 complex direct both the configuration and polymerization of actin in the Drosophila blastoderm. SCAR embryos in metaphase contain more than half the actin of wild-type embryos, yet this substantial amount of actin often fails to form even a discontinuous network of metaphase furrows. Instead, actin remains in aberrant surface structures that are not normally found at the surface of mitotic embryos. These observations suggest that SCAR plays a role in actin redistribution, perhaps through a local Arp2/3-dependent polymerization event that triggers a global cell cycle–dependent change in actin organization. This role of SCAR in the Drosophila embryo may be analogous to the reorganization of actin structures that occurs in other contexts, such as during cytokinesis or at the leading edge of migrating cells (Zallen, 2002).

The WAVE regulatory complex links diverse receptors to the actin cytoskeleton

The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. This study has identified a large family of potential WRC ligands, consisting of approximately 120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sarah and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. These findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans (Chen, 2014).

A consensus peptide motif, WIRS, specifically binds to a unique surface formed by the Sra and Abi subunits of the WRC. Strict conservation of the binding surface suggests that this interaction is broadly important to metazoans. The WIRS motif defines a novel class of WRC ligands that contains ~120 diverse membrane proteins. Genetic data further show that mutating the WIRS binding site of the WRC in Drosophila disrupts actin cytoskeleton organization and egg morphology during oogenesis, leading to female sterility, and also disrupts development of the visual system. In summary, these data characterize a widespread and conserved interaction that may link numerous membrane proteins to the WRC and the actin cytoskeleton (Chen, 2014).

The WIRS binding surface is contributed by both the Sra and Abi subunits of the WRC and therefore is only present in the fully assembled complex. Consequently, the WIRS interaction is unique to the intact WRC and cannot occur with individual subunits or subcomplexes. This may have important functional implications because, in cells, individual WRC subunits may form complexes with other proteins. For example, Sra1 binds the fragile-X mental retardation syndrome protein FMRP, along with the translation initiation factor eIF4E, using an interaction surface that is normally buried within the WRC. Moreover, Abi has been shown to interact with other proteins independent of its assembly into the WRC, including another member of the Wiskott-Aldrich syndrome protein WASP and the Diaphanous-related formin. Finally, the Nap1 ortholog Hem1 was suggested to exist in large complexes distinct from the WRC. These various complexes likely have distinct cellular functions. For example, the Sra1-FMRPeIF4E complex regulates mRNA localization and protein translation, and the Abi complexes were shown to regulate the actin cytoskeleton in processes distinct from those regulated by the WRC. Therefore, the multisubunit nature of the WIRS binding site may provide a mechanism to specifically regulate the intact WRC (Chen, 2014).

WIRS proteins can directly recruit the WRC to membranes, likely in cooperation with the other classes of WRC ligands. WIRS proteins may also have additional effects on the biochemical activity of the WRC. For example, this study has demonstrated that, although the minimal WIRS motif does not activate the WRC, sequences flanking the motif can potentiate (as in PCDH10) or inhibit (as in PCDH17) activity of the WRC in vitro. Therefore, WIRS proteins may exert different effects on the activity of the assembly, again likely in cooperation with other WRC ligands such as Rac1 or kinases. Alternatively, WIRS proteins could act as a scaffold and modulate WRC activity by coordinately recruiting the complex and other ligands. For example, the cytoplasmic tail of the NMDA receptor subunit NR2B could potentially corecruit cyclin-dependent kinase 5 (Cdk5) and the WRC to facilitate phosphorylation and consequent activation of WAVE. In fact, many WIRS-containing proteins are thought to function as scaffolds, including APC, Ankyrin, WTX/Amer1, Shroom, and Shank. Finally, many WIRS proteins are cell-cell adhesion receptors, which are often densely clustered at the plasma membrane. Such clustering would locally concentrate the WRC, a process known to increase the activity of WASP proteins toward the Arp2/3 complex (Chen, 2014).

Finally, WIRS/WRC interactions themselves are likely regulated. In fact, the data suggest that the WIRS motif (F-x-T/S-FX- X) could be modulated by phosphorylation. High-affinity binding requires Thr or Ser at the third position of the WIRS motif. No other residues examined were tolerated. Thus, it is likely that Thr/Ser phosphorylation at this position would block binding as well. Indeed, phosphorylation of various WIRS sites has been identified not only in global phosphoproteome studies but also by site-specific mutagenesi. Together, these various mechanisms could bring a large range of regulatory dynamics to locally tune WRC activity and consequent actin assembly in vivo (Chen, 2014).

The conservation of the WIRS binding surface in virtually all metazoans suggests that the WIRS/WRC interaction is broadly important and unique to animals because it is absent from other eukaryotes, including protists, fungi, and plants. It is notable that the WIRS binding surface is found even in nonmetazoan choanoflagellates, suggesting that WIRS/WRC interactions appeared more than 700 million years ago in an early ancestor that predates metazoans. Choanoflagellates are considered to be the closest living relatives to metazoans because they encode many metazoan-specific protein domains, including various cell adhesion molecules and proteins enriched in the nervous system. Although choanoflagellates are generally considered unicellular organisms, they can form simple colonies, leading to the possibility that the WIRS interaction arose to maintain multicellularity. However, this interaction may not be strictly necessary for multicellularity, as the WIRS binding surface is not found in the placozoan T. adhaerens, a primitive, amoeboid- like metazoan that lacks tissues or organs but is made up of distinct cell types. Moreover, a significant number of nonadhesion proteins also contain WIRS motifs, indicating that the WIRS interaction likely developed additional functions (Chen, 2014).

In this study, the search was limited to proteins whose WIRS motifs were conserved in four of seven representative species. Among the ~120 WIRS proteins, some display high conservation of their WIRS motifs. These include netrin receptors and ROBO proteins, whose WIRS motifs are conserved from human to C. elegans, despite a significant divergence in the overall sequences of their cytoplasmic tails. The WIRS motifs of many other proteins, including protocadherins and neuroligins, are conserved in all vertebrates examined (from human to zebrafish). It is noted that, by using conservation as a criterion in the search, other bona fide WIRS ligands that are less conserved might have been missed (Chen, 2014).

This study has demonstrated biological functions of WIRS/WRC interactions in animals by using Drosophila oogenesis as a model system. Defects observed by disrupting the WIRS binding surface, which resulted in defective egg morphology, disrupted actin cytoskeleton, and female sterility, resemble defects that arise from knocking out the WRC, suggesting that the WIRS interaction plays a major role in regulating WRC function during oogenesis in flies. Additionally, it was observed that the WIRS binding site is also important to the WRC in its non-cell-autonomous function of regulating photoreceptor axonal targeting in developing optic lobes. It is believed that many more WIRS-mediated regulatory functions are yet to be discovered. In support of this assertion, in C. elegans, WIRS-mediated interaction of the neuronal adhesion receptor SYG-1 with the WRC has been shown to regulate actin assembly at presynaptic sites in the neuromuscular junction of the egglaying motor neuron HSN and consequently is critical in initiating both synaptogenesis and axonal branching. It has been proposed that WIRS/WRC interactions are of general and diverse importance to animals throughout development (Chen, 2014).

Future studies are needed to reveal which specific WIRScontaining ligands are important to particular processes. Prior data in the literature suggest candidate WIRS proteins during oogenesis. Two membrane-associated proteins, P08630 (Tec29 tyrosine kinase) and Q9VCX1 (locomotion defects protein, Loco), both contain WIRS motifs and have been shown to regulate nurse cell dumping. Loco was also found to regulate the cortical actin cytoskeleton in glia. Phenotypic analysis also reveals an opposite oogenesis defect, which is similar to those observed in kugelei mutants deficient for dFAT2, another WIRS-containing protein. It remains to be determined whether these proteins or others are directly linked to the WRC during this process (Chen, 2014).

A variety of evidence also exists in the literature, suggesting functional roles of the WIRS interaction in other biological processes. In addition to PCDH10 and PCDH19, the WIRS proteins DCC and ROBO and the epithelial sodium channel ENaC (γ subunit) have been genetically linked to the WRC. DCC and ROBO differentially regulate the abundance and subcellular localization of the WRC to control the actin cytoskeleton in C. elegans embryonic epidermis. The WRC and Rac1 were found to be essential in regulating the activity of ENaC. The current data suggest that these genetic interactions may be due to direct physical interactions of WIRS motifs with the WRC. The functions of many other WIRS proteins, only a few of which have been previously linked to the actin cytoskeleton (e.g., glutamate receptor NR2B and the postsynaptic cell adhesion molecule Neuroligin1), may also depend on an interaction with the WRC. As a notable example, a 21 amino acid sequence of the Neuroligin1 cytoplasmic tail harboring a WIRS motif (PGIQPLHTFNTFTGGQNNTLP, WIRS bold is required for presynaptic terminal maturation (Chen, 2014).

Although it is still very premature to link WIRS/WRC interactions to any disease, several cases are suggestive. For example, seven cases of epilepsy and mental retardation in females (EFMR) were reported to arise from truncations of the cytoplasmic tail of PCDH19, all resulting in the loss of its WIRS motif. Additionally, partial truncation of the DCC cytoplasmic tail, along with its WIRS motif, caused congenital mirror movement in four affected members of a three generation Italian family. Finally, a point mutation (S1359C) that disrupts the WIRS site (LDSFES, S1359) in the adenomatous polyposis coli (APC) protein was associated with three unrelated cases of hepatoblastoma (Chen, 2014).

In summary, this study has defined and characterized a large family of potential WRC ligands unique to metazoans. A large and diverse set of membrane proteins comprises this class, many with important biological functions. These findings provide a mechanistic framework to understand how these proteins signal downstream to the actin cytoskeleton via direct interaction with the WRC and how their mutations may ultimately lead to disease (Chen, 2014).

Ataxin-7 and Non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization

Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE regulatory complexes (WRC), which control actin cytoskeleton assembly. There, Non-stop countered polyubiquitination and proteasomal degradation of WRC subunit SCAR. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop augmentation increased protein levels, and directed subcellular localization, of SCAR, decreasing cell area and number of protrusions. In vivo, heterozygous mutation of SCAR did not significantly rescue knockdown of Atxn7, but heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR (Cloud, 2019).

Purification of Atxn7-containing complexes indicated that Atxn7 functions predominantly as a member of SAGA. In yeast, the Atxn7 orthologue, Sgf73, can be separated from SAGA along with the deubiquitinase module by the proteasome regulatory particle. Without Sgf73, the yeast deubiquitinase module is inactive. In higher eukaryotes, Atxn7 increases, but is not necessary for Non-stop/USP22 enzymatic activity in vitro. In Drosophila, loss of Atxn7 leads to a Non-stop over activity phenotype, with reduced levels of ubiquitinated H2B observed (Mohan, 2014). In this study, purification of Non-stop revealed the active SAGA DUBm associates with multi-protein complexes including WRC and Arp2/3 complexes separate from SAGA. SCAR was previously described to be regulated by a constant ubiquitination/deubiquitination mechanism. SCAR protein levels increased upon knockdown of Atxn7 and decreased upon knockdown of non-stop. Decreases in SCAR protein levels in the absence of non-stop required a functional proteasome (Cloud, 2019).

Conversely, overexpression of Non-stop in cells led to increased SCAR protein levels and this increased SCAR protein colocalized to subcellular compartments where Non-stop was found. Nuclear Arp2/3 and WRC have been linked to nuclear reprogramming during early development, immune system function, and general regulation of gene expression. Distortions of nuclear shape alter chromatin domain location within the nucleus, resulting in changes in gene expression. Nuclear pore stability is compromised in SAGA DUBm mutants, resulting in deficient mRNA export. Similarly, mutants of F-actin regulatory proteins, such as Wash, show nuclear pore loss. Non-stop may contribute to nuclear pore stability and mRNA export through multiple mechanisms (Cloud, 2019).

When the basis for this unexpected regulatory mechanism was examined, a series was uncovered of WIRS motifs conserved in number and distribution between flies and mammals. These sequences functionally modulate Non-stop ability to increase SCAR protein levels. Point mutants of each WIRS resulted in less SCAR protein per increase in Non-stop protein. WIRS mutant Non-stop retained the ability to incorporate into SAGA, indicating these are separation of function mutants (Cloud, 2019).

Overall, these findings suggest that the cell maintains a pool of Non-stop that can be made available to act distally from the larger SAGA complex to modulate SCAR protein levels (see Non-stop regulates SCAR protein levels and location). In yeast, the proteasome regulatory particle removes the DUBm from SAGA. In higher eukaryotes, caspase-7 cleaves Atxn7 at residues which would be expected to release the DUBm, although this remains to be shown explicitly. The mechanisms orchestrating entry and exit of the DUBm from SAGA remain to be explored (Cloud, 2019).

Activation of Arp2/3 by WASp is essential for the endocytosis of Delta only during cytokinesis in Drosophila

The actin nucleator Arp2/3 generates pushing forces in response to signals integrated by SCAR and WASp. In Drosophila, the activation of Arp2/3 by WASp is specifically required for Notch signaling following asymmetric cell division. How WASp and Arp2/3 regulate Notch activity and why receptor activation requires WASp and Arp2/3 only in the context of intra-lineage fate decisions are unclear. This study found that WASp, but not SCAR, is required for Notch activation soon after division of the sensory organ precursor cell. Conversely, SCAR, but not WASp, is required to expand the cell-cell contact between the two SOP daughters. Thus, these two activities of Arp2/3 can be uncoupled. Using a time-resolved endocytosis assay, it was shown that WASp and Arp2/3 are required for the endocytosis of Dl only during cytokinesis. It is proposed that WASp-Arp2/3 provides an extra pushing force that is specifically required for the efficient endocytosis of Dl during cytokinesis (Trylinski, 2019).

In animal cells, a thin cortex of actin filaments is dynamically regulated to produce the force required for basic cellular processes, such as motility, cytokinesis, and endocytosis. This regulation involves the nucleation of branched actin filaments by the actin-related proteins 2/3 (Arp2/3) complex (Goley, 2006, Pollard, 2007, Rotty, 2013). By itself, Arp2/3 is weakly active, and nucleation-promoting factors (NPFs) are needed to stimulate its nucleation activity. Thus, when and where actin-based pushing forces are produced in the cell depends on the localization and activity of the NPFs. Wiskott-Aldrich syndrome protein (WASP) family proteins are the best-studied NPFs. These are usually maintained in an autoinhibited state and can be activated at the membrane by small GTPases. Three WASP family members are known in Drosophila: WASp, SCAR/WAVE (suppressor of cyclic AMP repressor/WASp-family verpolin-homologous protein), and WASH (WASp and SCAR homolog). Genetic analysis indicates that SCAR is the primary NPF in Drosophila, since the loss of SCAR activity leads to developmental and cellular defects that are similar to those seen upon the disruption of Arp2/3 activity, whereas WASH has a non-essential function during oogenesis, and WASp is only required for specific Notch-mediated fate decisions following asymmetric cell divisions in muscle, brain, and sensory organ lineages. This function of WASp is mediated by Arp2/3, since the loss of the Arp3 and Arpc1 subunits of the Arp2/3 complex leads to WASp-like cell fate defects. How WASp and Arp2/3 regulate Notch signaling is unclear. In addition, given the ubiquitous expression of WASp and the functional pleiotropy of Notch, it is unclear why WASp is only required for Notch signaling in the context of asymmetric cell division (Trylinski, 2019).

Notch receptor activation requires a pulling force to expose an otherwise buried cleavage site in the extracellular domain of Notch, the cleavage of which eventually produces the Notch intracellular domain (NICD). Previous studies have shown that endocytosis of the Notch ligands provides a strong enough pulling force to direct receptor activation. Since WASp and Arp2/3 are known to increase the efficiency of endocytosis by nucleating branched filaments shortly after membrane ingression begins (i.e., when high forces are required), it is conceivable that WASp-stimulated Arp2/3 activity may facilitate receptor activation by regulating the endocytosis of the Notch ligand Delta (Dl). However, it was reported that the endocytosis did not depend on Arp3, clearly arguing against this model. It was proposed that Arp2/3 may instead regulate the transport of endocytosed Dl back to the apical membrane, where it would activate Notch. This model, however, is not supported by a recent photo-tracking analysis of fluorescent Notch receptors, showing that signaling takes place along the lateral membrane following asymmetric division. NICD was produced during cytokinesis from a subset of Notch receptors that are located basal to the midbody (Trylinski, 2017). Thus, how WASp-Arp2/3 positively regulates Notch signaling is not known (Trylinski, 2019).

Early loss of Notch signaling in WASp and Arp3 mutants did not merely result from a defect in pIIa-pIIb contact expansion at cytokinesis. Contact expansion involves the activation of Arp2/3 by Rac and SCAR, but not by WASp, and SCAR and Rac are dispensable for Notch activation during cytokinesis and pIIa specification. Thus, Arp2/3 has separable functions in contact expansion and Notch signaling at cytokinesis. Instead, this detailed analysis of the endocytosis of Dl revealed that WASp is required for the efficient endocytosis of Dl during cytokinesis, but not afterward. This specific requirement of WASp and Arp2/3 for endocytosis during cytokinesis only may explain its specific requirement in Notch-mediated intra-lineage decision (Trylinski, 2019).

This study showed that Arp2/3 has two separable activities in asymmetric cell divisions: Arp2/3 promotes the rapid expansion of the new cell-cell contact and stimulates the endocytosis of Dl from this cell-cell contact to regulate intra-lineage fate decisions by Notch. These two activities involve distinct NPFs. SCAR, downstream of Rac, promotes the formation of a dense F-actin network around the midbody to generate a force that regulates cell-cell contact between sister cells and facilitates withdrawal of the membranes of the neighboring cells. SCAR, however, is largely dispensable for Notch receptor activation, suggesting that the force required for contact expansion is not key for Notch receptor activation. In contrast, WASp is required for Notch signaling but is dispensable for contact expansion during cytokinesis. While these two functions of Arp2/3 are separable, a functional interplay is possible, if not likely. For instance, Rac and SCAR may facilitate the activity of WASp in Dl endocytosis through the recruitment of Arp2/3 along the pIIa-pIIb interface (Trylinski, 2019).

Before the present study, WASp-mediated activation of Arp2/3 was thought to regulate the intracellular trafficking of internalized Dl, not its endocytosis. This model assumed that Dl signals at the apical membrane, which seems unlikely since it was recently shown that NICD originates from the lateral membrane during cytokinesis (Trylinski, 2017). Using a time-resolved endocytosis assay, this study showed that WASp-mediated activation of Arp2/3 is required to promote the endocytosis of Dl during cytokinesis, but not afterward. The specific time window during which the activities of WASp and Arp3 are required may explain why this requirement had previously been missed. In analogy to the role of WASp in yeast, it is proposed that WASp is recruited at sites of Dl endocytosis to form a branched actin network that provides an inward pushing force onto the invaginated membrane. This would increase the efficiency of endocytosis-hence the rate of the force-dependent activation of Notch. Accordingly, WASp would play a modulatory role, which is critical within a defined time window. Consistent with this view, the WASp mutant bristle phenotype can be suppressed by lowering the threshold for NICD levels in flies with reduced levels of the CSL co-repressor Hairless (Trylinski, 2019).

Why are WASp and Arp3 required only during cytokinesis for the endocytosis of Dl? WASp is likely to have a general function in endocytosis in Drosophila, as in other organisms, and may therefore regulate the endocytosis of many cargoes, including Dl, throughout development. Consistent with a general function of WASp, it is ubiquitously expressed and is not specifically upregulated in sensory lineages. However, the role of WASp-activated Arp2/3 in endocytosis is essential, at the organismal level, only in the context of intra-lineage decisions regulated by Notch. This specificity may be explained by when Notch signals, namely, at the end of mitosis. It is well established that clathrin-mediated endocytosis is shut down at mitosis and is progressively restored during cytokinesis. One mechanism contributing to this inhibition throughout mitosis is increased membrane tension. Since an increased requirement for actin is observed in cells in which membrane tension is high, it is speculated that the endocytosis of Dl more critically depends upon actin regulation by WASp during cytokinesis due to increased membrane tension. In other words, it is proposed that the pushing force provided by WASp-induced F-actin is needed for the efficient endocytosis of Dl to counteract the increased membrane tension associated with mitosis. Thus, while WASp and Arp2/3 likely play a general role in endocytosis, their activities become critical for the mechanical activation of Notch only when the inhibition of endocytosis needs to be overcome in late mitosis (Trylinski, 2019).

The requirement of WASp for Notch receptor activation is symmetric to those of Epsin, a conserved endocytic adaptor that helps generate the force for membrane invagination during endocytosis. Epsin is generally required for ligand endocytosis and Notch signaling in flies and mammals, with the exception of Notch-mediated intra-lineage decisions, as revealed by the development of sensory bristles in epsin mutant clones. It is speculated that the inhibition of Epsin at mitosis, possibly via its phosphorylation by CDK1/Cdc2, renders necessary the extra pushing force provided by WASp for the efficient endocytosis of Dl (Trylinski, 2019).

In summary, a model is proposed whereby the activity of WASp-Arp2/3 generally increases the efficiency of endocytosis and becomes specifically required only during cytokinesis, when Dl activates Notch to mediate intra-lineage decisions. This model may be general and apply to mammalian tissues where Notch is known to regulate intra-lineage decisions. N-WASp, the ubiquitously expressed WASp in mammals, is required for the maintenance of skin progenitor cells and hair follicle cycling in the mouse, and Notch plays a critical role in the self-renewal of skin stem cells. Whether Notch signaling is regulated by N-WASp in this context remains to be examined (Trylinski, 2019).

GENE STRUCTURE

cDNA clone length - 2832

Bases in 5' UTR - 259

Exons - 5

Bases in 3' UTR - 731

PROTEIN STRUCTURE

Amino Acids - 613

Structural Domains

A search of the sequenced Drosophila genome identified a single Scar/WAVE homolog (SCAR, corresponding to transcription unit CG4636) that maps to cytogenetic band 32C4-5 on the second chromosome. The structure of the SCAR transcript was determined by sequencing three ESTs from the Berkeley Drosophila Genome Project database. The 2,184 nucleotide SCAR transcript is predicted to encode a 613 amino acid protein possessing the major hallmarks of Scar/WAVE proteins (Zallen, 2002).

date revised: 25 October 2023

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