In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. The gene kette (correctly termed Hem-protein, or simply Hem) is required for the normal projection of the VUM axons and interference with kette function disrupts glial migration. In spite of the fact that glial migration is disrupted in kette mutants, both the axon guidance and glial migration phenotypes have their origin in midline neuron expression and not in midline glial expression. Axonal projection defects are found for many moto- and interneurons in kette mutants. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The Hem/Kette protein is homologous to the transmembrane protein HEM-2/NAP1 (Nck-associated protein) evolutionary conserved from worms to vertebrates. In the CNS, the membrane protein Kette could be participating directly in the neuron-glial interaction at the midline, where it could act as a signal to direct glial migration. Alternatively, Kette could serve as a receptor of possibly glial-derived signals during VUM growth cone guidance. The experimental data suggest that Kette transduces information to the neuronal cytoskeleton, which is in agreement with a receptor function (Hummel, 2000).
In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2-SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins Rac1 and Cdc42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant Cdc42 or RAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated Rac1 transgene. These data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding (Hummel, 2000).
Most of the Kette protein is found in the cytoplasm where it colocalizes with F-actin to which it can bind via its N-terminal domain. Some Kette protein is localized at the membrane and accumulates at focal contact sites. Loss of Kette protein results in the accumulation of cytosolic F-actin. Actin dynamics crucially depend on the ability of the protein to switch from a monomeric (G-actin) to a filamentous form (F-actin). Polymerization of F-actin starts with the de novo nucleation of an actin trimer, a process that occurs relatively slowly and requires the action of the Arp2/3 complex (see Drosophila Arp2/3 component Suppressor of profilin 2). Subsequent elongation is fast and cells have to prevent spontaneous actin polymerization by expressing a variety of actin-binding proteins such as profilin. The nucleation activity of the Arp2/3 complex in turn is regulated by a set of activators, such as the members of the Wasp (Wiskott-Aldrich syndrome protein) and Wave (Scar - FlyBase) families. Wave, which does not bind Cdc42, is trans-inhibited through its association with members of the Kette family: Sra1 (specifically Rac associated 1) and Abi (Abelson-interactor). Rac1 binding, presumably to Sra1, relieves the inhibitory function of this complex (Bogdan, 2003 and references therein).
To date it is still unclear how Kette could regulate the organization of the actin cytoskeleton in vivo. Since Kette has been predicted to be an integral membrane protein with six transmembrane domains, it might serve as a receptor recruiting Nck/Dock or Rho-GTPases to the membrane. Biochemical and genetic evidence reveals that Kette is found predominantly in the cytosol. Only a small amount of Kette is recruited to the plasma membrane. In vivo as well as in tissue culture models, Kette protein colocalizes with F-actin, and co-sedimentation assays reveal a direct interaction with F-actin. Within the membrane, Kette accumulates at the insertion sites of large F-actin bundles, suggesting that targeted localization of Kette may be required for its function. Loss of Kette protein leads to a Scar/Wave-dependent accumulation of F-actin within the cell. Ectopic expression of wild-type or different truncated Kette proteins in tissue culture cells or during Drosophila development does not affect F-actin formation or viability. However, expression of membrane-tethered Kette efficiently induces ectopic bundles of F-actin in a process depending on Wasp but not on Scar/Wave. These data indicate that Kette fulfils a novel role in regulating F-actin organization by antagonizing Wave and activating Wasp-dependent actin polymerization (Bogdan, 2003).
Thus Kette is able to modulate the activity of both Wasp and Scar/Wave and thus contribute to the regulation of F-actin dynamics. Wasp and Wave together with Cdc42 and Rac1 control different aspects of cortical actin dynamics. Cdc42 and Wasp are required for filopodia formation, whereas dominant-negative Wave disrupts the Rac1-dependent formation of branched network of F-actin bundles required to form lamellipodia. Wave localizes to membrane ruffles induced by activated Rac1 and Wasp accumulates in microspikes containing bundled F-actin (Bogdan, 2003 and references therein).
A large 500 kDa complex comprising Nap1/Kette, PIR121/Sra1, Abi, HSPC300 and Wave silences the otherwise constitutive activity of Wave in stimulating actin polymerization (Eden, 2002). The 500 kDa complex is stabilized by direct protein-protein interactions that have been demonstrated between Nap1/Kette and Abi, and Nap1/Kette and PIR121/Sra1 (also called Gex2). The exact binding partners of Wave are presently unknown (Bogdan, 2003 and references therein).
Two modes of Wave activation have been demonstrated in vitro: (1) activated Rac1 is able to bind to PIR121/Sra1 and can relieve Wave inhibition by dissociating the Nap1/Kette, PIR121/Sra1 and Abi sub-complex (Eden, 2002); (2) PIR121/Sra1 is able to bind to the first SH3 domain of SH2SH3 adapter Nck, which is sufficient to activate the Wave complex (Eden, 2002). In vivo these two mechanisms may work at the same time to fully activate Wave (Bogdan, 2003 and references therein).
In agreement with the work of Eden (2002) disruption of Kette function leads to an excess formation of cytoplasmic F-actin. However, expression of even very high levels of wild-type Kette protein do not evoke any mutant phenotype. Thus, in wild type cells the inactive Wave complexes are already formed and Kette overexpression does not result in an additional sequestering of Wave into the silencing complexes and/or an incorporation of additional Kette into these complexes. In addition, since Kette requires Sra1 function to bind to membrane-associated adapters such as Nck, excess cytosolic Kette will not be able to reorganize subcellular Wave distribution (Bogdan, 2003).
Further support for the notion that Kette mediates repression of Scar/Wave activity stems from genetic analyses. Embryos lacking zygotic kette function display a characteristic CNS phenotype, whereas loss of zygotic Scar/Wave expression does not affect embryonic nervous system development. The kette mutant phenotype, which is due to defects in neurite outgrowth (Hummel, 2000), is significantly suppressed by reducing the dose of Scar/Wave expression. This demonstrates that in wild-type embryos, Kette acts as a negative regulator of Scar/Wave. Similar results are obtained when Kette and or Scar/Wave expression is reduced in Drosophila S2 cells. These experiments also reveal that Scar/Wave is required for the normal subcellular distribution of Kette, which may, however, be an indirect effect caused by the disruption of the F-actin actin cytoskeleton (Bogdan, 2003).
Kette protein localizes to the plasma membrane where it accumulates in focal adhesion contacts. A prime candidate that may mediate recruitment of Kette to the membrane is the SH2 SH3 adapter protein Nck which, besides binding of the Sra1/Kette/Abi complex, also recruits numerous other proteins to focal contact sites. Among these is Wasp, which binds to the third SH3 domain of Nck (Bogdan, 2003 and references therein).
kette interacts with dock, which encodes the Drosophila Nck homolog (Hummel, 2000). Membrane recruitment of Kette is sufficient to activate actin polymerization in the cell cortex mediated by Wasp. How is this brought about? One explanation might be that recruitment of Kette to the membrane disintegrates the inhibitory Wave complex, independent of the Nck/Sra1 association. This would then lead to an excess of Wave activity and subsequently to an excess of actin polymerization. However, the genetic data clearly show that membrane bound Kette functions independent of Scar/Wave but depends on Wasp (Bogdan, 2003).
Wasp usually adopts an auto-inhibited conformation and is activated after Cdc42, Nck binding or phosphorylation. A structure-function analysis of the Drosophila Wasp has demonstrated that the Cdc42-binding domain is not necessary for function, suggesting that alternative pathways, such as phosphorylation can activate Wasp. Kette might be a part of such an alternative pathway; a genetic interaction between kette and wasp in the regulation of actin dynamics has been demonstrated. Regulation of Wasp by Kette is not mediated by direct protein-protein interaction, but probably involves Abi that is able to link Kette and Wasp. The Abl interactor (Abi) protein localizes to sites of actin polymerization at the tips of lamellipodia and filopodia and has been implicated in the cytoskeletal reorganization in response to growth factor stimulation. As a positive regulator of the non-receptor tyrosine-kinase Abelson (Abl) Abi may bring Abl into position to phosphorylate and thus activate Wasp. Abl is known to phosphorylate many proteins regulating focal adhesion and F-actin dynamics and overexpression of activated Abl induces F-Actin formation in Cdc42-independent manner. Some tyrosine kinases are activate by phosphorylation of Wasp; however, direct phosphorylation of Wasp by Abl remains to be demonstrated (Bogdan, 2003 and references therein).
Further support of the model that suggests in vivo Kette activates Wasp but suppresses Wave comes from the phenotypic analyses of Drosophila kette, wasp and scar/wave mutants. Mutations in kette have been isolated due to defects in commissure formation in the embryonic CNS (Hummel, 2000). If Kette acts via activating Wasp, similar phenotypes are expected following disruption of either gene. This is indeed the case and loss of zygotic and maternal Wasp function results in a kette-like embryonic CNS phenotype (Bogdan, 2003 and references therein).
In agreement with the proposed function of Kette in regulating both, Wasp and Wave, is its subcellular distribution. Whereas the majority of Kette is present in the cytoplasm to keep Wave in its inactive state (Eden, 2002) some is present in the leading edge of lamellipodia-like structures. However, the highest amounts of Kette are present at the insertion points of large F-actin bundles where N-Wasp is also present. Kette might be recruited to these focal adhesion sites via Sra1/Nck; via Wasp, it could enhance the formation of F-actin bundles (Bogdan, 2003 and references therein).
It is presently unclear just how Wasp activity results in straight F-actin bundles, whereas Wave stimulates the formation of a meshed F-actin network. In the cytosol, Kette may act as a scaffold protein that keeps Wave close to F-actin and recruits additional factors to F-actin such as Profilin, which not only binds to Kette but also enhances actin nucleation. Thus, Kette could promote the formation of a meshed F-actin network characteristic for lamellipodia. At the membrane other proteins may interact with Kette and in this respect it is interesting to note that the F-actin crosslinking protein Filamin, which plays an important role in filopodia formation, also binds to Kette. This suggests that Kette, in addition to regulating Wasp and Wave, may also contribute to the decision whether filopodia or lamellipodia are formed (Bogdan, 2003).
Actin polymerization is a key process for many cellular events during development. To a large extent, the formation of filamentous actin is controlled by the WASP and WAVE proteins that activate the Arp2/3 complex in different developmental processes. WAVE function is regulated through a protein complex containing Sra1, Kette and Abi. Using biochemical, cell biological and genetic tools, this study shows that the Abi protein also has a central role in activating WASP-mediated processes. Abi binds WASP through its carboxy-terminal domain and acts as a potent stimulator of WASP-dependent F-actin formation. To elucidate the biological function of abi in Drosophila, bristle development, a process known to require wasp function, was studied. Reduction of abi function leads to a loss of bristles similar to that observed in wasp mutants. Activation of Abi results in the formation of ectopic bristles, a phenotype that is suppressed by a reduction of wasp activity but is not affected by the reduction of wave function. Thus, in vivo Abi may set the balance between WASP and WAVE in different actin-based developmental processes (Bogdan, 2005). In a mammalian system Abi also binds and activates N-WASP, suggesting a functional conservation of this central player (Innocenti, 2005).
Developmental processes involving movement and cell shape changes are based on a highly dynamic reorganization of the actin cytoskeleton. The Arp2/3 complex is an important regulator of actin polymerization. Its activation is mediated by members of the Wiskott-Aldrich syndrome proteins (N-WASP/WAVE). WASP and N-WASP (neural WASP) are specific effectors of Cdc42 that are characterized by a CRIB (Cdc42/Rac interactive binding) domain and an NH2-terminal WASP homology domain. By contrast, WAVE proteins lack these sequence motifs but possess a highly specific WAVE homology domain (WHD) and transduce Rac signalling to the Arp2/3 complex (Bogdan, 2005).
The in vivo roles of WASP and WAVE proteins are as different as their molecular architectures. This has been clearly established both in vertebrates and Drosophila. Whereas WAVE acts as the main Arp2/3 regulator during axonal growth, oogenesis and eye development, WASP is required for cell fate decisions during sensory organ development. This last function is independent of Cdc42 but is mediated through Arp2/3 (Bogdan, 2005).
To activate WASP, an intramolecular inhibition has to be released by factors such as Cdc42, phosphatidylinositol-4,5-bisphosphate, or several SH3-domain-containing proteins. In contrast, WAVE proteins are constitutively active in vitro. Activity is regulated by a multi-protein complex comprising Kette (NAP1, HEM), Sra-1 (CYFIP, PIR121), Abi (E3b1) and HSPC300. Initial work had suggested that WAVE1 is trans-inhibited and cannot stimulate Arp2/3 nucleation activity in vitro. This finding was supported by genetic data, showing that Kette or PIR121 antagonize WAVE function. However, new data clearly show that a reconstituted pentameric WAVE complex is constitutively active in stimulating Arp2/3 function and remains stable, even after Rac stimulation. This argues against a trans-inhibition model of WAVE function and raises the question as to whether in vivo additional proteins or post-translational modifications are required to control WAVE activity (Bogdan, 2005).
Surprisingly, Sra1 and Kette are also involved in the regulation of WASP. These unforeseen genetic interactions prompted the elucidation of the functional relationship of these genes. Biochemical, cell biological and genetic data presented in this study clearly identify Abi as a central player in orchestrating the regulation of WASP, and thus define a new key component in directing cytoskeletal dynamics (Bogdan, 2005).
The Abi protein has a relative molecular mass of 52,000, and was first identified as a substrate of the Abelson kinase. During embryonic development, Abi shows a prominent coexpression with known members of the WAVE complex, Kette and Sra-1. It is maternally expressed and later in development becomes concentrated in the nervous system. Abi contains a C-terminal SH3 domain, which can interact with the Abelson kinase and the amino-terminal WAVE-interacting domain (WAB domain), which is required for binding and recruiting WAVE (Bogdan, 2005).
In addition, Abi binds Kette and WASP. Yeast two-hybrid experiments show that a deletion of the N-terminal 85 amino acids of the Drosophila Abi protein (AbiDeltaN) abrogates its interaction with WAVE but does not affect the interaction with Kette or WASP. Conversely, the C-terminal SH3 domain of Abi is necessary and sufficient for WASP interaction, and deletion of this domain blocks the interaction with WASP but not with WAVE. These deletion constructs also show that Abi binds Kette through its central domain (Bogdan, 2005).
To further test the interaction between Abi and WASP, glutathione S-transferase (GST) pull-down experiments were performed. Affinity purification of Abi from S2R+ cells using GST-WASP coupled to glutathione-Sepharose beads results in the copurification of Kette, which directly binds Abi, and Sra-1, which binds Kette. Significant amounts of WAVE were also found to be present in the protein complex bound to GST-WASP. Disruption of F-actin by Latrunculin A does not affect this coprecipitation. Furthermore, in WAVE pull-down experiments, some WASP protein could be detected in the precipitate. As expected, Abi is able to coprecipitate WAVE as well as WASP. Since WASP cannot bind directly to WAVE in a yeast two-hybrid assay, these findings suggest that Abi is in a position to coordinate the activity of two main regulators of the Arp2/3 complex (Bogdan, 2005).
As described for mammalian cells, endogenous WAVE is distributed relatively uniformly at the leading edge in Drosophila S2R+ cells. Abi and WASP show a more dotted expression pattern. Thus, Abi, WASP and WAVE are all localized at the leading edge of the cell, as reported for N-WASP and WAVE2 in lamellipodia of mouse myoblasts (Bogdan, 2005).
To determine the functional relevance of the proteins, RNA interference (RNAi) experiments were performed. RNAi-mediated silencing of abi, kette or sra-1 in S2 cells leads to a collapse of all lamellipodia-like structures and induces a starfish-like morphology. Reduction of WASP expression results in a more complex phenotype and the most sensitive phenotypic trait appeared to be the cell size. Twenty per cent of the cells showed a normal F-actin pattern but were consistently smaller than control cells. In 55% of the cells, lamellipodia appeared to collapse, leaving isolated F-actin-rich cell extensions. A requirement of the mammalian N-WASP for lamellipodia formation has also been observed in mammalian cell lines. Twenty-five per cent of the cells showed a starfish-like phenotype, which is similar to the abi knockdown phenotype. These effects are dependent on the wasp RNAi dose and require efficient protein silencing (Bogdan, 2005).
In contrast to the loss-of-function phenotype, even high levels of Abi expression did not affect cell morphology and F-actin organization. Ectopically expressed Abi is distributed similarly to the endogenous protein, but increased levels are associated with F-actin. It has been shown that membrane recruitment of Kette or Sra-1 results in their activation and induces WASP-dependent phenotypes in vivo. Expression of membrane-tethered Abi (AbiMyr) or of a N-terminal deletion variant lacking the WAVE interaction domain Abi (AbiDeltaN) results in extensive filopodia formation in about 50% of the transfected cells. However, deletion of the C-terminal SH3 domain from AbiMyr, which mediates binding to WASP, abrogates this effect. If AbiMyr acts through WASP, then a membrane-tethered WASP should also induce the formation of filopodia-like structures. Indeed, WASPMyr expression induces a regularly spaced array of filopodia-like structures in about 80% of the cells. Whereas wild-type S2R+ cells are characterized by membrane ruffling, both AbiMyr and WASPMyr induce small and dynamic cell extensions. In contrast, reduction of abi function by RNAi leads to less dynamic structures and finally cell collapse. To test whether the AbiMyr phenotype depends on WASP or WAVE, RNAi experiments were performed in cells expressing AbiMyr. In both cases the knockdown phenotype of WAVE or WASP is epistatic to the AbiMyr-induced phenotype, indicating that in S2R+ cells WAVE either performs vital functions, or that WAVE-dependent formation of lamellipodia is a prerequisite for AbiMyr-induced filopodia formation (Bogdan, 2005).
Because Abi binds WASP and can induce WASP-dependent phenotypes, whether the regulation of F-actin formation can be reconstituted in vitro was tested. Drosophila WASP and Abi proteins expressed in Escherichia coli were purified and their ability to activate mammalian Arp2/3 was tested in an actin polymerization assay. Drosophila WASP has only a small effect on Arp2/3-dependent actin polymerization kinetics by itself. The addition of Abi significantly increases this basal activity in a dose-dependent manner (Bogdan, 2005).
These findings prompted similar experiments in vivo. The expression of different Abi transgenes were induced using the Gal4-UAS system. Overexpression of AbiMyr in wing imaginal discs results in a significant elevation in the level of F-actin. The en-Gal4 driver allows the anterior compartment of the wing imaginal disc to be used as a negative control. Upon expression of high levels of AbiMyr, F-actin fibres are rearranged and the free G-actin pool appears to be reduced. When WASP function is present, F-actin fibres extend into AbiMyr-expressing processes. The elevation in actin polymerization was neither observed upon overexpression of wild-type Abi nor upon overexpression of AbiMyr lacking the C-terminal SH3 domain, suggesting that both membrane localization and interaction with WASP are required for F-actin formation. To further test the requirement for WASP, a C-terminal truncation (WASPDeltaCA) was used that still contains the proline-rich sequences binding to Abi but lacks sequences required for Arp2/3 complex binding. Expression of WASPDeltaCA results in a wasp-like mutant phenotype. Coexpression of membrane-tethered Abi and the dominant-negative WASPDeltaCA construct clearly suppressed ectopic F-actin formation, indicating that AbiMyr stimulates actin polymerization via WASP. Similarly, the effects caused by AbiMyr expression could be suppressed by reducing the wasp gene dose, clearly showing that Abi positively regulates WASP activity in vitro and in vivo. Expression of WASPDeltaCA alone does not have any effect on F-actin or G-actin distribution. The specificity of F-actin and G-actin detection was demonstrated by Latrunculin A treatment (Bogdan, 2005).
The functional relevance of abi was examined in the developing fly, focusing on the development of the sensory organs. Drosophila harbours only one gene encoding a WASP-like protein. Homozygous zygotic wasp mutants survive until adulthood and show a loss of bristles. Further analyses showed that WASP governs cell-fate decisions of specific external sensory organs that develop from proneural clusters. Expression of the dominant-negative WASPDeltaCA by the sca-GAL4 driver, which activates expression in the proneural clusters, leads to a loss of thoracic bristles similar to but not as extreme as is seen in wasp mutant flies. Likewise, reduction of abi function by in vivo expression of double-stranded abi RNA results in a similar bristle phenotype. In addition, the bristles are shorter and thinner compared with the wild type. In contrast, expression of AbiMyr resulted in the formation of additional bristles, independent of the GAL4 driver used (ptc-GAL4) and the wave gene dosage. Expression of an AbiMyr protein lacking the N-terminal WAVE interaction domain still induces the formation of extra bristles. These experiments show that Abi does not act through WAVE during Drosophila bristle development. Moreover, heterozygous loss of wasp function or coexpression of the dominant-negative waspDeltaCA transgene partially suppresses the formation of ectopic bristles induced by AbiMyr, which is most clearly seen for the notopleural bristles. By contrast, deletion of the C-terminal WASP-binding domain of Abi abrogates the ability of AbiMyr to induce bristles. Thus, in vivo Abi acts through WASP (Bogdan, 2005).
During development of Drosophila sensory organs, a single cell is selected out of a group of equivalent proneural cells (proneural cluster) by lateral inhibition to become a sensory organ precursor (SOP). The SOP undergoes a series of stereotyped asymmetric cell divisions giving rise to the different cells comprising an individual sensory organ. Zygotic wasp mutants show defects in asymmetric SOP division and lack bristles. Because a WASP protein lacking the CA domain is not able to rescue this phenotype, Arp2/3-dependent processes are involved in establishing cell fate (Bogdan, 2005).
To test directly whether Abi affects asymmetric cell division or promotes the allocation of neural fate the number of SOPs was determined using neuralized-lacZ or hindsight expression. Wild-type wing imaginal discs are characterized by a stereotypic patterning of SOPs. Expression of abi RNAi in proneural clusters results in a loss of many SOPs, which is similar to the wasp mutant phenotype. In contrast, expression of AbiMyr in proneural clusters leads to the formation of additional hindsight-expressing cells, indicating the formation of additional SOPs; all dorsocentral, scutelar and notopleural SOPS are affected; on average, three (instead of one) SOPs are found (Bogdan, 2005).
In summary, these findings place the Kette-Sra-1-Abi complex at a key position coordinating the formation of F-actin by regulating WAVE and WASP proteins. Furthermore, Abi-WASP is involved in SOP specification. The formation of SOPs is regulated by two main antagonistic pathways. Whereas Notch restricts the formation of neural cells, activation of the epidermal growth factor receptor (EGFR) promotes SOP formation. WASP forms a complex with EGFR and transmits Nck-dependent signals from EGFR to polarize cortical actin filaments. In turn, cytoskeletal rearrangements lead to receptor clustering and their subsequent activation. These experiments highlight the possibility that reverse signalling may occur in vivo and point to the interwoven relationship between receptor signalling and actin dynamics that is mediated by Abi and WASP. Thus, Abi functions as a central regulator that directs WASP- or WAVE-dependent developmental processes, presumably with the help of additional factors (Bogdan, 2005).
Drosophila bristle development is dependent on actin assembly, and prominent actin bundles form against the elongating cell membrane, giving the adult bristle its characteristic grooved pattern. Previous work has demonstrated that several actin-regulating proteins are required to generate normal actin bundles. This study addresses how two actin regulators, capping protein, a barbed end binding protein, and the Arp2/3 complex, a potent actin assembly nucleator that acts downstream of WASp, function to generate properly organized bundles. As predicted from studies in motile cells, it was found that capping protein and the Arp2/3 complex act antagonistically to one another during bristle development. However, these proteins do not primarily act directly on bundles, but rather on a dynamic population of actin filaments that are not part of the bundles. These nonbundle filaments, termed snarls, play an important role in determining the number and spacing of the actin bundles. Reduction of capping protein leads to an increase in snarls, which prevents actin bundles from properly attaching to the membrane. Conversely, loss of an Arp2/3 complex component leads to a loss of snarls and accumulation of excess membrane-attached bundles. These results indicate that in nonmotile cells dynamic actin filaments can function to regulate the positioning of stable actin structures. In addition, the results suggest that the Arpc1 subunit may have an additional function, independent of the rest of the Arp2/3 complex (Frank, 2006).
These results establish that the Arp2/3 complex and capping protein have opposing actions on dynamic actin structures, called snarls, in Drosophila bristle cells. In the absence of the Arp2/3 complex, no snarls are apparent, whereas when capping protein is reduced, snarls persist longer during development. Combinations of mutations that affect both proteins are more similar to wild type in number and persistence of snarls, as would be expected if the two activities need to be balanced for proper regulation of actin assembly. Because most other studies of capping protein and the Arp2/3 complex have utilized in vitro assays or motile cells in culture, the observation that these regulators function similarly in nonmotile cells in vivo is important, albeit not surprising. What is surprising, however, is that the main structural features important for the morphology of these cells, the large cortical actin bundles, are not the direct target of these activities, despite the fact that their organization and positioning is strongly affected by altered amount of these regulators (Frank, 2006).
This study presents evidence that early in bristle development capping protein and the Arp2/3 complex control actin assembly not in actin bundles, but rather in the actin snarls that are present between these bundles. It is hypothesized that snarls influence the proper positioning of the cortical actin bundles by competing with them for binding sites on the membrane. When the level of snarls is severely reduced (in homozygous arp3 mutant pupae), excess actin bundles associate with the membrane. This results in adult bristles that are shorter than wild type and have more, smaller grooves in their cuticle. In the presence of too many snarls (in transheterozygous cpb mutant pupae), actin bundles are unable to form against the membrane and instead develop internally. Because they are not stabilized by attachment to the membrane, they become irregularly sized and highly variable in number. This results in a cell membrane that is inadequately supported by actin bundles. Adult bristles that have numerous defects, such as irregular groove patterns and smooth regions in the cuticle, as well as bends, splits, and protrusions in the shaft are produced (Frank, 2006).
Actin snarls are dynamic and it has been suggested that their 2-min half-life is due to their failure to be stabilized either by cross-linking to other filaments or by forming stable attachments to the membrane. It has been argued that those filaments that do not become stabilized are turned over, and thus the actin bundles develop in a Darwinian 'survival of the fittest' manner. The snarls have been proposed to be nonproductive actin structures. This study proposes that the snarls have a function in forming properly shaped bristles: they are important for proper positioning of bundles. Snarls may have additional functions as well. They cause protrusions in the membrane between the bundles and lie beneath the regions that will become the ridges in the cuticle of the adult bristle (the actin bundles lie below the grooves). Thus the Arp2/3 complex–directed actin assembly in snarls may push the membrane out, much in the way that this complex causes membrane protrusion at the leading edge of motile cells. This region of the bristle membrane is then kept free from actin bundles so that the proper number and size of bundles are formed. When too many bundles form, cuticle is secreted in a disorganized manner. Thus it may be important that some regions of the membrane are kept clear of actin bundles to allow for proper secretion of chitin. Alternatively, actin filaments in the snarls could be directly involved in membrane deposition that occurs during cell extension and/or in the secretion of cuticle (patches of cuticulin are evident on developing bristles as early as 36 h APF). Snarls may be analogous to actin patches in Saccharomyces cerevisiae that are turned over very rapidly, contain the Arp2/3 complex and capping protein, and are involved in endocytosis and cell wall synthesis (Frank, 2006).
The interplay seen between actin bundles and snarls in bristles is reminiscent of that seen between actin cables and patches in S. cerevisiae. Yeast cells lacking functional capping protein have diminished actin cables and excess patches. Conversely, long-term lack of Arp3 results in cells that have lost patches and accumulate actin bundles. The antagonistic relationship between capping protein and the Arp2/3 complex, as well as their opposing effects on actin patches and bundles, might thus be a common theme in the regulation of the actin cytoskeleton (Frank, 2006).
Mutations in arp3 and wasp, as well as deficiencies that remove arpc4 and arpc5, all suppress the cpb bristle phenotype as would be expected from the opposing biochemical functions of the Arp2/3 complex (filament assembly nucleator) and capping protein (prevents further assembly of actin filaments). So it is surprising to find that mutations in arpc1 have the opposite phenotype. This is an especially unexpected result given that previous work has demonstrated that adult bristles completely lacking Arpc1 or Arp3 have an identical mild phenotype of more, smaller grooves. It is tempting to suggest that there is a genetic background issue at play here. It is believed that this is unlikely, however, because two different arpc1 alleles isolated in independent labs in different genetic backgrounds gave the same result (Frank, 2006).
One explanation for the enhancement seen by mutations in arpc1 is that in the absence of this subunit, a complex forms that in some way acts as a neomorph. Arpc1 contains the WASp interaction capability of the complex, so complexes lacking this subunit might be unactivatable, and thus might block the interaction between actin filaments and other important binding proteins. Although this possibility out cannot be ruled out, it is thought unlikely because of the following: Arpc1 (also referred to as p41) and Arpc5 (p16) subunits form an interacting pair. They find that when complexes are formed in the absence of Arpc5, Arpc1 also fails to assemble into the complex. If this neomorph model were correct (and assuming fly Arp2/3 complex components behave in the same way as their human homologues), it would be expected that reduction of Arpc5 would lead to loss of Arpc1 from the complex and cause enhancement of the cpb phenotype. However, this is not what was observed. A deficiency chromosome lacking the arpc5 region in fact suppressed the cpb bristle phenotype (Frank, 2006).
To explain arpc1 enhancement of bundle displacement observed in cpb mutants, it is noted that the difference in phenotype between cpb6.15/cpbF19 and cpb6.15 +/cpbF19 arpc1Q25st is only apparent at late developmental times. In both genotypes early in development, bundles are displaced from the membrane. However, in cpb6.15/cpbF19 at late times, bundles often are associated with the membrane, whereas in cpb6.15 +/cpbF19 arpc1Q25st they rarely are. Interestingly, in cpb null epithelial clones, bundles also remain displaced. These similar phenotypes suggest that capping protein and Arpc1 both participate in a late membrane-attachment process. Thus, it is suggested that in cases where bundles form not associated with the membrane initially, capping protein and Arpc1 promote bundle attachment late in development. The ability of some bundles to associate with the membrane in cpb6.15/cpbF19 bristles is likely due to the fact that in this genotype capping protein is not completely eliminated but rather is reduced to 48% of the wild-type level. When capping protein is completely absent or when both capping protein and Arpc1 are reduced in amount, then this late membrane attachment does not occur. Interestingly, capping protein localization on bundles starts at ~42 h APF, around the time when the effect of capping protein and Arpc1 on bundle membrane association is manifest. Under normal circumstances, this late bundle attachment activity is not essential, because bundles are already associated with the membrane. Similarly, in cases where snarls are reduced and excess bundles form (arp3, and presumably, arpc1 homozygous) this late attachment activity is not needed because the bundles form initially against the membrane. Obviously, other as yet unidentified proteins must be important for membrane association at early times (Frank, 2006).
Capping protein has previously been observed to be required for attachment of actin filaments to the Z disks during myofibrillogenesis; a role in membrane attachment in bristles would be consistent with this function. It has been suggested that Arpc1 (therein referred to as p41-Arc) might serve as a link to the membrane through its WD repeats binding to pleckstrin homology domains, which could in turn bind to phosphatidyl inositol 4,5-bisphosphate. Additionally, Arpc1 binds to the activating domain of WASp, which is itself activated at the membrane (Frank, 2006).
It is suggested that Arpc1 has a function outside the Arp 2/3 complex. Although work in yeast demonstrating an unique function for Arpc1 was later shown to be an experimental artifact, recent studies in the moss Physcomitrella patens has revealed that removal of Arpc1 results in different phenotypes than removal of Arpc4. Thus, it will be important to explore this phenomenon of potential differential Arp2/3 complex subunit functions further in the future (Frank, 2006).
Tissue morphogenesis requires stereotyped cell shape changes, such as apical cell constriction in the mesoderm and cell intercalation in the ventrolateral ectoderm of Drosophila. Both processes require force generation by an actomyosin network. The subcellular localization of Myosin-II (Myo-II) dictates these different morphogenetic processes. In the intercalating ectoderm Myo-II is mostly cortical, but in the mesoderm Myo-II is concentrated in a medial meshwork. Spacial constriction is repressed by JAK/STAT signalling in the lateral ectoderm independently of Twist. Inactivation of the JAK/STAT pathway causes germband extension defects because of apical constriction ventrolaterally. This is associated with ectopic recruitment of Myo-II in a medial web, which causes apical cell constriction as shown by laser nanosurgery. Reducing Myo-II levels rescues the JAK/STAT mutant phenotype, whereas overexpression of the Myo-II heavy chain (also known as Zipper), or constitutive activation of its regulatory light chain, does not cause medial accumulation of Myo-II nor apical constriction. Thus, JAK/STAT controls Myo-II localization by additional mechanisms. Regulation of actin polymerization by Wasp, but not by Dia, is important in this process. Constitutive activation of Wasp, a branched actin regulator, causes apical cell constriction and promotes medial 'web' formation. Wasp is inactivated at the cell cortex in the germband by JAK/STAT signalling. Lastly, wasp mutants rescue the normal cortical enrichment of Myo-II and inhibit apical constriction in JAK/STAT mutants, indicating that Wasp is an effector of JAK/STAT signalling in the germband. Possible models are discussed for the role of Wasp activity in the regulation of Myo-II distribution (Bertet, 2009).
Myo-II subcellular localization controls different cell shape changes such as cell constriction or intercalation. The data shed new light on the mechanisms of the subcellular localization of actomyosin networks in the early Drosophila ectoderm. VL ectodermal cells intercalate via the cortical recruitment of Myo-II at AJs, which drives polarized junction remodelling. This contrasts with the behaviour of immediately adjacent cells in the mesoderm, which undergo apical constriction and recruit Myo-II into a medial apical web. The data indicate that the cortical enrichment of Myo-II in ectodermal intercalating cells is not a 'default pathway', and requires at least activity of the JAK/STAT pathway. Indeed, in JAK/STAT pathway mutants, Myo-II is aberrantly recruited in a medial apical meshwork and cells consequently undergo apical constriction. This is surprising, as apical constriction is normally only observed in mesodermal ventral cells and is considered to be a unique attribute owing to their selective expression of Twist and Snail. Twist and Snail induce expression of the ligand Fog in the ventral cells only, which activates RhoGEF2, Rok and Myo-II. It also regulates expression of the transmembrane protein T48, which participates in the apical recruitment of RhoGEF2 and contributes to apical constriction. However it is not clear whether activation of the RhoGEF2 pathway is sufficient to drive the apical medial recruitment of Myo-II. This study shows that apical constriction is not simply induced in mesodermal cells by Fog, but is also prevented in ectodermal cells by activity of the JAK/STAT pathway and that this is essential for germ-band extension (GBE). In JAK/STAT pathway mutants, ectodermal cells undergo apical constriction despite the absence of ectopic Twist expression. Note, however, that apical constriction is not as rapid in these mutants as in mesodermal cells, so Twist and Snail accelerate or render more efficient the capacity to apically constrict. Moreover, the fact that Wasp mediates JAK/STAT function in the ectoderm but is not required in the mesoderm indicates that the mechanisms promoting medial Myo-II in mesoderm cells are likely to be different (Bertet, 2009).
These findings provide a novel opportunity to investigate the regulation of cortical or medial Myo-II localization in the ectoderm. The data document two novel features of this regulation (Bertet, 2009).
MRLC (Sqh) phosphorylation by the RhoGEF2 and the Rok pathway are both necessary for apical constriction; lowering the dose of RhoGEF2, Rho or Rok suppress the apical constriction observed in upd mutants. However, neither constitutive activation of this pathway by expression of a phosphomimetic form of Sqh, ShqE20E21, which rescues Rok inhibition, nor overexpression of MHC (Zip) is sufficient to promote medial accumulation of Myo-II. The medial accumulation of Myo-II requires additional regulation apart from the activation of Myo-II. Since RhoGEF2 and Rok are key regulators of Myo-II, this suggests that activation of the RhoGEF2/Rok pathway is necessary but not sufficient to explain medial Myo-II accumulation and apical constriction (Bertet, 2009).
This analysis of the JAK/STAT mutant phenotypes indicates a key role of Wasp in this process. Wasp is shown to be necessary for medial Myo-II accumulation, at least in ectodermal cells, and very strong activation of Wasp at the cortex (myrWasp) also causes medial Myo-II accumulation. Moreover, although Wasp is normally downregulated in VL ectodermal cells, in JAK/STAT pathway mutants Wasp is strongly recruited and hence activated at the plasma membrane, which suggests that JAK/STAT signalling represses the membrane activation of Wasp. Importantly, lowering the dose of Wasp maternally suppresses medial accumulation of Myo-II in upd mutants, and restores prominent accumulation at the cortex, as in wild-type embryos. Consistent with this, ectopic apical constriction is completely rescued in these double mutant embryos (Bertet, 2009).
Dia and Wasp play different roles in the regulation of Myo-II localization. Consistent with previous data, Dia controls the amount of apical Myo-II, but the specific localization of Myo-II at the cortex or in the medial network is not affected by loss of Dia. Dia promotes polymerization of non-branched filaments, and might control the formation of a good substrate for the stable association of Myo-II minifilaments. The fact that in dia heterozygotes the amount of apical Myo-II is reduced indicates that the amount of actin filaments might be limiting and controlled. Indeed, more F-actin is detected at the cortex of intercalating cells, preceding by a few minutes the enrichment of Myo-II. The role of Wasp is more surprising and unique; it is shown to mediate specifically repression of medial Myo-II accumulation and, hence, cell constriction in the germband. Because activation of Wasp leads to activation of medial web formation and reduction of Wasp dosage rescues cortical Myo-II in JAK/STAT mutants, it is concluded that Wasp controls an essential feature of Myo-II subcellular localization that is essential for the regulation of apical constriction. How does Wasp control Myo-II localization? Two non-exclusive models. In the first model, Wasp controls actin branching through activation of the Arp2/3 complex. Because Wasp has been implicated in endocytosis via Arp2/3 in Drosophila, Wasp could promote Myo-II web formation indirectly by regulating endocytosis of a surface protein required to anchor the medial actomyosin network at the membrane, such as E-cadherin. Consistent with this, downregulation of E-cadherin by RNAi disrupts the faint medial Myo-II pool. In mesodermal cells, E-cadherin appears to anchor the strong medial Myo-II pool. In the second model, Wasp might act more directly via the regulation of actin network architecture and its impact on the dynamic interactions between the medial web and the cortex, and thereby might affect the steady-state distribution of Myo-II exchanging between these two pools. Although Wasp uniquely mediates Myo-II regulation via JAK/STAT in the ectoderm and not in the mesoderm, regulation of Arp2/3 might be more generally implicated in the control of Myo-II regulation (Bertet, 2009).
Although wasp is an important mediator of JAK/STAT function in the ectoderm, it is unlikely to be the only one. Indeed wasp mutants rescue the cortical accumulation of Myo-II and apical constriction in upd mutants, but GBE is still strongly affected; it was noticed that cortical Myo-II distribution was not properly polarized in the plane of the epithelium. This suggests that other subcellular processes are also perturbed in the mutant. The fact that a reduction of Myo-II levels suppresses the upd defects indicates that the overall dosage of Myo-II is important as well. Identifying the transcriptional targets of JAK/STAT might shed light on its complex regulatory role during embryonic morphogenesis (Bertet, 2009).
Finally, although this work identifies an important regulator of Myo-II network subcellular distribution in epithelial cells, it is still not clear what regulates the polarized distribution of Myo-II at the cortex (Bertet, 2009).
JAK/STAT signalling controls a number of developmental processes. Importantly, this pathway has been implicated in diverse morphogenetic processes, such as convergent extension movements in the zebrafish embryo, hindgut elongation in Drosophila embryos, which probably involves intercalation movements as well, and posterior spiracle morphogenesis in Drosophila embryos. JAK/STAT signalling also controls border cell migration. The data indicate that JAK/STAT signalling plays an important and hitherto unappreciated morphogenetic function in gastrulating embryos. These data document evidence that JAK/STAT controls, via Wasp, a morphogenetic switch based on the regulation of medial or cortical Myo-II distribution. Interestingly, dorsal cells do not undergo apical constriction in JAK/STAT mutants. Indeed, dorsal cells exhibit neither cortical nor medial web Myo-II and are thus unable to participate in profound tissue remodelling. It appears that DV patterning provides a first general subdivision within the embryonic epithelium whereby Myo-II is globally repressed dorsally, and activated laterally and ventrally. Cortical or medial web distribution then results from the combinatorial input of Fog and JAK/STAT (Bertet, 2009).
The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. The formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, was found to be essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, it was demonstrated that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together these data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease (Deng, 2015).
This study provides the first evidence that Dia is essential for Drosophila myoblast fusion. Dia is expressed in all myoblasts and is recruited to the myoblast fusion site. The spatial and temporal distribution of Dia at the fusion site parallels that of the F-actin focus, which forms the core of an invasive podosome. This actin rich podosome is critical for FCM invasion of the FC/Myotube during fusion. In keeping with its expression pattern, Dia is essential for myoblast fusion progression: both loss and gain of Dia function lead to a fusion block. Under both conditions, the integrity of the F-actin focus and hence the invasive podosome is compromised; myoblasts expressing DiaDN fail to form the focus, whereas myoblasts expressing DiaCA have many filopodia and have a diffuse organization of F-actin, both of which contribute to a failure in invasive podosome formation and fusion. Dia activity is required after FC/Myotube and FCM recognition and adhesion but upstream of Arp2/3 activity. It is required, in parallel with PI(4,5)P2 signaling, to build a functional F-actin focus at the fusion site. These experiments further indicate that Dia activity is critical for actin dynamics at the fusion site, which, in turn, regulate fusion progression. Moreover, the aberrant F-actin organization at the fusion site in both loss and gain of function is also due to altered localization of the Arp2/3 regulators, SCAR and WASp. Taken together, these data support a role for the formin Dia in a critical first step of actin polymerization at the fusion site, downstream of cell-cell recognition and adhesion, and link its activity to the formation of F-actin foci, required for myoblast fusion (Deng, 2015).
Actin remodeling is critical for myoblast fusion, but Arp2/3 was the only known actin polymerization factor that was shown to be necessary for myoblast fusion. This study now shows that the formin Dia is also required during myoblast fusion. Whereas Arp2/3 preferably binds to pre-existing actin filaments and generates uncapped F-actin, formins nucleate F-actin both de novo and from the barbed ends of pre-existing actin filaments. Thus, Dia can generate actin filaments de novo, which Arp2/3 can bind or elongate (Deng, 2015).
This study also shows that the level of Dia activity is critical for myoblast fusion. Too much actin polymerization leads to too many filopodia and absence of an invasive podosome with its characteristic F-actin core. Too little polymerization leads no actin focus and no podosome formation. FRAP data with DiaCA also hint at whether a limited pool of actin is available for the actin polymerization factors during myoblast fusion. Despite the high rates of actin turnover with expression of DiaCA, the final fluorescence levels of actin returns to the same value as in controls. Additional actin monomers are not recruited to the site, even with high levels of polymerization activity. Interestingly, the rate of actin turnover has also been measured in mutants that affect Arp2/3 activity: specifically, mutations in blow, which regulates the Arp2/3 NPF WASp, show lower rates of actin exchange than in controls, due to a reduced exchange rate for WASp on the barbed ends of actin at the fusion site. Together these data suggest future experiments aimed at examination of whether rates of actin polymerization regulated by both Dia and Arp2/3 are optimized for the available actin pool and tightly controlled for myoblast fusion to properly occur (Deng, 2015).
Both cooperative and antagonistic functions between Dia and Arp2/3 have been reported. This study demonstrates that the coordinated and cooperative activities of these two actin polymerization factors leads to the formation of the F-actin focus. With the exception of sltr/Dwip/vrp mutants that form a focus of wild-type size, single mutants in the Arp2/3 NPF pathways, WASp and SCAR, lead to enlarged foci; however, double mutants in WASp and SCAR pathways do not form foci. This is the same phenotype that is seen in myoblasts expressing the DiaDN. The data support Dia activity being upstream of WASp and SCAR activation of Arp2/3 at the fusion site. This suggests that, at the fusion site, Dia initially provides the necessary context upon which Arp2/3 can act and not vice versa, as has been suggested in other contexts in which linear actin filaments emerge from Arp2/3 based structures. Nevertheless, both sets of actin regulators are necessary for F-actin focus formation that provides the core of the invasive podosome. Neither Dia nor Arp2/3 alone are sufficient (Deng, 2015).
The interplay between Dia and Arp2/3 at the fusion site is also reflected by the localization studies. Too little or too much Dia activity resulted in improper localization and, by extension, improper activity of Arp2/3 NPFs. How could Dia regulate this localization? One possibility is that Dia indirectly regulates Arp2/3 localization. Dia could nucleate linear actin filaments, which then would provide the necessary substrate for recruitment, maintenance and /or activation of Arp2/3 and its regulators, such as the WASp-WIP complex. Another possibility is that Dia, through its interactions with members of the SCAR/WAVE complex such as Abi, may directly localize and/or maintain the localization of Arp2/3 regulators, which are then activated at the fusion site. Abi has been reported to bind directly with Dia in vitro, and this interaction is required for the formation and stabilization of cell-cell junctions. Dia likely changes the localization and integrity of the SCAR/WAVE complex by competitively binding to the N-terminal part of Abi, dissociating Kette/Nap1 from the complex, and thus changing the stability and localization of SCAR/WAVE. It has also been established that the recognition and adhesion receptor, Sns, is capable of recruiting the Arp2/3 NPFs, such as WASp, to the fusion site. While Sns is still clustered at the fusion site in DiaDN and DiaCA, its recruitment activity appears not sufficient for focus formation capable of supporting an invasive podosome (Deng, 2015).
This study has shown that localization of Arp2/3 NPFs is affected in Dia loss and gain of function. In addition to this spatial control, another important way of controlling Arp2/3 activity is through activation of the NPFs via small GTPases. SCAR is activated through Rac-dependent dissociation from SCAR inhibitory complex. WASp is activated by binding to Cdc42, which releases it from auto-inhibited state. This study did not examine the localization of these activated GTPases. However, previous work has shown that PI(4,5)P2 signaling is required for proper localization of activated Rac at the fusion site. How the localization and activity of small GTPases at the fusion site contribute to the spatial and temporal interplay between Dia and Arp2/3 regulation of actin polymerization requires further investigation (Deng, 2015).
It remains unresolved how Dia itself is recruited to the fusion site. The data suggest that the recognition and adhesion receptors Duf and Sns would be involved either directly or indirectly in recruiting Dia to the fusion site, as embryos that fail to express either of these adhesion receptors fail to recruit Dia to the fusion site. In addition, recent data from Drosophila epithelial tubes indicate that PI(4,5)P2 serves as a localization cue for Dia. Previous work has shown that PI(4,5)P2 accumulates at the fusion site after FC-FCM recognition and adhesion; sequestering of PI(4,5)P2 results in a significant fusion block. Therefore, whether PI(4,5)P2 regulates Dia localization at the fusion site was tested. Dia was found to be recruited to the fusion site in the PI(4,5)P2 sequestered myoblasts, suggesting that, in this context, PI(4,5)P2 signaling is not required for Dia localization. These data provide possible explanations for why in PI(4,5)P2 sequestering embryos, smaller actin foci are detected: the localized Dia may be sufficient to recruit low levels of Arp2/3 and its NPFs, which, upon activation, lead to the formation of small F-actin foci. Nevertheless, in the absence of PI(4,5)P2 signaling, Dia that is recruited to the fusion site is not sufficient to produce functional actin focus, capable of directing a fusion event. Recent work also indicates that charged residues in the N- and C-termini of mDia1 are sufficient both for mDia's clustering of PI(4,5)P2 and its own membrane anchorage. This interaction between mDia1 and PI(4,5)P2, in turn, regulates mDia1 activity. Whether such a mechanism is in play at the myoblast fusion site needs to be further investigated (Deng, 2015).
A working model is proposed for the interplay between the actin regulators during myoblast fusion. Dia is recruited to the fusion site upon engagement of the recognition and adhesion receptors by a yet-to-be determined mechanism. It is proposed that PI(4,5)P2 signaling at the fusion site regulates the localization and activation of downstream targets such as Rho-family of small GTPases. These small GTPases lead to the activation of Dia. Activated Dia, in turn, polymerizes linear actin filaments and, in combination with the recognition and adhesion receptors and PI(4,5)P2, recruits the Arp2/3 NPFs, SCAR and WASp. Activation of these Arp2/3 NPFs at the fusion site would be accomplished by small GTPases such as Rac. These, in turn, would activate Arp2/3, leading to branched actin and formation of the F-actin focus and the invasive podosome. Whether the Arp2/3 NPFs such as SCAR/WAVE would negatively regulate Dia to downregulate linear actin polymerization, as suggested for mDia2 in cell culture, or whether Dia competes with WASp for barbed end binding remains to be investigated. However, these mechanisms would underscore a switch from linear F-actin filopodium formation to the linear and branched F-actin invasive podosome-like structure that is necessary for fusion (Deng, 2015).
The actin focus formed at the fusion site is an F-actin rich, invasive podosome-like structure that has been suggested to provide a mechanical force for FCMs to invade the FC/Myotube. Similar invasive actin structures named invadosomes have been seen in different cell types, such as podosomes in macrophages and invadopodia in cancer cell. Arp2/3 is known to play a key role in invadosome formation, and recent studies have revealed the involvement of formins in developing invadosomes. The current data indicate that specific temporal and spatial interactions between the formin Dia and Arp2/3 are required for the actin focus and invasive podosome formation. The data thus provide new mechanistic insights for the interplay of Arp2/3 and Formins during invadosome formation in these contexts (Deng, 2015).
Wiskott-Aldrich Syndrome proteins (WASp) serve as important regulators of cytoskeletal organization and function. These modular proteins, which are well-conserved among eukaryotic species, act to promote actin filament assembly in response to cues from various signal transduction pathways. Genetic analysis has revealed a requirement for the single Drosophila
homolog, WASp, in cell-fate decisions governing specific neuronal lineages. This unique developmental context was used to assess the contributions of established signaling and cytoskeletal partners of WASp. Biochemical and
genetic evidence is presented that, as expected, Drosophila WASp performs its developmental role via the Arp2/3 complex, indicating
conservation of the cytoskeletal aspect of WASp function in vivo. In contrast, association with the key signaling molecules CDC42 and PIP2 is not an essential requirement, implying that activation of WASp function in vivo depends on additional or alternative signaling pathways (Tal, 2002).
Evidence presented in this study suggests that the role of WASp in cell fate determination in neural lineages involves established cytoskeletal partners of WASp, and in particular, the Arp2/3 protein complex. Binding
studies demonstrate a capacity for WASp to directly associate
with monomeric actin via WA, the C-terminal cytoskeleton-interacting
domain present in all WASp and WASp-related
proteins. In parallel, the WA domain of WASp is shown to interact with components of the Arp2/3 complex, the primary downstream target of signal transduction pathways operating through WASp family proteins. The in vivo significance of these associations, which are characteristic of WASp elements in general, is demonstrated by a dual genetic approach. The final
30 residues at the C-terminal end of the WA domain of WASp
prove necessary for rescue of WASp mutant phenotypes, while
mutations in the Arp2/3 complex subunit Arpc1 lead to
cell-fate transformations and neuronal excess during sensory
organ development, a distinct, WASp-like phenotype. Taken
together with the binding studies, these genetic observations
imply that engagement of the cytoskeletal machinery via the C-terminal WA domain is an essential aspect of WASp function in vivo (Tal, 2002).
Several additional inferences can be drawn from the
reported results, regarding the mechanism by which the
cytoskeleton-interacting domain of WASp operates. Significant function is retained after removal of the extreme C-terminal 15 residues, corresponding to the A (acidic) portion of the WA domain. This observation suggests
that the remaining WA sequences, comprising the so-called central (C) domain, contribute significantly to the functional interaction with Arp2/3, and is in good keeping with a recent study highlighting the importance of the C domain in WASp-based activation of the actin-nucleating
activity of Arp2/3. A second noteworthy
aspect is the inability of the full WASp WA domain to
rescue WASp mutant phenotypes on its own, even though
biochemical studies have repeatedly demonstrated constitutive
Arp2/3 activation by the isolated WA domains of
various WASp and WASp-related proteins. This
observation implies that N-terminal regions absent from
the truncated protein play important in vivo roles, beyond
their established capacity to relieve a self-inhibitory conformation.
These may include proper localization of the
activated protein to specific cellular sites of function (Tal, 2002).
The cellular mechanism by which WASp influences lineage
decisions during Drosophila development remains unknown.
The data presented here strongly suggest that the
functional requirement for WASp is mediated via the Arp2/3
complex, thereby implying that WASp-dependent cell-fate
specification involves reorganization of the actin-based
cytoskeleton. It remains to be seen just how this intriguing
connection between the cytoskeletal machinery and a key
developmental mechanism is carried out (Tal, 2002).
In contrast to the demonstration of a functional connection
in vivo between WASp and the established cytoskeletal
partners of WASp proteins in general, the data
suggest that association with the major established activators
of WASp, the small GTPase CDC42 and the
phosphoinositide PIP2, is not essential for the developmental
roles carried out by the Drosophila WASp homolog.
Characterization of the prototype WASp as a
CDC42-binding protein is a longstanding observation, and a functional connection between CDC42 activation of WASp elements and reorganization of the actin cytoskeleton via Arp2/3 has been firmly established. Association with PIP2 has gained prominence as
an alternative activating mechanism of WASp, while
optimal activation is achieved by the combined action of
both signaling molecules. The Drosophila WASp protein interacts with both CDC42 (in its activated state) and PIP2, and this association
maps to the well-conserved domains identified and characterized
as the CDC42 and PIP2 binding sites in WASp.
Elimination of these sites, however, does not interfere
with the ability of WASp transgenic constructs to rescue
WASp mutant phenotypes, suggesting that association with these elements, either separately or in combination, is not an essential aspect of WASp function during Drosophila development (Tal, 2002).
Several issues are raised by these unexpected observations
and warrant further discussion. One issue is the
basis for evolutionary conservation of the activator binding
sites, despite the absence of an essential developmental
role. This situation is not without precedence, and may indicate that the conserved sites function in a relatively subtle context, which the
phenotypic studies have failed to identify. A second, cardinal
issue is the implications these findings have for
understanding the WASp molecular pathway. In particular,
the possibility that elements other than CDC42 and PIP2
contribute significantly to WASp activation must be considered.
Elements of tyrosine-kinase signaling pathways constitute
possible alternative candidates, since several such molecules
have been shown to associate with and activate
WASp. This contribution could act in concert with the
functions performed by CDC42 and PIP2, but may well
provide the primary activating signal in this particular in
vivo setting. These observations thus suggest caution in
drawing inferences from in vitro studies, and underscore
the need for further work, with particular emphasis on
genetic screens designed to identify additional, physiological
activators of the WASp pathway (Tal, 2002).
Dock, the Drosophila ortholog of Nck, is an adaptor protein that is known to function in axonal guidance paradigms in the fly including proper development of neuronal connections in photoreceptor cells and axonal tracking in Bolwig's organ. To develop a better understanding of axonal guidance at the molecular level, proteins in a complex with the SH2 domain of Dock were purified from fly Schneider 2 cells. A protein designated p145 was identified and shown to be a tyrosine kinase with sequence similarity to mammalian Cdc-42-associated tyrosine kinases. It was demonstrated that Drosophila Ack (DAck) can be co-immunoprecipitated with Dock and the sorting nexin DSH3PX1 from fly cell extracts. The domains responsible for the in vitro interaction between Drosophila Ack and Dock were identified, and direct protein-protein interactions between complex members were established. It is concluded that DSH3PX1 is a substrate for DAck in vivo and in vitro, and one of the major in vitro sites of DSH3PX1 phosphorylation was found to be Tyr-56. Tyr-56 is located within the SH3 domain of DSH3PX1, placing it in an important position for regulating the binding of proline-rich targets. It was demonstrated that Tyr-56 phosphorylation by DAck diminishes the DSH3PX1 SH3 domain interaction with the Wiskott-Aldrich Syndrome protein while enabling DSH3PX1 to associate with Dock. Furthermore, when Tyr-56 is mutated to aspartate or glutamate, the binding to Wiskott-Aldrich Syndrome protein is abrogated. These results suggest that the phosphorylation of DSH3PX1 by DAck targets this sorting nexin to a protein complex that includes Dock, an adaptor protein important for axonal guidance (Worby, 2001).
nwk (nervous wreck), a temperature-sensitive paralytic mutant, causes excessive growth of larval neuromuscular junctions (NMJs), resulting in increased synaptic bouton number and branch formation. Ultrastructurally, mutant boutons have reduced size and fewer active zones, associated with a reduction in synaptic transmission. nwk encodes an FCH and SH3 domain-containing adaptor protein that localizes to the periactive zone of presynaptic terminals and binds to the Drosophila ortholog of Wasp (Wsp), a key regulator of actin polymerization. wsp null mutants display synaptic overgrowth similar to nwk and enhance the nwk morphological phenotype in a dose-dependent manner. Evolutionarily, Nwk belongs to a previously undescribed family of adaptor proteins that includes the human srGAPs, which regulate Rho activity downstream of Robo receptors. It is proposed that Nwk controls synapse morphology by regulating actin dynamics downstream of growth signals in presynaptic terminals (Coyle, 2004).
Several lines of evidence suggested that Nwk might interact with Wsp: (1) the SH3a domain of Nwk is between 35% and 45% identical to the three SH3 domains of Dock. Nck, a mammalian homolog of Dock, binds to Wasp via its SH3 domains. (2) Bzz1p, the yeast ortholog of Nwk, binds to Las17p, the yeast ortholog of Wasp (Soulard, 2002), via its SH3 domains. (3) wsp mutants have a morphological phenotype similar to nwk at larval NMJs (Coyle, 2004).
To evaluate whether Nwk and Wsp interact biochemically, affinity chromatography was performed using GST-fusion proteins containing each of the two Nwk SH3 domains. The construct containing the SH3a domain, but not the one containing the SH3b domain, precipitated Wsp immunoreactivity from Drosophila head homogenates with high affinity (Coyle, 2004).
A yeast two-hybrid protein binding assay further indicated that Nwk and Wsp can interact directly. Two-bait constructs expressing either full-length Nwk or a subfragment consisting of the two SH3 domains plus the C terminus stimulates transcription of reporter genes in yeast cells cotransformed with a full-length Wsp prey. Colonies grew rapidly on selectable media and expressed β-gal. In contrast, bait constructs containing the FCH and ARNEY domains of Nwk but lacking the SH3 domains did not activate reporter genes when cotransformed with Wsp prey, indicating that the SH3 domains are required for binding with Wsp. Single transformation of any Nwk bait or Wsp prey alone did not stimulate transcription of reporter genes at substantial levels (Coyle, 2004).
A previous study of Drosophila wsp mutants revealed defects in sensory cell proliferation during PNS development, but a role at NMJs had not been investigated (Ben-Yaacov, 2001). Therefore, to determine whether endogenous Nwk and Wsp are capable of interacting in vivo, the distribution of Wsp at the NMJ was examined by immunocytochemistry. Wsp was found to be localized diffusely at synaptic boutons in irregular patches. Postsynaptically, Wsp is associated with the subsynaptic reticulum (ssr), which was revealed by colocalization with the ssr marker, Dlg, and by reduction of immunoreactivity in pak mutants that reduce the ssr. There is also a significant presynaptic component of Wsp immunoreactivity that is most evident in thin (0.25 μm) optical sections through the center of boutons. Wsp is clearly present both inside the bouton as well as in association with the surrounding ssr. Although the presynaptic distribution of Wsp is not continuous throughout the periactive zone, regions of overlap with Nwk can be observed in thin optical confocal sections through double-labeled boutons. Thus, Nwk and Wsp colocalize in spatially restricted regions of the periactive zone. Nwk is not required for Wsp localization since Wsp immunoreactivity is not obviously disrupted in nwk mutants (Coyle, 2004).
Wasp promotes the nucleation and branching of F-actin. Phalloidin staining revealed that F-actin is enriched around synaptic boutons and generally colocalizes with Wsp. The overall abundance and distribution of F-actin appear normal in wsp mutants, indicating that Wsp is not the only factor promoting actin polymerization at the synapse. The Drosophila genome contains one other wsp family member, scar, whose function may overlap with wsp, although its role at synapses has not been investigated. Thus, loss of Wsp may have only a limited, localized effect on actin dynamics and architecture without completely abolishing the cortical cytoskeleton (Coyle, 2004).
Regulation of growth cone and cell motility involves the coordinated
control of F-actin dynamics. An important regulator of F-actin formation is
the Arp2/3 complex, which in turn is activated by Wasp and Wave. A complex
comprising Kette/Nap1, Sra-1/Pir121/CYFIP, Abi and HSPC300 modulates the
activity of Wave and Wasp. This study presents the characterization of
Drosophila Sra-1 (specifically Rac1-associated protein 1).
sra-1 and kette are spatially and temporally co-expressed,
and both encoded proteins interact in vivo. During late embryonic and larval
development, the Sra-1 protein is found in the neuropile. Outgrowing
photoreceptor neurons express high levels of Sra-1 also in growth cones.
Expression of double stranded sra-1 RNA in photoreceptor neurons
leads to a stalling of axonal growth. Following knockdown of sra-1
function in motoneurons, abnormal neuromuscular junctions were noted, similar to
what was determined for hypomorphic kette mutations. Similar mutant
phenotypes were induced after expression of membrane-bound Sra-1 that lacks
the Kette-binding domain, suggesting that sra-1 function is mediated
through kette. Furthermore, both proteins
stabilize each other and directly control the regulation of the F-actin
cytoskeleton in a Wasp-dependent manner (Bogdan, 2004).
Wasp proteins are auto-inhibited, whereas the Wave proteins are trans-inhibited. Both usually require small G proteins of the Rho family for activation. In the case of Wasp, activated, GTP-bound Cdc42 binds to the CRIB (Cdc42/Rac Interactive Binding) domain of Wasp, releasing the auto-inhibition and thereby leading to the activation of the Arp2/3 complex. However, a structure-function analysis of the Drosophila Wasp has demonstrated that the Cdc42-binding domain is not strictly necessary for function, suggesting that alternative pathways, such as phosphorylation can activate WASP. Indeed, some tyrosine kinases have been shown to activate Wasp by phosphorylation (Bogdan, 2004 and references therein).
In contrast to Wasp, Wave is not auto-inhibited. It is kept in an inactive state through association with a protein complex comprising Kette/Nap1, Sra1 (specifically Rac associated 1, Sra-1; also called CYFIP/p140Sra-1, from here on called Sra-1 according to FlyBase) and the Abelson-interactor protein (Abi) (Eden, 2002). Upon dissociation or conformational changes of this complex, Wave is assumed to be active. Thus, Kette or Sra-1 should antagonize Wave function. This is supported by genetic studies in Dictyostelium and Drosophila. Cell culture experiments show that Wave is degraded in a Ubiquitin-dependent manner following disruption of the Sra-1/Kette complex. These latter findings are likely to reflect the fast inactivation of Wave once activated. In vitro, activation of Wave can be mediated by Rac1 or SH3 domains, which presumably bind to Sra-1 (Bogdan, 2004 and references therein).
The Sra-1/Kette protein complex is not only required to negatively regulate the activity of Wave but is also able to activate Wasp function at the membrane. The interaction of Kette and Wasp is not direct but is likely to be mediated by the Abi, which can bind to both Kette and Wasp. Interestingly, the Nck adapter protein is also able to bind to Wasp via its third SH3 domain. Thus, Sra-1, which can bind to the first SH3 domain of Nck is a good candidate to locate the Sra-1/Kette complex to the membrane close to Wasp (Bogdan, 2004 and references therein).
To test whether the mutant phenotypes of sra-1 and kette are alike as predicted, and whether Sra-1 indeed acts through Kette to regulate actin dynamics, a functional characterization of Sra-1 during Drosophila development was conducted. Sra-1 and Kette are both required for axonal growth and perform common functions during formation and maturation of neuromuscular junctions (NMJ). Analysis of temporal and spatial distribution of the Sra-1 protein shows a prominent co-expression with Kette. Both proteins are maternally expressed and later in development become concentrated in the developing nervous system (CNS). Sra-1 is highly expressed in growth cones and neuromuscular synapses. Direct interaction of Sra-1 and Kette depends on a short C-terminal domain of the Sra-1 protein. Expression of a Sra-1 variant lacking the C-terminal domain leads to a dominant-negative phenotype that can be suppressed by expression of an activated Kette protein. In tissue culture cells as well as in vivo Sra-1 function is required for F-actin organization. Further genetic analyses demonstrate that Sra-1 function at the membrane depends on the presence of Wasp (Bogdan, 2004).
In addition to the CNS phenotypes, mutations in sra-1 and
kette both lead to synaptic defects that are characterized by an
overall reduction in size of the neuromuscular junction, as well as the
induction of supernumerary buds in sra-1 and kette mutant
synaptic boutons. It is known that new boutons often arise from existing ones
by asymmetrical budding or symmetrical division, which in
turn requires an intact regulation of the actin cytoskeleton.
The increased number of branches as well as the bulged appearance of the
synaptic boutons after depletion of kette or sra-1 function
may reflect their function in regulating wasp.
Indeed wasp mutants display synaptic phenotypes similar to those of
sra-1 and kette. Recently, it has been found that the adaptor protein
Nervous wreck (Nwk) binds Wasp and is also required for normal synapse
morphology. Thus, Nwk might act as a scaffolding protein in the synapse
assembling a Wasp activation complex comprising Sra-1, Kette and Abi (Bogdan, 2004).
As in Drosophila, mutations in several of the vertebrate orthologs
of the above mentioned genes are associated with learning deficits,
demonstrating the pivotal importance of F-actin dynamics for precise neuronal
function (Bogdan, 2004).
Rapid remodeling of the F-actin cytoskeleton is mostly brought about by the
Arp2/3 complex, which in turn is activated by members of the Wasp and Wave
protein families. Wasp as well as Wave are potent F-actin nucleation factors.
Obviously within the cell their activity must be tightly regulated. Whereas
Wasp is auto-inhibited, Wave is trans-inhibited and requires the inhibiting
Sra-1 Kette protein complex. Upon dissociation of this complex or conformational changes within the complex, Wave is active and presumably remains active until it is
degraded via ubiquitination. This latter mechanism, which is frequently used in
regulating the effective concentration of active proteins, ensures that
Wave activity lasts for only a short time period (Bogdan, 2004).
Wave is not the only protein of the complex that is
degraded upon disruption of the protein complex. Depletion of Kette not only
leads to a loss of Wave but also of Sra-1. Vice versa, depletion of Sra-1
leads to a loss of Kette. Thus, ultimately the stability of all proteins of
the inhibitory Sra-1 complex appears to be interdependent (Bogdan, 2004).
Kette can activate Wasp-mediated F-actin
formation. Sra-1 function also depends on Wasp. In
both cases, the membrane localization of Kette or Sra-1 is essential,
indicating that in vivo regulation of membrane recruitment of Sra-1 and Kette
is important for function. The data presented in this work also suggest that
membrane-bound Sra-1 or Kette proteins are both able to activate Wasp
independently of each other (Bogdan, 2004).
Vertebrate homologues of Sra-1 and Kette were first identified in a complex
with the SH2 SH3 adapter Nck. The N-terminal SH3 domain of Nck is thought to bind to Sra-1, evoking a model where Nck recruits Sra-1 and the associated Kette protein to the membrane. kette and dock
which encodes the Drosophila Nck homologue interact during axonal
pathfinding. Cell signaling and cell adhesion leads to the activation of
a number of receptor systems which in turn mediate anchorage-dependent
recruitment of adapter proteins such as Nck. Nck in turn
is able to connect cell-surface receptors via different signal cascades to the
F-actin cytoskeleton (Bogdan, 2004 and references therein).
However, in vivo Nck cannot mediate all aspects of Sra-1/Kette function;
the complete loss of maternal and zygotic Nck results in similar but not
identical phenotypes when compared with kette or sra-1
loss-of-function phenotypes. In the developing synapse, the function of Nck in
recruiting Sra-1 and Kette to the membrane may be fulfilled by Nwk, which as
Nck also binds Wasp. Interestingly, both adaptor proteins are involved in Slit
Robo signaling, where they may mediate different biological effects. This
suggests that combinatorial and tissue specific factors are assembled in
response to specific cues to activate Wasp in different cell types or
compartments (Bogdan, 2004).
Formation of syncytial muscle fibers involves repeated rounds of cell fusion between growing myotubes and neighboring myoblasts. Wsp, the Drosophila homolog of the WASp family of microfilament nucleation-promoting factors, is an essential facilitator of myoblast fusion in Drosophila embryos. D-WIP (termed Verprolin 1 in FlyBase), a homolog of the conserved Verprolin/WASp Interacting Protein family of WASp-binding proteins, performs a key mediating role in this context. D-WIP, which is expressed specifically in myoblasts, associates with both the WASp-Arp2/3 system and with the myoblast adhesion molecules Dumbfounded and Sticks and Stones, thereby recruiting the actin-polymerization machinery to sites of myoblast attachment and fusion. This analysis demonstrates that D-WIP recruitment is normally required late in the fusion process, for enlargement of nascent fusion pores and breakdown of the apposed cell membranes. These observations identify cellular and developmental roles for the WASp-Arp2/3 pathway, and provide a link between force-generating actin polymerization and cell fusion (Massarwa, 2007).
The evolutionarily conserved Arp2/3 protein complex is the primary microfilament-nucleating machinery in eukaryotic cells. To perform its diverse cellular roles, the complex must first be activated by nucleation-promoting factors (NPFs), such as members of the WASp and WAVE/SCAR protein families. These elements serve as essential mediators, linking signal-transduction pathways and Arp2/3-based actin polymerization. Actin polymerization triggered by this system is translated into forces that drive a variety of key cellular functions, including cell locomotion, motility of membrane-bound particles within cells, and formation of endocytic vesicles (Massarwa, 2007).
A major challenge in the field is the assignment of physiological roles to this potent cellular machinery during the development of multicellular organisms. While genetic approaches in model organisms have shown promise in this regard, the numerous and sometimes overlapping roles assigned to the Arp2/3 system often prove difficult to separate. Previous work has shown that Wsp, the Drosophila WASp homolog, acts as an Arp2/3 activator in restricted developmental contexts, thus allowing for characterization of Arp2/3 function in vivo. This approach was used to reveal an unexpected involvement of the WASp-Arp2/3 system in myogenesis. Specifically, this system is shown to play a distinct role in myoblast fusion during Drosophila embryogenesis (Massarwa, 2007).
Somatic muscle fibers in the mature Drosophila embryo are comprised of multinucleated cells that form by multiple rounds of fusion between two distinct myoblast subpopulations. After the initial specification of the mesoderm, each embryonic trunk hemi-segment contains ~30 'founder cell' myoblasts, which will direct muscle formation and differentiation, and a large number of fusion-competent myoblasts (FCMs). Founder cells possess the information necessary for determining the identity and size of the individual somatic muscles, while the FCMs serve as a repository that will add cytoplasmic bulk to each muscle fiber (Massarwa, 2007).
Recognition and association of founder cells and FCMs are based on heterotypic interactions between differentially expressed immunoglobulin superfamily cell-surface proteins. Founder cells express Dumbfounded (Duf) and the closely related Roughest (Rst), which serve as attractants for FCMs. Physical association between Duf/Rst and the FCM-specific protein Sticks and Stones (SNS) provides a key step in myoblast adhesion and alignment of the myoblast cell membranes. Founder cells initially fuse with one or two FCMs, leading to the formation of bi-/trinuclear muscle precursors. A second, major phase of muscle growth then ensues, in which the precursor myotubes undergo successive rounds of fusion with multiple FCMs. In addition to the cell-adhesion molecules, genetic approaches have revealed a number of elements that contribute to various steps of the fusion process, including transcription factors, signaling molecules, and cytoskeleton-associated proteins (Massarwa, 2007).
This study demonstrates that function of the WASp-Arp2/3 system is essential for the second phase of myoblast fusions, between maturing myotubes and FCMs, and acts after formation of fusion pores in the double membrane of the apposed cells. Recruitment of the WASp-Arp2/3 system to founder cell-FCM attachment sites is achieved via D-WIP, a Drosophila homolog of the Verprolin/WASp Interacting Protein (Vrp/WIP) family. Functional associations with members of this protein family constitute an evolutionarily conserved feature of WASp activity. D-WIP is specifically expressed in myoblasts and associates with the cell-surface proteins that mediate adhesion between founder cells and FCMs, thereby establishing a critical link between the cellular machineries that govern fusion and microfilament dynamics. These findings present a novel tissue context for the involvement of the Arp2/3 system in physiological events and extend the functional applications of the forces generated by actin polymerization to a central process of tissue morphogenesis (Massarwa, 2007).
This study has identified an exceptional and highly cell-type-specific mode for regulating the Arp2/3 system. Functional selectivity in this system is usually achieved via spatial and temporal control over the operation of signal-transduction pathways and the resulting production of potent activating elements for the relevant Arp2/3 nucleation-promoting factor. In contrast, it is the restricted expression of D-WIP in the FCMs that confines Wsp-mediated triggering of Arp2/3 activity to the fusing myoblasts of Drosophila embryos. Transcriptional control over D-WIP expression, governed directly or indirectly by the Lame Duck (Lmd) transcription factor, thus provides a means for translating embryonic patterning schemes into distinct and specific cellular activities, which can profoundly influence cell morphology (Massarwa, 2007).
The structural basis for the interaction between D-WIP and Wsp is consistent with the established principles of Vrp/WIP-WASp protein association, which rely on an interaction between an ~25 residue long peptide from the extreme C-terminal region of Vrp/WIP proteins and the WH1/EVH1 N-terminal region of WASp proteins. Most critical residues within these domains are conserved in the Drosophila homologs. Moreover, genetic data and S2 cell localization observations strongly implicate these domains in mediating physical association between the two proteins (Massarwa, 2007).
By virtue of its association with the cell-surface adhesion proteins Duf and SNS, expressed in founder cells and FCMs, respectively, D-WIP may impose a common functionality on these distinct myoblast types. Yet to be determined, however, is the nature of the interaction between D-WIP and the myoblast-attachment machinery, and whether this interaction is constitutive or is dependent upon founder cell-FCM contact. Colocalization in both developing embryonic muscles and aggregated S2 cells, as well as the coimmunoprecipitation of D-WIP and Duf, underlies the suggestion of a physical association, but whether this association is direct requires further investigation (Massarwa, 2007).
The lack of significant sequence homology between the cytoplasmic portions of the Duf and SNS proteins, and the comparatively tighter correspondence between D-WIP and SNS localizations, may be indicative of distinct modes of association between D-WIP and the two types of adhesion proteins. It is interesting to note in this context that mammalian Nephrin, which shares structural and sequence similarities with SNS, employs direct binding of its cytoplasmic portion to the adaptor protein Nck, as a means of establishing a functional link to the actin-based cytoskeleton (Massarwa, 2007).
WASp-family proteins are thought to reside in an auto-inhibited conformation, which prevents productive interaction with Arp2/3 and is alleviated only by binding of signaling molecules. Scenarios consistent with a recruiting role for Vrp/WIP proteins have been described, including involvement of WASp in actin-based motility of intracellular pathogens and in cytoskeletal remodeling of the immune synapse. However, Vrp/WIP proteins on their own fail to stimulate, or may even inhibit, WASP-based Arp2/3 activation (Martinez-Quiles, 2001: Ho, 2004), implying a requirement for additional activating elements. The observation that WspMyr, a membrane-tethered form of Wsp, can partially compensate for loss of D-WIP function is consistent with an exclusive recruitment role for D-WIP. However, it should be born in mind that an additional step of Wsp activation may be required after its recruitment. Since the results of phenotypic rescue experiments further imply that established activators of WASp-type proteins such as CDC42 and PIP2 do not operate in this context, the identity of an independent Wsp activator during myoblast fusion, if one indeed exists, is currently unknown (Massarwa, 2007).
Activation of the Arp2/3 complex promotes the generation of branched networks of polymerizing actin filaments, in close proximity to both the cell surface and to internal cell membranes. The physical force liberated by this energetically favorable process can be harnessed to push against, or otherwise influence, membrane behavior. A key challenge stemming from the experimental observations is to identify the mechanism by which Arp2/3-based force production contributes to the progress of myoblast fusion (Massarwa, 2007).
The detailed TEM-level description of Drosophila myoblast fusion has stipulated a series of events, including formation of pores next to sites of accumulated electron-dense material along the apposed myoblast membranes, vesiculation/fragmentation of the membranes between the pores, and removal of the residual membrane material. Analysis of the D-WIP and Wsp mutant phenotypes demonstrates a requirement for the Arp2/3 system at a relatively late stage of the fusion process, after formation of the initial fusion pores (Massarwa, 2007).
Much of what is known about the mechanisms driving cell-cell (including myoblast) fusion relates to recognition and adhesion between pairs of cells and construction of initial fusion pores, while the more advanced processes of pore enlargement and the eventual establishment of full cytoplasmic continuity between the fusing cells remain mostly unexplored. The demonstration of a requirement for the cellular actin-polymerization machinery at these stages holds the promise of establishing a mechanistic basis for these late events (Massarwa, 2007).
Several possible mechanisms can be proposed for the manner by which polymerization-based forces drive fusion to completion, after initial pore formation. Pore enlargement during membrane fusion poses considerable energy requirements, which Arp2/3-based polymerization seems well suited to satisfy. The 'pushing' forces inherent in this cellular machinery can be applied to the contours of nascent fusion pores, thereby ensuring their continuous expansion. Alternatively, myoblast membranes may be broken down by vesiculation, akin to endocytosis. Detailed genetic and cellular studies have demonstrated essential roles for the Vrp/WIP-WASp-Arp2/3 machinery during endocytosis of clathrin-coated vesicles in budding yeast, and mechanistic interpretations of the forces involved have been put forward. In keeping with previous discussions of these issues, it is tempting to suggest that electron-dense structures, common to the contact sites of myoblasts in both Drosophila and vertebrate species, may provide a structural framework through which polymerization-based forces exert their influence. Finally, a role for the Arp2/3 machinery can be invisioned in an even more advanced step in the fusion process, namely, the final removal of residual, vesiculated membrane material from the disrupted sites of membrane contact to create full cytoplasmic continuity (Massarwa, 2007).
In summary, these observations linking myoblast cell-surface adhesion proteins in Drosophila embryos with the WIP/WASp module suggest a mechanism through which the conserved cellular machinery promoting force production via microfilament nucleation can be harnessed to drive muscle fiber formation to completion. Future studies will determine the finer mechanistic details of the cellular mechanism employed in this instance, and the degree to which this link can be generalized to myogenesis in vertebrate species, as well as other processes of cell fusion (Massarwa, 2007).
Myoblast fusion is an essential step during muscle differentiation. Previous studies in Drosophila have revealed a signaling pathway that relays the fusion signal from the plasma membrane to the actin cytoskeleton. However, the function for the actin cytoskeleton in myoblast fusion remains unclear. This study describes the characterization of solitary (sltr), a component of the myoblast fusion signaling cascade. sltr encodes the Drosophila ortholog of the mammalian WASP-interacting protein. Sltr is recruited to sites of fusion by the fusion-competent cell-specific receptor Sns and acts as a positive regulator for actin polymerization at these sites. Electron microscopy analysis suggests that formation of F-actin-enriched foci at sites of fusion is involved in the proper targeting and coating of prefusion vesicles. These studies reveal a surprising cell-type specificity of Sltr-mediated actin polymerization in myoblast fusion, and demonstrate that targeted exocytosis of prefusion vesicles is a critical step prior to plasma membrane fusion (Kim, 2007).
Characterization of Sltr provides a number of novel, and somewhat unexpected, findings concerning the role of the actin cytoskeleton in myoblast fusion. In contrast to the widespread expression of WIP in mammals, Sltr is specifically expressed in developing muscles and, moreover, only in fusion-competent myoblasts. Such cell-type specificity is unexpected, given that Sltr is the only WIP homolog in Drosophila. As a positive regulator of actin polymerization, Sltr is recruited to sites of fusion by the fusion receptor Sns and is required for the formation of F-actin-enriched foci at these sites. EM studies suggest that these actin-rich foci may provide directionality for the trafficking of prefusion vesicles, which are routed to ectopic membrane sites in the fusion-competent cells in sltr mutant embryos. It is suggested that targeted exocytosis of prefusion vesicles represents a critical step leading to plasma membrane fusion (Kim, 2007).
The identification of Antisocial/Rolling pebbles as a founder cell-specific protein that mediates signaling from the fusion receptor Duf to the actin cytoskeleton suggests the existence of a fusion-competent cell-specific protein(s) with an analogous function. The current work suggests that Sltr represents such a molecule. Not only is Sltr recruited to sites of fusion by the fusion-competent cell-specific receptor Sns, likely mediated by the small adaptor protein Crk, it also brings the actin polymerization machinery to these sites by binding to WASP and G-actin. As a result, Sltr colocalizes with F-actin-rich foci at sites of fusion, and is required for the formation of these actin foci in fusion-competent cells. Thus, like Ants/Rols7 in founder cells, Sltr is a fusion-competent cell-specific protein that links the fusion receptor with the actin cytoskeleton (Kim, 2007).
How does Sltr regulate actin polymerization? In vitro and in vivo assays demonstrate that this activity is mediated by the WH2 and WBD domains of Sltr, which bind to actin and WASP, respectively. In Drosophila S2 cells, overexpression of Sltr, but not mutant forms lacking the WH2 or WBD domains, leads to profound changes in actin cytoskeleton organization characterized by the formation of F-actin-filled microspikes. Likewise, the ability of Sltr to rescue sltr mutant embryos requires the WH2 and WBD domains. These observations suggest that both actin and WASP binding contribute to Sltr function. Interestingly, while the first WH2 domain of Sltr binds to G-actin, the second WH2 domain and its flanking region interact with F-actin. Thus, the actin-binding activity of Sltr serves a dual role -- it not only provides a pool of monomeric G-actin (in addition to the G-actin recruited by WASP) at sites of fusion but also stabilizes the newly formed actin filaments. The importance of the WASP-binding activity in Sltr function is further supported by the observation that RNAi knockdown of WASP abolished the ability of Sltr to induce microspikes in S2 cells, and that WASP itself is required for myoblast fusion in Drosophila. How WASP activity is regulated in myoblast fusion remains to be determined. Although mammalian WASP is known to be activated by the small GTPase Cdc42, this is unlikely the case in Drosophila myoblast fusion, as expression of a dominant-negative Cdc42 does not cause any fusion defects. Future experiments are required to identify the specific WASP activating factor(s) in myoblast fusion (Kim, 2007).
Myoblast fusion is a multistep process that includes cell recognition, adhesion, alignment, and membrane merger. To initiate the fusion process, fusion-competent cells that reside in a deeper mesodermal layer in the embryo need to migrate and extend filopodia toward founder cells that are in close contact with the ectoderm. Because the actin cytoskeleton is required for both cell migration and filopodia formation, one may predict that the actin cytoskeleton plays a role in these early events of myoblast fusion. Indeed, mutations in mbc and rac, components of the Mbc --> Rac --> WAVE pathway, produce a large number of round-shaped fusion-competent cells, suggesting a potential defect in myoblast migration and/or filopodia formation (Kim, 2007).
This study has identified a novel function of the actin cytoskeleton in a later step during myoblast fusion. This function is mediated by the Crk --> Sltr --> WASP pathway, which is independent of that of Mbc --> Rac --> WAVE; the recruitment of Sltr by the fusion receptor Sns is not affected by either mbc or rac. Using light microscopy, F-actin-rich foci have been observed localized to sites of fusion during myoblast adhesion, raising the possibility that localized actin polymerization at these sites may be functionally important for fusion. It was also demonstrated that the F-actin-rich foci are organized by the fusion receptor and actin cytoskeleton regulators, since they are absent in sns and sltr mutant fusion-competent cells. Taken together, these observations suggest a potential link between the fusion receptor, actin polymerization, and the membrane fusion machinery (Kim, 2007).
EM studies provide further insights into how localized actin polymerization may contribute to myoblast fusion by uncovering a relationship between the actin-rich foci and prefusion vesicles. The prefusion vesicles are of exocytic origin and are transported to the plasma membrane via the microtubule network. Immuno-EM analyses further revealed two classes of vesicles, one coated with and the other devoid of actin, at different subcellular locations. While naked vesicles are farther away from the actin foci, actin-coated vesicles are either within the foci or have reached the plasma membrane. That all vesicles at the membrane are actin-coated suggests that they have transited through actin foci and thus become distinct from the naked vesicles. Although the trafficking of these vesicles was not followed in real time due to the lack of vesicle-specific markers, the 'snapshots' provided by the EM analyses are most consistent with a model that actin foci at sites of fusion provide cortical capturing sites for the prefusion vesicles. This model is further supported by the transient nature of the actin-rich foci, which disintegrate in the cortical region after the prefusion vesicles have paired along the membrane in mature myoblasts. This model provides a plausible explanation for the mistargeting of vesicles in sltr mutant embryos - in the absence of actin foci at the prospective fusion sites, prefusion vesicles are randomly routed to the plasma membrane, resulting in their accumulation between adjacent founder and fusion-competent cells as well as between neighboring fusion-competent cells (Kim, 2007).
It is intriguing that in mature myoblasts, the prefusion vesicles that have aligned at the plasma membrane are all actin-coated, concurrent with an absence of the actin-rich patches at the cell cortex. Could actin coating of the prefusion vesicles, as well as disintegration of the actin-rich foci in mature myoblasts, play a role in vesicle-membrane fusion? The failure of vesicle-plasma membrane fusion in sltr mutant myoblasts with either too little (in fusion-competent cells) or prolonged accumulation of (in founder cells) actin is consistent with this possibility. While definitive answers to these questions await future investigation, it is worth noting that actin is required for yeast vacuole fusion and that actin has recently been identified as a component of neuronal synaptic vesicles (Kim, 2007).
An important future direction is to identify the biochemical composition of the prefusion vesicles. The involvement of such vesicles in myoblast fusion is not unique to Drosophila, as similar vesicles with electron-dense material have been reported in quail myoblast cultures and the L6 rat muscle cells. It is conceivable that these vesicles may deliver an unknown fusogen, or proteins/chemicals that stimulate fusogen activity, to the fusion sites, which ultimately leads to the fusion of apposing cell membranes. Because the actin cytoskeleton has also been implicated in other types of cell-cell fusion events, including fusion of human macrophages and viral-induced cell-cell fusion, it is speculated that targeted exocytosis of prefusion vesicles might represent a general step in myoblast fusion from Drosophila to mammals and perhaps in other cell-cell fusion events as well (Kim, 2007).
Regulation of synaptic morphology depends on endocytosis of activated growth signal receptors, but the mechanisms regulating this membrane-trafficking event are unclear. Actin polymerization mediated by Wiskott-Aldrich syndrome protein (WASp) and the actin-related protein 2/3 complex generates forces at multiple stages of endocytosis. FCH-BIN amphiphysin RVS (F-BAR)/SH3 domain proteins play key roles in this process by coordinating membrane deformation with WASp-dependent actin polymerization. However, it is not known how other WASp ligands, such as the small GTPase Cdc42, coordinate with F-BAR/SH3 proteins to regulate actin polymerization at membranes. Nervous Wreck (Nwk) is a conserved neuronal F-BAR/SH3 protein that localizes to periactive zones at the Drosophila larval neuromuscular junction (NMJ) and is required for regulation of synaptic growth via bone morphogenic protein signaling. This study shows that Nwk interacts with the endocytic proteins dynamin and Dynamin associated protein 160 (Dap160) and functions together with Cdc42 to promote WASp-mediated actin polymerization in vitro and to regulate synaptic growth in vivo. Cdc42 function is associated with Rab11-dependent recycling endosomes, and this study shows that Rab11 colocalizes with Nwk at the NMJ. Together, these results suggest that synaptic growth activated by growth factor signaling is controlled at an endosomal compartment via coordinated Nwk and Cdc42-dependent actin assembly (Rodal, 2008).
Nwk interacts with the endocytic machinery and activates Wsp/Arp2/3 actin polymerization together with Cdc42 to regulate synaptic growth upstream of growth factor signaling. Mapping these interactions and activities provides a critical framework for determining the mechanism by which endocytic accessory proteins and the cytoskeleton control membrane deformation during endocytosis (Rodal, 2008).
Nwk activates Wsp/Arp2/3 actin polymerization via its SH3a domain, and Nwk-SH3b is not required for Wsp binding or activation, but is required for the residual Wsp-inhibitory activity of Nwk when SH3a function is abolished. This activity may be more pronounced on endogenous Wsp, which is more tightly autoinhibited than recombinant WASp, raising the possibility that Nwk-SH3b could potently regulate Nwk-SH3a-dependent activation of Wsp. Thus, ligands of Nwk-SH3b are in a position to serve as activators of Nwk and Wsp/Arp2/3 actin polymerization. Nwk-SH3b is required for interactions between Nwk and Dap160, which is an excellent candidate for acting upstream of Nwk, because dap160 mutants exhibit synaptic overgrowth and temperature-sensitive seizures like those of nwk mutants, and Nwk is mislocalized in dap160 NMJs. Recently, it was reported that the fragment of Dap160 containing its last two SH3 domains is required for interaction with full-length Nwk in Drosophila extracts, leading to the hypothesis that the C terminal proline-rich region of Nwk mediates these interactions (O'Connor-Giles, 2008). The current results show instead that interactions between purified Nwk{Delta}C (i.e., Nwk lacking the C terminus) and both endogenous full-length Dap160 as well as purified Dap160 SH3 domain-containing fragment depend on Nwk SH3b. Two possible interpretations can reconcile these results. Nwk SH3b may interact with a noncanonical SH3 domain-binding site in the intervening sequences between the Dap160 SH3 domains. Alternatively, Nwk SH3b may function in an intramolecular interaction within Nwk that is required to expose one of several proline-rich sequences in the N-terminal region Nwk for interaction with Dap160 SH3 domains. Thus, it is concluded that Nwk SH3b is important for Dap160-Nwk interactions via an indirect or noncanonical mechanism. Further experiments will be needed to identify the Nwk-binding site on Dap160 and to confirm activity of Dap160 on Nwk in vitro (Rodal, 2008).
Nwk-SH3a is required for interactions of Nwk with both dynamin and Wsp. Other F-BAR/SH3 family members have been postulated to link dynamin and Wsp by multimerization via their F-BAR domains (Itoh, 2006; Tsujita, 2006; Shimada, 2007), but endogenous complexes containing Wsp and dynamin have only been demonstrated for the F-BAR/SH3 protein syndapin (Kessels, 2006). Nwk could thus be in a position to bring dynamin and Wsp together. It has not been possible to coimmunoprecipitate endogenous Wsp and Nwk using the available antibodies. However, dynamin immunoprecipitates contain Nwk but not Wsp, suggesting that Nwk-SH3a may switch associations between dynamin and Wsp. Another interpretation is that Wsp and dynamin binding are restricted to separate populations of Nwk molecules, and that the SH3a domain thus acts in two parallel biochemical pathways (Rodal, 2008).
In vivo analysis reflects the complexity of these SH3 domain interactions. SH3a and SH3b of Nwk have both separate and overlapping functions in regulating synaptic growth, perhaps reflecting the multivalent nature of interactions in the Nwk network. [In addition to binding Nwk, Dap160 binds to both dynamin and to Wsp.] Furthermore, the fact that mutation of both SH3 domains together (Nwk-SH3a*b*) produces additional dominant effects suggests that a non-SH3 ligand of Nwk is inappropriately titrated away from its function after mutation of Nwk SH3 domains. An excellent candidate ligand is the membrane itself, because the Nwk F-BAR domain has the potential to bind to and tubulate phospholipid bilayers. Determining the specific order and regulation of F-BAR/SH3 domain protein interactions with competing SH3 domain ligands and with the membrane will be important for uncovering the molecular mechanisms of these proteins during endocytosis (Rodal, 2008).
NMJ overgrowth with an excess of satellite boutons is a hallmark of endocytic mutants. Nwk interacts with the endocytic machinery and cdc42 and nwk mutants exhibit overproliferation of satellite boutons. A prominent function of endocytosis in nerve terminals is the recycling of synaptic vesicles. However, nwk single mutants and cdc42; nwk double mutants show no detectable defect in endocytosis of synaptic vesicles. One interpretation of this result is that receptor endocytosis is more sensitive to perturbation than synaptic vesicle recycling. However, given the documented function of Cdc42 and Wsp in endosomes, it is more likely that Nwk functions in a later step of endocytic traffic. Importantly, although the synaptic vesicle endocytosis defects in shi (dynamin) and dap160 reflect the function of these molecules in the internalization step of endocytosis, synaptic overgrowth in these mutants could arise from defects at later steps of endocytic traffic, because dynamin functions in a variety of membrane-trafficking events, ranging from Golgi traffic to endosome traffic (van Dam and Stoorvogel, 2002Go; Kessels et al., 2006Go) (Rodal, 2008).
The endosomal system is organized into subdomains defined by specific members of the Rab GTPase family and adopts distinct morphology and ultrastructure in different cell types. Thus, functionally conserved Rab subdomains provide a unifying approach to understanding structurally diverse membrane systems. Rab11 controls the function of the recycling endosome in directing traffic to the cell surface and colocalizes with Nwk in periactive zones at the Drosophila NMJ [although it can occasionally be observed in larger puncta]. Like cdc42 and nwk mutants, rab11 mutants have a profound defect in synaptic growth, exhibiting excessive satellite boutons. Cdc42 and WASp have recently been implicated in recycling endosome function. Thus, periactive zones may be the synaptic representation of the recycling endosome, with Cdc42 and Nwk controlling actin polymerization-dependent traffic of signaling complexes at this Rab11-positive compartment. Whether Cdc42 functions as a signal-responsive element in this compartment or forms part of the constitutive machinery for membrane traffic remains uncertain (Rodal, 2008).
The TGF-β/BMP family member Gbb activates downstream signals that may be the critical targets of Nwk/Cdc42-mediated endocytosis in synaptic growth. Indeed, recent work has shown that Gbb signaling is required for synaptic overgrowth in nwk mutants, phosphorylation of the Gbb signaling target Mothers against decapentaplegic (Mad) is upregulated in nwk mutants, and Nwk biochemically interacts with the intracellular domain of the Gbb receptor Tkv. However, other signaling pathways could equally be regulated by Nwk/Cdc42-mediated endocytosis, lead to upregulation of phosphorylated Mad, and contribute to the synaptic overgrowth in cdc42; nwk mutants. One candidate pathway is the presynaptic component of the Wnt/Wg cascade, which may converge on Gbb/Mad regulation in the synapse as observed in other tissues. It has not been possible to detect any change in the steady-state localization of candidate cargoes in synaptic boutons in nwk or cdc42 mutants, suggesting that Nwk and Cdc42 are not required for the gross morphology of endosomes, but instead contribute to the rate of cargo trafficking through this compartment. Determining the specific signaling pathways, receptors, and their activation states in recycling endosomes will require tools to measure the activity and rates of traffic of specific receptors in situ (Rodal, 2008).
Nwk is conserved from insects to higher vertebrates, and the mammalian genome encodes two Nwk homologs, which have not yet been characterized. However, Cdc42 and WASp-induced actin polymerization have been implicated in synapse formation in Aplysia sensory neurons and in mammalian hippocampal cultures. These reports suggest that the direct consequence of activating these proteins was the formation of filopodia that mature into synapses. An alternative hypothesis, consistent with the established function of Cdc42 and WASp family members in generating force for intracellular membrane traffic rather than in filopodial formation, is that synaptic growth regulatory functions of Cdc42 and WASp depend on endosomal traffic of signaling complexes by a similar mechanism to Drosophila Nwk-Wsp-induced synapse formation (Rodal, 2008).
Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. This study shows that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. The Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. Altogether These results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue (Leibfried, 2008).
Cdc42 has been implicated in the regulation of polarity establishment in the early Drosophila embryo. The function was shown to be dependent upon the interaction of Cdc42 with the Baz-Par6-aPKC complex that promotes the exclusion of Lgl through Lgl phosphorylation by aPKC. However, the role of Cdc42 in epithelial tissue is unlikely to depend only on its regulation of aPKC because aPKC was shown to be dispensable for apico-basal polarity establishment in the Drosophila embryo. The role of Cdc42 in mammalian epithelial cells has so far been examined by the expression of constitutively active and dominant-negative forms of Cdc42, and such an examination has led to conflicting results in establishing the exact role of Cdc42 in apico-basal polarity maintenance. Nonetheless, they point toward an important role of Cdc42 in the regulation of polarized trafficking. The possible role of Cdc42 in polarized trafficking in epithelial cells was further strengthened by the identification of Cdc42 and the Par complex as regulators of endocytosis in both mammalian cells and C. elegans. Nevertheless, the precise role of Cdc42 and the Par complex in the regulation of endocytosis has remained poorly understood except in migrating cells in which the Par complex was shown to inhibit integrin endocytosis via Numb (Leibfried, 2008).
Cdc42 and its effector Drosophila Cip4 have been found to regulate E-Cad endocytosis and that their loss of function is associated with the formation of long tubular endocytic structures similar to what is observed upon blocking Dynamin function. It is therefore proposed that in Drosophila epithelial cells, Cdc42 controls the early steps of E-Cad endocytosis via Cip4. Because Cdc42, aPKC, and Par6 loss of function are associated with similar defects in E-Cad and Cip4 localization, a simple model is favored, in which the loss of aPKC or Par6 activity disrupts Cdc42 localization or activity and in turn prevents Cip4 function (Leibfried, 2008).
The identified role of PCH family of protein stems in part from the biochemical analysis of Toca-1 as a regulator of actin polymerization. Toca-1 is necessary to activate actin polymerization and actin comet formation downstream of PIP2 and Cdc42 in a WASp-dependent manner (Ho, 2004). On the basis of elegant biochemical assays, Toca-1 was further shown to be necessary to alleviate the WIP inhibitory activity on WASp, in order to allow efficient Arp2/3 activation by WASp (Ho, 2004). Toca-1 was proposed to play an essential role in the fine spatial and temporal regulation of actin polymerization in both cell migration and vesicle movement. Cip4 has been implicated in microtubule organizing center (MTOC) polarization in immune natural killer cells (Banerjee, 2007), a process in which Cdc42 and the Par complex are also involved. Importantly, because Cip4 was shown to bind microtubules, the interaction between Cdc42 and Cip4 might indicate that Cip4 might also be an effector of Cdc42-Par complex in the regulation of MTOC polarization (Leibfried, 2008).
In mammalian cells, regulation of endocytic-vesicle formation has been proposed to be dependent upon both branched actin-filament formation and Dynamin. The role of WASp and Arp2/3 in the regulation of E-Cad endocytosis may therefore indicate that Cip4, which is also known to form dimers, can promote vesicle scission by recruiting Dynamin and promoting actin polymerization via WASp. Therefore, it is proposed that Cip4 and WASp act as a link between Cdc42-Par6-aPKC and the early endocytic machinery to regulate E-Cadherin endocytosis in epithelial cells (Leibfried, 2008).
In the nervous system, neurons form in different regions, then they migrate and occupy specific positions. RP2/sib, a well-studied neuronal pair in the Drosophila ventral nerve cord (VNC), has a complex migration route. This study shows that the Hem protein, via the WAVE complex, regulates migration of GMC-1 and its progeny RP2 neuron. In Hem or WAVE mutants, RP2 neuron either abnormally migrates, crossing the midline from one hemisegment to the contralateral hemisegment, or does not migrate at all and fails to send out its axon projection. Hem regulates neuronal migration through stabilizing WAVE. Since Hem and WAVE normally form a complex, the data argues that in the absence of Hem, WAVE, which is presumably no longer in a complex, becomes susceptible to degradation. It was also found that Abelson tyrosine kinase affects RP2 migration in a similar manner as Hem and WAVE, and appears to operate via WAVE. However, while Abl negatively regulates the levels of WAVE, it regulates migration via regulating the activity of WAVE. The results also show that during the degradation of WAVE, Hem function is opposite to that of and downstream of Abl (Zhu, 2011).
Several studies have suggested that Hem dynamically regulates polymerization of F-actin. Hem can play a crucial role in linking extracellular signals to the cytoskeleton. On the other hand, Hem is also part of the WAVE complex and it may regulate the activity of the WAVE complex to promote polymerization of F-actin. The result that the migration defect in Hem mutants can be completely rescued by expression of WAVE from a transgene indicates that Hem regulates neuronal migration via WAVE (Zhu, 2011).
How Hem regulates WAVE is controversial. It has been argued that Hem (together with PIP212) inhibits WAVE in the WAVE complex. Upon activation by Rac1 or Nck, the WAVE complex dissociates releasing an active WAVE-HSPC300 to mediate actin nucleation. This conclusion was also supported by the findings that loss-of-function for Hem leads to an excess of F-actin in the cytosol. Moreover, a reduction in the WAVE gene dosage suppressed axon guidance defects in Hem mutant embryos. But, in vitro studies using Drosophila tissue culture cells argue that Hem protects WAVE from proteasome-mediated degradation. The current in vivo results are consistent with these studies and show that WAVE is protected by Hem and the above alternate model may be incorrect (Zhu, 2011).
The WAVE protein was first identified in Dictyostelium discoideum as a suppressor of mutations in the cAMP receptor (SCAR) but it is present in flies to humans. All WAVEs contain a N-terminal WHD/SHD (WAVE/SCAR homologue domain), a central
proline-rich region and a C-terminal VCA domain. WAVE protein regulates actin polymerization by mediating the signal of Rac to Arp2/3 in lamellipodia. It is involved in forming branched and cross-linked actin networks. Unlike WASp proteins, which are intrinsically inactive by autoinhibition and activated by directly binding to Cdc42, PIP2 etc., WAVE appears to be intrinsically active, at least in vitro.However, the majority of WAVE is in the 'WAVE complex' with four other proteins: Hem, Sra-1/PIR121/CYFIP, Abi and HSPC300/Brk1 (Zhu, 2011).
In the WAVE-complex, direct association between WAVE, Abi and HSPC300 represents the backbone of the complex. Hem binds to Sra-1 forming a sub-complex, which is able to bind to Rac through Sra-1. The interaction between Abi and Hem is what binds Hem and Sra-1 into the complex. Hem and Sra-1 are sequentially recruited to the WAVE complex. Free subunits and assembly intermediates of the WAVE-complex are usually not detected but supposedly degraded. Also, previous studies suggest that depletion of one component leads to degradation of others. Indeed, the current results, that in Hem mutants, the level of WAVE protein, but not the WAVE gene transcription, is drastically reduced supports this contention. Perhaps in the absence of Hem, WAVE complex is either not formed or partially formed, resulting in the degradation of WAVE and phenotypes such as mis-migration of neurons. When the levels of WAVE are supplemented using a WAVE transgene (UAS-WAVE), the migration defect in Hem mutants is promptly rescued (Zhu, 2011).
While a complete lack of WAVE (or Hem) function causes an arrest in the migration of RP2, a reduction in the levels of WAVE due to a reduction in the levels of Hem causes abnormal migration. For example, the lowest level of WAVE is seen in the Hem allele that has the strongest penetrance. Moreover, since this mis-migration defect is rescued by expressing WAVE from a transgene, it can be concluded that this mis-migration is also due to an effect on WAVE. It has been suggested that the WAVE-complex exists cytoplasmically and in membrane-bound forms. Through an interaction
with Rac, WAVE gets recruited to the lamellipodia where actin polymerization required for membrane protrusion is initiated and regulated. The integrity of the complex is critical for its proper localization since removal of either WAVE or Abi prevents its translocation to the leading edge of the lamellipodia. It is possible that a reduction in the levels of WAVE in Hem mutant embryos causes non-translocation of the WAVE complex to the membrane, causing a non/mis-migration of RP2 (Zhu, 2011).
These results show that WAVE protein exists as three different molecular weight forms. Treatment of the extract with phosphatase collapses these three forms into a single band, indicating that WAVE protein is phosphorylated, with varying degrees
of phosphorylation to yield different molecular weight species. Whether there are any changes in the three different forms with respect to their relative contributions in Hem and Abl mutants was examined. However, no changes were found in their relative contributions and the levels of all the forms were reduced in Hem mutants. Therefore, it may be that the reduction in all the forms, or that the reduction in one or two of the forms is responsible for the migration defect. In Abl mutants, the level of WAVE is modestly increased, which is the opposite to that of the effect of Hem on WAVE. Thus, it seems more likely that the activity of WAVE is affected in Abl mutants. Being a protein kinase, it was possible that Abl phosphorylates WAVE, thus affecting either its activity or level. However, no significant changes in were seen in the relative levels of the different phosphorylated forms of WAVE in Abl mutants. It has been shown in vitro that Abl is
recruited to WAVE by Abi following cell stimulation, triggering the translocation of Abl together with the WAVE complex to the leading edge of the membrane. Thus, Abl might affect WAVE activity, either directly or indirectly, via the translocation of the WAVE complex to the membrane of an actively migrating RP2. It is also possible that Abl affects migration in a pathway that does not involve WAVE (Zhu, 2011).
In contrast, the effect of loss-of-function for Abl on WAVE levels is more pronounced in older embryos. These results indicate that Abl directly or indirectly regulates the levels of WAVE. Furthermore, though modest, ectopic expression of Abl does down-regulate WAVE. Interestingly, the results also show that Hem regulation of WAVE levels is downstream of the Abl regulation of WAVE since the Hem; Abl double mutants had the same levels of WAVE as Hem single mutants. It seems likely that in the absence of Hem, WAVE protein gets degraded, resulting in the loss of migration or abnormal migration. Whereas in Abl mutants, the most likely scenario is that the activity of WAVE is affected, resulting in the same migration defect (Zhu, 2011).
The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 (see Drosophila Arp2/3 component Arpc1) pathways. This study utilized the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. Differential endocytosis of cell surface components was observed at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube (Haralalka, 2014: PubMed).
To identify mutant alleles of WASp, use was made of a large collection of recessive lethal and female-sterile mutations that fail to complement Df(3R)3450. Transgenic copies of an ~12-kb genomic fragment that includes WASp were introduced into the background of hemizygous mutant flies from these lines. Three lethal mutant alleles, later shown to form a complementation group, were rescued to viability in this manner. Furthermore, the morphological phenotypes characteristic of WASp mutant flies, were fully ameliorated in the rescued flies. Phenotypic rescue of these alleles was also achieved after expression of a UAS-WASp cDNA construct, under the control of various GAL4 drivers. Sequencing of PCR-amplified WASp genomic DNA from hemizygous mutant animals reveals that all three alleles contain small (10-15 bp), distinct intragenic deletions, resulting in predicted frameshifts in the Wsp primary protein sequence. In all three cases, the cytoskeleton-interacting COOH-terminal domain is lost, implying that protein function is severely compromised (Ben-Yaacov, 2001).
Hemizygous mutant WASp flies from all three lines complete nearly all stages of imaginal development, and die as young adults. Most commonly, WASp flies fail to fully eclose from the pupal case. Those that do can survive for a few days, but are lethargic and passive in their behavior. In general, WASp flies do not display any gross morphological abnormalities. However, these flies exhibit a pronounced lack of neurosensory bristles, external manifestations of sensory organs stereotypically positioned just underneath the entire cuticle of the adult fly. The bristle-loss phenotype is particularly apparent on the head capsule and abdomen. Significant but less severe effects are observed on the legs and thorax of the mutant flies, where the smaller microchaete bristles are primarily affected. The pattern of wing margin sensory bristles and wing blade nonsensory hairs is generally normal in WASp mutant flies. Noticeable features of the WASp phenotype, in addition to the marked reduction in bristle number, include loss of both the bristle shaft and bristle socket, occasional bristle duplications, and a normal morphology of those bristles which do form in the mutant flies. These observations suggest impairments in sensory organ development, rather then defects in bristle formation per se, as a probable underlying cause for the WASp phenotype. No major phenotypic distinctions were observed between flies hemizygous for the different alleles, or between hemizygous and transallelic combinations, suggesting that the phenotype described here approximates the full zygotic loss-of-function phenotype of WASp (Ben-Yaacov, 2001).
Microvilli are actin-based fingerlike membrane projections that form the basis of the brush border of enterocytes and the Drosophila melanogaster photoreceptor rhabdomere. Although many microvillar cytoskeletal components have been identified, the molecular basis of microvillus formation remains largely undefined. The Wiskott-Aldrich syndrome protein (WASp) is necessary for rhabdomere microvillus morphogenesis. WASp accumulates on the photoreceptor apical surface before microvillus formation, and at the time of microvillus initiation WASp colocalizes with amphiphysin and moesin. The loss of WASp delays the enrichment of F-actin on the apical photoreceptor surface, delays the appearance of the primordial microvillar projections, and subsequently leads to malformed rhabdomeres (Zelhof, 2004).
The identification of the major constituents and the description of the ultrastructure of microvilli have been known for years. However, how this structure is initiated and regulated remains a mystery. This phenotypic analysis of WASp function in Drosophila photoreceptor cells has provided several insights into the molecular mechanisms responsible for the formation of the microvillus core. Knowing that the primary constituent of the microvillus projection is F-actin, molecules were sought that could be candidates for coordinating the formation of the actin filaments. Using the SH3 domain of Amph as bait, another rhabdomeric protein, WASp, was isolated. This interaction is also conserved in Saccharomyces cerevisiae. The homologues of Amph (Rvs167p) and WASp (Las17p/Bee1p) also interact. Furthermore, WASp is an excellent candidate for initiating microvillus formation. WASp is the causative gene product of Wiskott-Aldrich syndrome and lymphocytes from Wiskott-Aldrich syndrome patients have a reduction in cell surface microvilli (Zelhof, 2004).
WASp is expressed and localizes to the apical surface before the appearance of microvillar apical folds and before the enrichment/rearrangement of F-actin in photoreceptor cells. More importantly, the loss of WASp function results in malformed rhabdomeres. Typically, WASp mutant rhabdomeres are misshapen and a small percentage are split; these are phenotypes not observed in wild-type photoreceptor cells. Knowing WASp has been identified as a key component in the specific subcellular localization and polymerization of actin, it is speculated that these phenotypes are a result of defects in establishing the F-actin cytoskeleton core (Zelhof, 2004).
The transformation of the apical surface into the rhabdomere is a highly coordinated event. After each apical surface involutes inward, there is an expansion of the apical surface down toward the retinal floor. As such, the examination of tangential sections through the depth of the photoreceptor cell represents a temporal profile of the photoreceptor apical surface as it is transformed into a rhabdomere. Inspection of markers for the initiation of microvillus formation (F-actin) and the separation and delimitation of each photoreceptor apical surface (Armadillo) in WASp mutant cells clearly demonstrates a temporal delay in rhabdomere formation compared with their neighboring wild-type counterparts. Furthermore, molecules that are implicated in the stabilization of the actin cytoskeleton core (Moesin), the cross-linking of each microvillus (Chaoptin), or the deformation of the overlying plasma membrane (Amph) are dependent on the presence of the primordial microvilli (confirmed from EM analysis) for their recruitment/stabilization to the apical surface, and thus, are epistatic to WASp function. In the case of Amph, the direct association with WASp may aid in the recruitment of Amph to the apical surface. Even though all the observations are consistent with the idea that WASp is coordinating the signal required for the transformation of the apical surface, surprisingly, WASp is not essential for the formation or growth of microvilli (Zelhof, 2004).
If WASp is responsible for integrating the signal for microvillus formation, what is the signal? Numerous studies have implicated a combination of upstream factors for the recruitment and activation of WASp, such as small Rho GTPases, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] phosphoinositide, and SH3-containing proteins that bind the proline-rich domain of WASp. The data are suggestive, but not conclusive, that Cdc42 may play a role in the activation of WASp. A similar delay in F-actin enrichment and subsequent recruitment of rhabdomeric proteins is observed in cdc42 mutant cells. This idea directly conflicts with the result that the Rho GTPase binding domain of WASp is not required for the rescue of viability in WASp mutants. However, the binding site for PI(4,5)P2 is also dispensable, and the data demonstrate that the proline-rich domain is not necessary to rescue viability. Given that each individual domain in vivo is expendable, this may be indicative that any one or a combination of the three domains can be sufficient for the recruitment and activation of WASp. In rhabdomere biogenesis, all three potential ways of activating WASp are present. The SH3 domain of Amph binds WASp. Mutation of Cdc42 results in defects in rhabdomere morphogenesis, and there is a coincident accrual of PI(4,5)P2 on the photoreceptor apical surface during the initiation of microvilli formation. Only through the combination of in vitro activation studies, further in vivo WASp structure-function studies, and deciphering the role of PI(4,5)P2 metabolism in microvillus biogenesis will the contribution of each regulatory domain of WASp in microvillus initiation be able to be clarified (Zelhof, 2004).
Finally, it is evident that a second pathway for initiating F-actin formation is present. WASp is not essential for microvillus formation, and clearly, F-actin accumulates and functional microvilli do form in WASp mutant cells. The molecular basis of this second pathway is unknown. One possible mechanism could involve a p21-activated kinase (PAK). Besides a role in cell differentiation and proliferation, PAK proteins have been implicated in the regulation of the cytoskeleton. Mbt (mushroom bodies tiny), a PAK, has been implicated in photoreceptor morphogenesis. mbt mutant photoreceptor cells have malformed rhabdomeres, and Cdc42 is required for its correct localization in photoreceptor cells. That Cdc42 may be responsible for the activation of more than one pathway would be consistent with the fact that photoreceptor cell differentiation, and in particular rhabdomere formation, is more severely affected in cdc422 mutant cells compared with WASp mutant cells. In addition to a broader role of Cdc42, a second member of the WASp/WAVE family, SCAR, exists in Drosophila. SCAR is required for patterning of the eye ommatidium and external morphology. Thus, SCAR activity could account for the assembly of actin observed in WASp mutant cells. However, it will be necessary to genetically separate the early requirement of SCAR in ommatidial patterning before examining its contribution to microvillus initiation. Overall, this work has now defined a budding network of molecules required for microvillus initiation and provided a framework in which the mechanisms of microvillus biogenesis and regulation can be further elucidated (Zelhof, 2004).
The protrusion of two distinct actin-containing organelles, lamellipodia and filopodia, is thought to be regulated by two parallel pathways: from Rac1 through Scar/WAVEs to lamellipodia, and from Cdc42 through N-WASP to filopodia. This hypothesis was tested in Drosophila, which contains a single gene for each of the WASP subfamilies, SCAR and WASp. Targeted depletion of SCAR or WASp by dsRNA-mediated interference was performed in two Drosophila cultured cell lines expressing lamellipodial and filopodial protrusion. Knockdown was verified by laser capture microdissection and RT-PCR, as well as Western blotting. Morphometrical, kinetic and electron microscopy analyses of the SCAR-depleted phenotype in both cell types revealed strong inhibition of lamellipodial formation and cell spreading, as expected. More importantly, filopodia formation was also strongly inhibited; this is not consistent with the parallel pathway hypothesis. By contrast, depletion of WASp did not produce any significant phenotype, except for a slight inhibition of spreading, showing that both lamellipodia and filopodia in Drosophila cells are regulated predominantly by SCAR. A new, cascade pathway model of filopodia regulation is proposed in which SCAR signals to lamellipodia and then filopodia arise from lamellipodia in response to additional signal(s) (Biyasheva, 2004).
Depletion of SCAR from S2R+ cells is correlated with the development of a strong, penetrant and highly reproducible phenotype, which reflected the declining ability of cells to form lamellipodia and spread over the substratum. Although some heterogeneity inevitably accompanies the development of the phenotype (probably due to uneven dsRNA uptake), the progression clearly occurs from the appearance of slight irregularity of the lamellipodial outline to the formation of long narrow processes to virtual failure of a cell to spread (Biyasheva, 2004).
The formation of linear processes reminiscent of filopodia during SCAR depletion could have resulted from the undepleted activity of WASp leading to the induction of filopodia, as is predicted by the model of two parallel signaling pathways. However, a detailed analysis of these aberrant processes showed that they are more like extremely narrowed residual lamellipodia than filopodia. The curved shape, uneven thickness and kinetic behavior of these processes are distinct from those seen during normal filopodial protrusion. Structurally, the processes of SCAR-depleted cells do not contain bundles of long parallel filaments that are characteristic of filopodia. Instead, they are filled with a sparse dendritic network like that seen in lamellipodia. The weak presence of the lamelipodial marker Arp3 and absence of the filopodial marker Ena in these processes also suggests that they are not filopodia. Thus, the depletion of SCAR in S2R+ cells does not induce filopodia, but produces narrow aberrant processes, probably by a combination of residual SCAR activity and retraction elsewhere along the cell perimeter. These findings in Drosophila cells are in agreement with the previous work in Dictyostelium, where SCAR-deficient cells manifested abnormal cell size and morphology, and instead of normal broad lamellipodia formed 'pseudopodia-like extensions' depleted of F-actin (Biyasheva, 2004).
The formation of narrow lamellipodia following depletion of SCAR provides an additional insight into the function of SCAR. Scar/WAVEs in mammalian and Drosophila cells are localized to the extreme edge of protruding lamellipodia. Possibly, SCAR acts cooperatively in the process of protrusion. That is, protrusion at the leading edge may be more probable at sites of a pre-existing protrusion. A consequence of SCAR depletion would be that protrusion would become progressively restricted to smaller domains (Biyasheva, 2004). In contrast to the striking effect of SCAR depletion, WASp depletion did not generate a phenotype in S2R+ cells aside from a minor, albeit statistically significant, reduction in the cell projected area. One possible explanation for the lack of a significant phenotype might be the incomplete knockdown of WASp, only 75% depletion of WASp was achieved, as compared with greater than 95% depletion of SCAR. However, considering that several diverse assays were used for phenotypic analysis, it is believed that if WASp made a significant contribution to protrusive behavior, a phenotype would have been detectable (Biyasheva, 2004).
Taken together, the results using S2R+ cells indicate that SCAR is the major regulator of protrusive activity in these cells. It is necessary for the assembly of the actin dendritic network, formation of lamellipodia and cell spreading onto the substratum. By contrast, WASp makes little, if any, contribution to actin-based protrusion in these cells (Biyasheva, 2004).
The lack of filopodia formation in S2R+ cells after SCAR inhibition may be explained by an intrinsic deficiency of the filopodial machinery in these cells. Therefore, to test the role of SCAR in filopodial formation, BG2 cells were used -- these are, in addition to lamellipodia, very rich in filopodia, similar to other cells of neuronal origin (Biyasheva, 2004).
The parallel signaling hypothesis predicts that depletion of the lamellipodial regulator, SCAR, will not affect filopodia, which, according to the model, would be induced by WASp. By contrast, the cascade model predicts that SCAR depletion will inhibit not only lamellipodia, but also filopodia. The results support the latter prediction. It was confirmed that SCAR in BG2 cells, as in S2R+ cells, regulates lamellipodia. Indeed, depletion of SCAR in BG2 cells inhibits lamellipodia, leading to decreased spreading and altered shape similar to results obtained with S2R+ cells. By EM criteria, the lamellipodial dendritic network in BG2 cells was significantly reduced; only a few small patches of the dendritic network remained at some cell edges (Biyasheva, 2004).
The conclusive evidence helped in distinguishing between two alternative models of regulation expected from effects of SCAR depletion on filopodia formation. The results clearly show that SCAR RNAi in BG2 cells inhibits not only lamellipodia, but also filopodia. Although light microscopic evaluation of static cultures per se gave an impression of filopodia sustaining in SCAR-depleted BG2 cells, more detailed analyses, namely, EM structural assay and a quantitative kinetic analysis of filopodial initiation during cell spreading, revealed that filopodia formation, in fact, was strongly inhibited. These results allowed the parallel pathway model to be ruled out and validate the cascade pathway model for actin-based protrusions in Drosophila cell lines (Biyasheva, 2004).
In contrast to SCAR, depletion of WASp in BG2 cells did not result in any changes in cell morphology, kinetics of protrusion or cytoskeletal architecture except for a slight inhibition of spreading, similar to what was observed in S2R+ cells. BG2 cells after WASp depletion retained not only lamellipodia, but also numerous perfectly looking filopodia indistinguishable from those in control cells. These results show that WASp does not play a distinctive role for filopodia formation, thus further corroborating the priority of a cascade pathway model over the parallel pathway model (Biyasheva, 2004).
Regulation of lamellipodia and filopodia The requirement of SCAR activity in Drosophila cells for both lamellipodial and filopodial protrusion is fully consistent with the idea that filopodial initiation in motile cells occurs downstream of lamellipodial protrusion, as suggested by previous studies. Therefore, a model stipulating a two-step cascade regulation of filopodial protrusion in Drosophila cells is proposed. SCAR activates the Arp2/3 complex which then induces assembly of the dendritic network in lamellipodia. The lamellipodium by itself represents an important part of the protrusive machinery, but it is suggested that the lamellipodium also provides a foundation for subsequent reorganization into filopodial bundles. Such reorganization would be dependent on additional as-yet-unspecified signals. Indirect evidence in support of the cascade regulation model comes from in vivo studies of Drosophila. In mutations removing all three Drosophila isoforms of Rac, both lamellipodia and filopodia are abolished during dorsal closure. Such a result would not be predicted if Rac signaled only to lamellipodia and a parallel pathway existed for filopodia (Biyasheva, 2004).
In general, these results and interpretations of the roles of SCAR and WASp at the cellular level are consistent with conclusions drawn from studies on Drosophila development in vivo: SCAR mutations result in more severe phenotypes and affect many structures, whereas mutations in WASp had very little phenotype. SCAR, rather than WASp, is the major mediator of Arp2/3 function during Drosophila oogenesis and in the development of the central nervous system. SCAR and Arp2/3 are essential for proper spatial distribution of nuclei during cortical divisions, as well as for metaphase furrow formation and ring canal formation. Overall actin levels are lower in SCAR and Arp2/3 mutant cells. By contrast, WASp function seems to be restricted to specific processes in development, such as the proper execution of asymmetrical cell divisions in neural lineages or in bristle formation in ommatidia. Nevertheless, WASp performs its role via the Arp2/3 complex. Although WASp does not seem to contribute to protrusive activity in the Drosophila cell lines tested in these experiments, consideration of mutant developmental phenotypes suggests that WASp can activate the Arp2/3 complex in certain cellular contexts and induce downsteam events that are dependent on dendritic nucleation of actin filaments (Biyasheva, 2004).
If WASP subfamily members are not essential for filopodia formation, then what are the signaling molecules for the second step of the postulated cascade pathway? A key element driving network reorganization into filopodial bundles appears to be a multiprotein complex at filopodial tips. It is proposed that the tip complex is a site at which the second step of the cascade signaling is directed. In favor of this hypothesis, IRSp53, an effector of Cdc42, induces filopodia by recruiting Mena, a protein that localizes to the filopodial tips and promotes the elongation of actin filaments by protecting them from capping. Also, a formin family protein Drf3 has recently been shown to be a downstream effector of Cdc42; it is localized to filopodial tips and plays a role in filopodia formation. The IRSp53 or Drf3 pathway or an as-yet-unidentified pathway, rather than N-WASP, may be responsible for Cdc42 signaling to filopodia. Irrespective of the specific molecules involved in the signaling pathway, the structural-kinetic analyses suggest that they share the property of targeting the filopodial tip complex to drive filopodial formation (Biyasheva, 2004).
Myoblast fusion takes place in two steps in mammals and in Drosophila. First, founder cells (FCs) and fusion-competent myoblasts (FCMs) fuse to form a trinucleated precursor, which then recruits further FCMs. This process depends on the formation of the fusion-restricted myogenic-adhesive structure (FuRMAS), which contains filamentous actin (F-actin) plugs at the sites of cell contact. Fusion relies on the HEM2 (NAP1) homolog Kette, as well as Blow and WASP, a member of the Wiskott-Aldrich-syndrome protein family. This study documents the identification and characterization of schwächling -- a new Arp3-null allele. Ultrastructural analyses demonstrate that Arp3schwächling mutants can form a fusion pore, but fail to integrate the fusing FCM. Double-mutant experiments revealed that fusion is blocked completely in Arp3 and wasp double mutants, suggesting the involvement of a further F-actin regulator. Indeed, double-mutant analyses with scar/WAVE and with the WASP-interacting partner vrp1 (sltr, wip)/WIP show that the F-actin regulator scar also controls F-actin formation during myoblast fusion. Furthermore, the synergistic phenotype observed in Arp3 wasp and in scar vrp1 double mutants suggests that WASP and SCAR have distinct roles in controlling F-actin formation. From these findings a new model was derived for actin regulation during myoblast fusion (Berger, 2008).
During the fusion process, the actin cytoskeleton of myoblasts is dynamically organized. Consistent with this, the center of the adhesion ring in FCs and FCMs contains filamentous actin (F-actin). The actin regulators kette and wasp are essential for myoblast fusion. Kette is the Drosophila homolog of HEM2 (NAP1), and can be found in a complex with SCAR (homolog of the mammalian WAVE) and is a member of the Wiskott-Aldrich-syndrome protein (WASP) family. Moreover, kette interacts genetically with blow during myoblast fusion. WASP family members possess a conserved VCA (verprolin homologous, cofilin homologous and acidic) domain, which is involved in the binding of G-actin and Arp2/3. The wasp3D3-035 allele lacks the CA domain of the VCA domain and thus neutralizes the function of maternal WASP. Furthermore, genetic data indicate that Kette antagonizes the function of WASP. The WASP-interacting partner Verprolin1 [Vrp1, also known as Solitary (Sltr) and Wip; the Drosophila homolog of the mammalian WIP], however, acts in a complex with WASP to ensure successful myoblast fusion (Kim, 2007; Massarwa, 2007; Berger, 2008 and references therein).
The branching of F-actin is initiated by the de novo nucleation of actin, triggered through the activity of the Arp2/3 complex. Alone, the Arp2/3 complex is inactive and requires a set of binding partners, including members of the WASP family, to become active. The binding of these proteins has been proposed to lead to a conformational change in the position of the seven subunits of the Arp2/3 complex, especially in the relative position of the subunits Arp2 and Arp3 (Berger, 2008).
This study describes an EMS-induced Arp3-null allele, Arp3schwächling, having an abnormal myoblast-fusion phenotype. Ultrastructural analysis of Arp3schwächling and wasp3D3-035 revealed the formation of a fusion pore in both mutants. Whereas in Arp3schwächling mutants the membrane between fusing myoblasts is removed, in wasp3D3-035 embryos, membrane breakdown is not completed. The finding that fusion in Arp3schwächling embryos is disrupted despite the formation of a fusion pore indicates that the pore does not expand in Arp3schwächling mutants. Therefore, to gain a deeper insight into the F-actin regulation of myoblast fusion, Arp3 wasp double-mutant embryos were examined, that show a complete block of myoblast fusion. This phenotype suggests that WASP is not the only actin regulator controlling F-actin polymerization during fusion. Indeed, studies on scar single mutants and scar vrp1 double mutants strongly imply that the first and the second fusion steps require a different set of F-actin nucleation factors (Berger, 2008).
The genetic data presented in this study provide new insights into the regulation of branching F-actin during myoblast fusion. There are three classes of proteins that initiate the polymerization of new actin filaments: the actin-related protein complex Arp2/3, the Formins and Spire. These protein classes are evolutionary conserved in most eukaryotes and promote new actin assembly by a distinct mechanism. The Arp2/3 complex is the only known protein complex that initiates new actin filaments branching off an existing filament. Upon cell-cell contact, F-actin is mostly present in the tip of the FCM and in the area of the FC/growing myotube to which the FCM is attaching. Analyses of Arp3, wasp and scar mutants indicate that branching F-actin is essential for myoblast fusion. Based on double-mutant analyses, it is postulated that the WASP-Vrp1 complex promotes branched F-actin formation positively during the second step of myoblast fusion, thereby allowing proposal of a new model for actin regulation during myoblast fusion (Berger, 2008).
This work emanated from the identification of an Arp3-null allele. Fusion is severely disrupted in Arp3schwächling mutants. However, stainings with anti-Duf clearly show that this is not due to a failure in cell-cell recognition and adhesion. For example, FCMs still attach to the site of the growing myotubes where Duf is expressed in Arp3schwächling and wasp3D3-035 mutant embryos. Interestingly, myoblasts also adhere successfully in Arp3schwächling wasp3D3-035 double mutants that fail to fuse completely. Duf serves to attract FCMs, which can migrate towards the Duf-expressing source. Hence, the ability of FCMs to migrate is a prerequisite for myoblast fusion. The migration of cells, however, also depends on actin-cytoskeleton regulation. Observations clearly show that the nature of the fusion arrest in Arp3schwächling and wasp3D3-035 single mutants, and Arp3schwächling wasp3D3-035 double mutants, is not due to either the inability of FCMs to migrate nor to a failure of FCs/myotubes and FCMs to recognize and adhere, but rather to a specific defect in cell-cell fusion (Berger, 2008).
Electron microscopy analyses on Arp3schwächling and wasp3D3-035 mutants further assisted in dissection of the process in which F-actin formation is required during myoblast fusion. The WASP-Vrp1 complex is involved in the formation of a fusion pore (Massarwa, 2007). In line with that study, mutations in wasp3D3-035 and wipD30 stop fusion during membrane breakdown, but not after pre-fusion-complex formation. The pre-fusion complex, which has been described to consist of 1.4 vesicles per pre-fusion complex, looks identical in both of the mutants as well as in wild-type embryos (Berger, 2008).
A model is presented for myoblast fusion derived from the data presented in this study. Studies on Arp3 mutants indicate that the polymerization-based force of branching F-actin is required beyond the stage of membrane breakdown. After membranes have been removed between fusing myoblasts, the FCMs must become integrated into the FC/growing myotube. Because Arp3schwächling mutants fail to fuse after membrane removal, it is proposed that F-actin is required to allow integration into the growing myotubes. After the Duf- and Sns-mediated recognition of FCs and FCMs, further essential proteins for myoblast fusion, e.g. Blow, become recruited to the point of fusion in FCMs. Vrp1 localizes in the tip of the filopodia of FCMs. The localization of WASP to the membrane depends on Vrp1 activity. Thus, the recruitment of WASP presumably leads to an increase of F-actin at the site of fusion. As a result, the formation of a fusion pore is initiated and expands until the FCM becomes finally pulled into the FC/growing myotube (Berger, 2008).
Morphological and statistical analysis on the nuclei of the DA1 muscle suggested that no fusion takes place in Arp3 wasp double mutants. Because wasp3D3-035 mutants should lack the ability to promote F-actin formation and stop fusion, like Arp3schwächling mutants, after precursor formation, this was a surprising result. Therefore whether the actin regulator SCAR additionally controls F-actin branching during myoblast fusion was examined. Double-mutant and epistasis experiments of scar and wasp revealed that this was indeed the case. The complete disruption of myoblast fusion in Arp3 wasp double-mutant embryos -- despite the presence of maternal wasp and Arp3 -- in conjunction with the finding that SCAR is required for myoblast fusion, led to a proposal that SCAR and WASP control different steps of myoblast fusion. So far, genetic data suggest that WASP is required only during the second fusion step. This is in line with previous data (Massarwa, 2007). It further implies that SCAR is essential for the first fusion step. However, myoblast fusion is not disrupted completely in scarδ37-null mutant embryos. The maternally provided gene product of scar is probably able to compensate for the loss of zygotic scar during myoblast fusion. Nevertheless, scar germline clones with reduced levels of maternal and zygotic SCAR protein show an enhanced myoblast-fusion phenotype. Because the loss of maternal scar disrupts oogenesis, it is not possible to eliminate the maternal component of scar completely. Hence, further experiments are required to determine whether SCAR controls the first fusion step alone or acts in functional redundancy with an additional factor (Berger, 2008).
Myoblast fusion in scar vrp1 double mutants was blocked completely. This might indicate that SCAR regulates the first fusion step together with the WASP-interacting partner Vrp1. The Vrp1 protein is expressed from stage 10 onwards, shortly before the first fusion step is initiated. Members of the Verprolin/WIP family have been reported to influence actin polymerization in a WASP-independent manner. It remains to be investigated whether this is also the case during Drosophila myoblast fusion (Berger, 2008).
In addition to regulating the first fusion step, the activity of SCAR might also be required for the second fusion step. Support for this notion comes from findings that the vertebrate homolog of Kette, HEM2, which is present in a complex with SCAR, is essential for myoblast fusion. Kette and WASP have antagonistic functions during myoblast fusion. Because the expression of WASP in FCs failed to rescue the wasp mutant phenotype, one could assume that the activity of WASP is only required in FCMs. It is therefore predicted that the polymerization of branching actin filaments might be regulated in a myoblast-type-specific manner (Berger, 2008).
In summary, these observations suggest for the first time that the cellular machinery leading to the formation of F-actin is controlled by a different set of nucleation-promoting factors during the first and second fusion steps. Future studies should now focus on the mechanistical details to reveal how these factors become activated in FCs and FCMs (Berger, 2008).
Myoblast fusion provides a fundamental, conserved mechanism for muscle fiber growth. This study demonstrates that the functional contribution of Wsp, the Drosophila homolog of the conserved actin nucleation-promoting factor (NPF) WASp, is essential for myoblast fusion during the formation of muscles of the adult fly. Disruption of Wsp function results in complete arrest of myoblast fusion in all muscles examined. Wsp activity during adult Drosophila myogenesis is specifically required for muscle cell fusion and is crucial both for the formation of new muscle fibers and for the growth of muscles derived from persistent larval templates. Although Wsp is expressed both in fibers and individual myoblasts, its activity in either one of these cell types is sufficient. SCAR, a second major Arp2/3 NPF, is also required during adult myoblast fusion. Formation of fusion-associated actin 'foci' is dependent on Arp2/3 complex function, but appears to rely on a distinct, unknown nucleator. The comprehensive nature of these requirements identifies Arp2/3-based branched actin polymerization as a universal mechanism underlying myoblast fusion (Mukherjee, 2011).
The universal nature of muscle fiber formation and growth via myoblast fusion suggests that common molecular mechanisms underlie the fusion process. The myogenic processes leading to the formation of adult Drosophila muscles provide a promising, yet generally unexplored, setting in which conserved elements of this type can be identified and characterized. This is particularly true for establishment of the prominent thoracic indirect flight muscles (IFMs), which exhibit similarities in their arrangement and program of differentiation to both Drosophila embryonic myogenesis and muscle formation in vertebrates, including mammals. Whereas a detailed genetic analysis of myoblast fusion has been undertaken for the Drosophila embryo, few, if any, mutants affecting the corresponding adult process have been reported. The current study has utilized a combination of genetic approaches to initiate progress along these lines via the study of the adult myogenic requirements for Wsp, the sole Drosophila WASp family member, and additional elements of the cellular actin polymerization machinery (Mukherjee, 2011).
A major finding of this study is that Wsp performs an essential role during adult fly myogenesis, which is crucial for the growth of muscle fibers via myoblast fusion. This requirement is highly specific to the fusion process, as other features and characteristics of adult myoblast development (e.g., the definition of a myoblast pool, myoblast proliferation levels and the different patterns of myoblast migration) appear unaffected in the absence of Wsp activity. The functional requirement for Wsp is comprehensive and universal in character. A complete arrest of myoblast fusion is observed in Wsp mutants for all classes of somatic muscle groups examined. Furthermore, reliance on Wsp activity during fusion is common to both of the major adult myogenic programs, namely the de novo construction of muscle fibers from individual myoblasts and fiber growth by fusion of myoblasts with persistent larval templates, as exemplified by the prominent dorsal-longitudinal muscle (DLM) flight muscles (Mukherjee, 2011).
Whereas de novo muscle fiber formation in the adult resembles the Drosophila embryonic program of myogenesis, in which Wsp was previously shown to mediate myoblast fusion, DLM growth presents a distinct paradigm. The incorporation of myoblasts into pre-existing fibers is characteristic of several aspects of skeletal muscle growth in vertebrates, including the repair of injured muscle by satellite cell-derived myoblasts and the initiation of the second wave of mammalian embryonic myogenesis. The essential and functionally conserved involvement of Wsp in diverse programs of Drosophila somatic muscle formation and growth leads to a proposal that the actin nucleation-promoting activity of WASp family proteins constitutes a fundamental aspect of the cell fusion events that accompany myogenesis (Mukherjee, 2011).
Spherical F-actin-rich structures closely associated with myoblast fusion pore formation during Drosophila embryogenesis have been recently described and extensively characterized. Current observation of very similar structures during the period of adult muscle growth via fusion now suggests that these foci are a universal feature of the myoblast fusion process. Although a variety of alterations in embryonic actin focus morphology have been described for various mutants, the molecular mechanism governing their formation is still unknown. A key lingering question is the identity of the actin nucleation machinery that is responsible for establishment of the foci. The Arp2/3 nucleation system is an obvious candidate for such a role, given the well-documented involvement of its different components in embryonic myoblast fusion. However, direct assessment of Arp2/3 complex function during embryogenesis is difficult because the maternal contribution of complex subunits, which is essential for oogenesis, is sufficient to overcome zygotic gene disruption. The capacity of RNAi to efficiently disrupt gene function during adult myogenesis thus provides a unique opportunity to study the contribution of the Arp2/3 complex to actin focus formation. The current data suggest that Arp2/3 indeed plays a crucial role in this process, as focus formation is significantly impaired upon expression of RNAi targeting two of the complex subunits (Mukherjee, 2011).
Such a requirement for Arp2/3 is somewhat surprising because actin foci persist or grow following separate or simultaneous disruption of the two major Arp2/3 nucleation-promoting factor systems centered on Wsp and SCAR. Furthermore, these behaviors closely match those reported following disruption of Wsp and SCAR system activity by a variety of means during embryogenesis. Although the WASp and SCAR systems continue to be regarded as primary nucleation-promoting factors for Arp2/3, a growing list of alternative nucleators has recently emerged, suggesting that a novel element might well activate Arp2/3 in this context. Identification of this novel NPF should provide genetic tools for determining the functional significance of the actin foci, which has so far remained elusive owing to the lack of information regarding the mechanism by which they are constructed (Mukherjee, 2011).
A central and ongoing issue is the mechanistic role played by Wsp during myoblast fusion. Several of the observations reported in this study are instructive in this context. The capacity to fully rescue the adult Wsp mutant phenotype by providing functional Wsp in either fibers or individual myoblasts suggests that interactions with cell type-specific factors are not a crucial mechanistic feature. It is therefore likely that the fusion-associated microfilament dynamics nucleated by Wsp are performed and utilized by mechanisms and molecular elements common to all muscle cells. This conclusion is particularly noteworthy considering the significance assigned to cell type-specific pathways in some models of embryonic myoblast fusion (Mukherjee, 2011).
As suggested in the embryo, localization to sites of fusion between muscle cell membranes appears to be an important aspect of Wsp activity, although the means by which Wsp is targeted to these sites during adult myogenesis is not clear. D-WIP appears to provide a key membrane-targeting function during embryonic myogenesis, but the current observations suggest that Wsp reaches adult fusion sites independently of D-WIP. Establishment of the mechanism by which Wsp localizes to adult myoblast fusion sites thus requires further study. These findings also leave open the role performed by D-WIP. A direct effect of D-WIP on cytoskeletal organization, as suggested during embryonic myoblast fusion, might be relevant in this context (Mukherjee, 2011).
The transfer of cytoplasmic material between attached, fusion-arrested muscle cells, which is a hallmark of the embryonic myoblast fusion phenotype in Wsp mutant embryos, is not observed following disruption of Wsp function during adult myogenesis. The basis for this difference is unknown and will require better elucidation of the particular aspects of the fusion process that rely on Wsp activity. One explanation is an expanded role for Wsp during adult myoblast fusion. An additional functional requirement prior to fusion pore formation might mask any subsequent participation in fusion pore expansion. Finally, it is of interest to note that, in the adult, disruption of either the Wsp or SCAR NPF systems leads to the strong arrest of myoblast fusion, similar to the embryonic scenario. These observations on adult myogenesis thus serve as a reaffirmation that the fusion process requires non-overlapping functional contributions from the two major Arp2/3 NPFs, even though they operate in close spatial and temporal proximity (Mukherjee, 2011).
In summary, the observations reported in This study generalize the involvement of the Arp2/3 complex and its associated cellular machinery during the construction of muscle fibers via myoblast fusion. In particular, a prominent and potentially universal role is assigned to the WASp NPF as a mediator of these events (Mukherjee, 2011).
Wiskott-Aldrich syndrome proteins (WASP) are nucleation promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR/WAVE, WASP and WASH, have been analyzed so far. This study characterizes WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, WHAMY specifically interacts with activated Rac1 through its two CRIB domains that are sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promotes exceptionally fast actin filament elongation, while it does not activate the Arp2/3 complex. Loss- and gain-of function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further imply synergistic functions between WHAMY and WASP during morphogenesis. Double mutants are late-embryonic lethal and show severe defects in myoblast fusion. Trans-heterozygous mutant animals show strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution (Brinkmann, 2015).
The actin cytoskeleton plays a central role in a number of different cellular functions, such as cell shape changes, cell motility and membrane trafficking. Members of the Wiskott–Aldrich syndrome protein (WASP) family are conserved nucleation-promoting factors (NPF) that activate the Arp2/3 complex, a major actin nucleator in eukaryotic cells. In mammals, the WASP protein family consists of eight different members: the two Wiskott-Aldrich syndrome proteins WASP and N-WASP (also known as WAS and WASL, respectively), the related WASP family Verprolin homologous proteins WAVE1–WAVE3 (also known as SCAR1–SCAR3 and WASF1–WASF3, the Wiskott–Aldrich syndrome protein and SCAR homolog WASH (also known as WASH1), and the WHAMM and JMY proteins. WASP proteins share a conserved C-terminal Arp2/3-complex-activating WCA module. This module consists of either one or multiple actin-monomer-binding WH2 (W) domains, a central domain (C) and an acidic (A) domain, which mediate Arp2/3 binding. Apart from the catalytic WCA module, WASP proteins often share a proline-rich region and a basic region, which bind SH3-domain containing proteins and acidic phosphoinositides, respectively. WASP proteins are regulated by similar molecular principles. Under resting conditions NPFs are primarily inactive and become activated upon binding of the Rho GTPases Cdc42 and Rac1. Additionally, a variety of factors further modulate proper activation and recruitment of WASP proteins (Brinkmann, 2015).
In Drosophila, only three WASP subfamily members have been described, namely WAVE, WASP and WASH (also known as CG13176). Insects like Drosophila have subsequently lost a WHAMM/JMY gene, although the common ancestor first arose in invertebrates. Genetic studies indicate that WAVE and WASP are the central activators of the Arp2/3 complex, differentially regulating most aspects of Arp2/3 function in Drosophila. These studies highlight distinct, but also overlapping cellular requirements of WAVE and WASP during development. WAVE function is in particular essential for cell shape and morphogenetic cell movements during development. By contrast, WASP function is needed for cell fate specification of sensory organ precursors (SOPs) and spermatid Both, WASP and WAVE are required for myoblast fusion (Brinkmann, 2015).
Loss of maternal and zygotic WASP results in late-embryonic lethality due to strong defects in cell fate decisions of neuronal cell lineages and myoblast fusion defects. Remarkably, animals lacking zygotic WASP function survive until early adulthood. Thus, maternally provided WASP protein is sufficient for proper embryonic and larval development. Mutant wasp flies show no strong morphological defects except a partial loss of sensory bristles. Loss of zygotic Arp2/3 function results in a similar, albeit stronger, neurogenic phenotype suggesting an involvement of additional factors in Arp2/3-dependent SOP development (Rajan et al., 2009). The loss of sensory bristles in wasp and arp2/3 mutants phenocopies Notch loss-of-function and is caused by a pIIa-to-pIIb cell fate transformation. This results in an excess of neurons at the expense of bristle sheath, shaft and socket cells. Recent work further suggests that the WASP–Arp2/3 pathway rather plays an important role in the trafficking of Delta-positive vesicles from the basal area to the apical cortex of the signal-sending pIIb cell (Brinkmann, 2015).
Remarkably, rescue experiments have implied that established activators of WASP, such as Cdc42 or phosphatidylinositol 4,5-bisphosphate (PIP2), are not required for WASP function, neither for the myoblast fusion process nor for SOP development. The identity of an independent activator that might act cooperatively to control Arp2/3 function in these contexts is unknown. This study presents a functional analysis of WHAMY, a new WASP-like protein that regulates cell motility of Drosophila blood cells but also synergizes with WASP during embryonic muscle formation and cell fate specification of adult SOPs (Brinkmann, 2015).
The identification of all WASP family homologs in all sequenced organisms allows a detailed phylogenetic analysis of the origin of diverse subfamilies evolving differential cellular functions. WASP proteins are multi-domain proteins. They share functions that are encoded by similar domains at the C-termini, whereas different N-terminal domains mainly define their diverse cellular processes. Gene duplication and domain shuffling are two important mechanisms driving novel and increasingly complex developmental programs during evolution. It is thought that this boost in domain shuffling is responsible for the apparent disconnection between greatly increased phenotypic complexity and a relatively small difference in gene number between humans and Drosophila (Brinkmann, 2015).
The whamy gene is an excellent example for how gene duplication and subsequent domain shuffling can create new gene functions after initial gene duplication. It arose through a duplication of wasp at the base of the genus Drosophila. Although the encoded protein has evolved a new function in cell motility, it also functions synergistically with WASP in muscle formation and sensory organ development. In the latter, WHAMY can even partially substitute for WASP, indicating that it has kept functionality following the duplication. This duality is reflected in the sequence of the WHAMY CRIB domains. As there is an overlap in function with WASP, selective pressure has been reduced since the duplication, leading to the observed increase of evolutionary rate. Following the duplication of the CRIB domains within WHAMY, a similar trend can be found. Whereas one domain has kept the function of binding to Cdc42-GTP, the other has lost the ability to interact. This is reflected in domain-specific conserved substitutions. The duplication of the wasp gene and subsequent subneofunctionalization of whamy might have occurred at the same time as the loss of a true WHAMM/JMY ancestor during insect evolution (Veltman, 2010). Like Drosophila WHAMY, the common ancestor of WHAMM/JMY proteins in invertebrates also lacks the characteristic C-terminal tryptophan residue in their VCA domains that is crucial for Arp2/3 binding and activation (Veltman, 2010). This further implies a primary Arp2/3-independent function of the common ancestor of invertebrate WHAMM/JMY proteins (Brinkmann, 2015).
WHAMY shows no Arp2/3-activating nucleation promoting factor (NPF) activity in vitro. However, different from WASP, WHAMY itself is able to promote fast elongation of linear actin filaments from actin-rich clusters. With respect to its activity, WHAMY resembles the WH2-domain containing Ena/VASP polymerases that actively drive processive actin-filament elongation and promote assembly of both lamellipodial and filopodia actin networks. Notably, Ena/VASP proteins are tetramers, and their oligomerization is mandatory to allow for polymerase activity in experiments in solution, as used in this study. Since fast filament elongation was exclusively observed from WHAMY clusters in total internal reflection fluorescence (TIRF) experiments, and consistent with the size exclusion chromatography experiments, it is proposed that WHAMY requires oligomerization to acquire actin polymerase activity. Concerning previously analyzed proteins of the WASP family, the filament elongation activity of WHAMY is therefore rather unique, and when compared to other fast actin polymerases, only the Drosophila formin Diaphanous achieves comparable high elongation activity in vitro. As evidenced from the pyrene data, the activity of WHAMY can further be increased by Rac1 (Brinkmann, 2015).
Rac1 seems to act on both the activity and the localization of WHAMY at lamellipodial tips. Both of the two CRIB domains of WHAMY bind equally to activated Rac1, and only loss of both CRIB domains abolishes Rac1 binding and the localization to the leading edge. Therefore, it currently remains unclear why WHAMY contains two CRIB domains and whether they differentially mediate distinct cellular functions. They might contribute to a local clustering of WHAMY and Rac1 at the leading edge. The most prominent Rac1 effector represents the WAVE regulatory complex (WRC) that drives Arp2/3-mediated branched actin nucleation. Rac1 directly binds and activates the WRC by allosterically releasing the bound Arp2/3-activating WCA domain of WAVE. Overexpression of WHAMY leads to a strong induction of filopodia, presumably due to the filament elongation activity of WHAMY. Additionally, competition between WHAMY and the WRC for Rac1 could disturb the balance between nucleation and elongation activity, and therefore might contribute to the observed overexpression phenotype. Different from WHAMY, WRC function is essential for lamellipodia formation and cell migration in most eukaryotic cells. By contrast, loss of WHAMY function does not impair lamellipodia formation but rather regulates cell spreading and contributes to cell motility (Brinkmann, 2015).
WHAMY does not compete but rather functions together with WASP in Drosophila morphogenesis. Previous studies have revealed that the major established activators of WASP, such as Cdc42 and PIP2, are not required for the function of WASP in sensory organ development or myoblast fusion. This observation already suggests that additional components, such as WHAMY, might act together with WASP in sensory organ development and myoblast fusion. Consistent with this, further reduction of whamy function in wasp mutants was found to phenocopy loss of arp2/3 function, resulting in an excess of neurons and a near absence of bristle sheath, shaft and socket cells. Rescue data further indicate that WHAMY can partially substitute for WASP function. Thus, WHAMY cooperates with WASP rather than acting redundantly in sensory organ development. Based on TIRF microscopy data, it is suggested that WHAMY might potentially generate mother filaments in close vicinity of Arp2/3 complex facilitating Arp2/3-mediated actin assembly (Brinkmann, 2015).
How might WHAMY and WASP act on actin dynamics during sensory organ development? Recent work suggests that the WASP–Arp2/3 pathway is not involved in Notch receptor endocytosis or its processing in the signal-receiving cell (pIIa) but rather plays an important role in the trafficking of Delta-positive vesicles from the basal area to the apical cortex of the signal-sending pIIb cell. This model also implies that recycled Notch ligands such as Delta and Serrate are active at apical junctions with actin-rich structures induced by WASP and the Arp2/3 complex, which in turn activate apical Notch receptor in pIIa. In vivo, WHAMY localizes at dynamic vesicles during sensory organ precursor formation and, together with WASP, becomes strongly enriched at apical junctions shortly after SOP division. Thus, a scenario is proposed in which WASP and WHAMY might act either on the assembly of actin-rich structures or directly promote apical trafficking of Delta through Rab11-recycling endosomes (Brinkmann, 2015).
A dynamic reorganization of the actin cytoskeleton into distinct cellular structures is also necessary to ensure successful myogenesis. Filopodial protrusions are crucial for the attachment of FCMs to the founder cell and growing myotube, and for the initiation of the fusion process. The recognition and adhesion of myoblasts depends on members of the immunoglobulin superfamily (IgSF) that are expressed specifically in myoblasts in a ring-like structure. The interaction of these proteins leads to the formation of a cell communication structure, which has been termed fusion-restricted myogenic adhesive structure (FuRMAS) or podosome-like structure. The cytodomains of the IgSFs trigger the activation of WAVE in founder cells, and of WAVE and WASP in FCMs. In FCMs, WAVE- and WASP-mediated Arp2/3 activation results in the formation of a dense F-actin focus that accumulates at the interface of adhering myoblasts. Electron microscopy studies have revealed that WASP is required for the formation of fusion pores at apposing myoblasts during embryonic and indirect flight muscle development. These fusion pores expand until full cytoplasmic continuity is achieved, and WASP has implicated to be required for fusion pore expansion. It has been discussed that WASP is required for the removal of membrane residuals during membrane vesiculation. WHAMY might contribute to this process, but the detailed mechanistic contribution of WHAMY in fusion pore formation needs to be addressed in future studies by ultrastructural analyses (Brinkmann, 2015).
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date revised: 30 December 2019
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