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).
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).
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).
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).
See the embryonic expression pattern of WASp at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
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).
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date revised: 25 August 2007
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