The actin nucleation factors JMY and WHAMM enable a rapid Arp2/3 complex-mediated intrinsic pathway of apoptosis

The actin cytoskeleton is a well-known player in most vital cellular processes, but comparably little is understood about how the actin assembly machinery impacts programmed cell death pathways. In the current study, we explored roles for the human Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation factors in DNA damage-induced apoptosis. Inactivation of each WASP-family gene revealed that two of them, JMY and WHAMM, are necessary for rapid apoptotic responses. JMY and WHAMM participate in a p53-dependent cell death pathway by enhancing mitochondrial permeabilization, initiator caspase cleavage, and executioner caspase activation. JMY-mediated apoptosis requires actin nucleation via the Arp2/3 complex, and actin filaments are assembled in cytoplasmic territories containing clusters of cytochrome c and active caspase-3. The loss of JMY additionally results in significant changes in gene expression, including upregulation of the WHAMM-interacting G-protein RhoD. Depletion or deletion of RHOD increases cell death, suggesting that RhoD normally contributes to cell survival. These results give rise to a model in which JMY and WHAMM promote intrinsic cell death responses that can be opposed by RhoD.     

WASP Family Proteins: Their Evolution and Its Physiological Implications

WASP family proteins control actin polymerization by activating the Arp2/3 complex. Several subfamilies exist, but their regulation and physiological roles are not well understood, nor is it even known if all subfamilies have been identified. Our extensive search reveals few novel WASP family proteins. The WASP, WASH, and SCAR/WAVE subfamilies are evolutionarily ancient, with WASH the most universally present, whereas WHAMM/JMY first appears in invertebrates. An unusual Dictyostelium WASP homologue that has lost the WH1 domain has retained its function in clathrin-mediated endocytosis, demonstrating that WASPs can function with a remarkably diverse domain topology. The WASH and SCAR/WAVE regulatory complexes are much more rigidly maintained; their domain topology is highly conserved, and all subunits are present or lost together, showing that the complexes are ancient and functionally interdependent. Finally, each subfamily has a distinctive C motif, indicating that this motif plays a specific role in each subfamily''s function, unlike the generic V and A motifs. Our analysis identifies which features are universally conserved, and thus essential, and which are branch-specific modifications. It also shows the WASP family is more widespread and diverse than currently appreciated and unexpectedly biases the physiological role of the Arp2/3 complex toward vesicle traffic.     

WASH, WHAMM and JMY: regulation of Arp2/3 complex and beyond

Arp2/3 complex mediates the nucleation of actin filaments in multiple subcellular processes, and is activated by nucleation-promoting factors (NPFs) from the Wiskott-Aldrich Syndrome family. In exciting new developments, this family has grown by three members: WASH, WHAMM and JMY, which extend the repertoire of dynamic membrane structures that are remodeled following Arp2/3 activation in vivo. These novel NPFs share an actin- and Arp2/3-interacting WCA module, and combine Arp2/3 activation with additional biochemical functions, including capping protein inhibition, microtubule engagement or Arp2/3-independent actin nucleation, none of which had been previously associated with canonical WCA-harboring proteins. Uncovering the physiological relevance of these unique activities will require concerted efforts from multiple disciplines, and is sure to impact our understanding of how the cytoskeleton controls so many dynamic subcellular events.     

Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation

Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity.     

Relative actin nucleation promotion efficiency by WASP and WAVE proteins in endothelial cells

The mammalian genome encodes multiple Wiskott-Aldrich syndrome protein (WASP)/WASP-family Verprolin homologous (WAVE) proteins. Members of this family interact with the actin related protein (Arp) 2/3 complex to promote growth of a branched actin network near the plasma membrane or the surface of moving cargos. Arp2/3 mediated branching can further lead to formation of comet tails (actin rockets). Despite their similar domain structure, different WASP/WAVE family members fulfill unique functions that depend on their subcellular location and activity levels. We measured the relative efficiency of actin nucleation promotion of full-length WASP/WAVE proteins in a cytoplasmic extract from primary human umbilical vein endothelial cells (HUVEC). In this assay WAVE2 and WAVE3 complexes showed higher nucleation efficiency than WAVE1 and N-WASP, indicating distinct cellular controls for different family members. Previously, WASP and N-WASP were the only members that were known to stimulate comet formation. We observed that in addition to N-WASP, WAVE3 also induced short actin tails, and the other WAVEs induced formation of asymmetric actin shells. Differences in shape and structure of actin-based growth may reflect varying ability of WASP/WAVE proteins to break symmetry of the actin shell, possibly by differential recruitment of actin bundling or severing (pruning or debranching) factors.     

Activation of nucleation promoting factors for directional actin filament elongation: Allosteric regulation and multimerization on the membrane

Nucleation promoting factors (NPFs) activate the Arp2/3 complex to produce branched actin filaments. Branched actin filaments are observed in most organelles, and specific NPFs, such as WASP, N-WASP, WAVEs, WASH, and WHAMM, exist for each organelle. Interestingly, Arp2/3 and NPFs are both inactive by themselves, and thus require activation. The exposure of the Arp2/3 activating region, the VCA fragment, is recognized to be a key event in the activation of the NPFs. Together, small GTPase binding, phosphorylation, SH3 binding, and membrane binding promote VCA exposure synergistically. The increase in the local concentration of NPF by multimerization is thought to occur with the combination of such activators, to maximally activate the NPF and confine the region of actin polymerization. The mechanism of uni-directional filament extension beneath the membrane also is discussed.     

Structural insights into WHAMM-mediated cytoskeletal coordination during membrane remodeling

The microtubule (MT) and actin cytoskeletons drive many essential cellular processes, yet fairly little is known about how their functions are coordinated. One factor that mediates important cross talk between these two systems is WHAMM, a Golgi-associated protein that utilizes MT binding and actin nucleation activities to promote membrane tubulation during intracellular transport. Using cryoelectron microscopy and other biophysical and biochemical approaches, we unveil the underlying mechanisms for how these activities are coordinated. We find that WHAMM bound to the outer surface of MT protofilaments via a novel interaction between its central coiled-coil region and tubulin heterodimers. Upon the assembly of WHAMM onto MTs, its N-terminal membrane-binding domain was exposed at the MT periphery, where it can recruit vesicles and remodel them into tubular structures. In contrast, MT binding masked the C-terminal portion of WHAMM and prevented it from promoting actin nucleation. These results give rise to a model whereby distinct MT-bound and actin-nucleating populations of WHAMM collaborate during membrane tubulation.     

γ-Tubulin localizes at actin-based membrane protrusions and inhibits formation of stress-fibers

γ-Tubulin serves as a template in the γ-TuRC machinery to nucleate microtubules. Curiously, γ-tubulin also interacts with Arp2/3, a complex that nucleates actin filaments, and with the Arp2/3 activator WASH. We previously reported that γ-tubulin and Arp2/3 colocalize at the centrosome, where WASH localizes. Here, we report that γ-tubulin localizes at actin-based membrane protrusions, where Arp2/3 operates. This was confirmed by the presence of tagged γ-tubulin at membrane protrusions in stimulated cells and by downregulation of γ-tubulin expression. Surprisingly, expression of tagged γ-tubulin dramatically inhibited the formation of stress-fibers, while having no effect on microtubules. This phenotype is similar to the disruption of stress-fibers by the overexpression of the WCA domain of WASH and other Wiskott–Aldrich syndrome (WAS) family members. We hypothesize that γ-tubulin regulates Arp2/3 and actin nucleation promoting factors such as WASH, explaining the similar effect of γ-tubulin expression and WCA domain expression on stress-fibers. The data presented here indicate that γ-tubulin has a profound relationship with actin filament dynamics.     

WASH has a critical role in NK cell cytotoxicity through Lck-mediated phosphorylation

Natural killer (NK) cells are important effector cells of the innate immune system to kill certain virus-infected and transformed cells. Wiskott–Aldrich Syndrome protein (WASP) and SCAR homolog (WASH) has been identified as a member of WASP family proteins implicated in regulating the cytoskeletal reorganization, yet little is known about its function in lymphocytes. Here we demonstrate that WASH is crucial for NK cell cytotoxicity. WASH was found to colocalize with lytic granules upon NK cell activation. Knockdown of WASH expression substantially inhibited polarization and release of lytic granules to the immune synapse, resulting in the impairment of NK cell cytotoxicity. More importantly, our data also define a previously unappreciated mechanism for WASH function, in which Src family kinase Lck can interact with WASH and induce WASH phosphorylation. Mutation of tyrosine residue Y141, identified here as the major site of WASH phosphorylation, partially blocked WASH tyrosine phosphorylation and NK cell cytotoxicity. Taken together, these observations suggest that WASH has a pivotal role for regulation of NK cell cytotoxicity through Lck-mediated Y141 tyrosine phosphorylation.Natural killer (NK) cells are the first defense line against viral infections and tumors.¹ NK cell-mediated lysis of target cells requires the formation of immunological synapse between NK cells and target cells and subsequent delivery of lytic granules containing perforin and granzymes.^{2, 3} The importance of the actin cytoskeleton in this process has been well documented.⁴ However, the precise mechanism of actin reorganization in NK cells remains to be elucidated.Wiskott–Aldrich syndrome protein (WASP) is the first identified member of an actin regulator family.⁵ WASP family proteins contain a C-terminal domain that binds to and activates the Arp2/3 complex for cytoskeleton remodeling.⁶ In the absence of WASP, cytotoxic activity of NK cells is defective owing to impaired immune synapse formation and perforin localization.⁷ It has also been shown that WASP may be important for integration of NK cell signaling, particularly for nuclear translocation of NFAT2 and NF-κB during the activating receptor NKp46-dependent activation.⁸WASP and SCAR homolog (WASH) has been discovered as a new WASP family member.⁹ Subsequent studies show that WASH interacts with multiple proteins, including FAM21, to form a large core complex and regulate actin dynamics.¹⁰ WASH localizes to sorting and recycling endosomes, where WASH complex activates Arp2/3-mediated actin polymerization and controls the production of transport intermediates from endosome.¹¹ Unlike other WASP family members, WASH has distinct N-terminal domains, termed WASH homology domain 1 (WHD1) and tubulin-binding region (TBR).¹² Moreover, WASH has been shown to regulate recycling of many surface receptors via endosomal trafficking in activated T cells.¹³In our previous works, we found embryonic lethality and extensive autophagy in WASH-deficient mice. WASH recruits RNF2 to ubiquitinate AMBRA1 and inhibits the ubiquitination of Beclin1, a well-known moderator in autophage.^{14, 15} Of interest, WASH is located in cell nucleus and participates in hematopoietic stem cell differentiation through recruiting NURF complex to c-Myc promoter.¹⁶ However, the role and mechanism of WASH in NK cell function remain poorly understood.In this study, we show that inhibition of WASH expression with RNA interference or an inducible gene targeting system severely impair NK cell cytotoxicity through blockade of lytic granule polarization. In addition, Src family kinase Lck can interact with and induce tyrosine phosphorylation of WASH protein in human NK cells. These analyses provide the cellular and molecular mechanisms involved in the regulation of WASH function during NK cell activation.     

WH2 and proline‐rich domains of WASP‐family proteins collaborate to accelerate actin filament elongation

WASP‐family proteins are known to promote assembly of branched actin networks by stimulating the filament‐nucleating activity of the Arp2/3 complex. Here, we show that WASP‐family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP‐family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline‐rich sequence that binds profilin–actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline‐rich sequences are required to support polymerase activity by (i) bringing polymerization‐competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin–actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP‐family proteins that create it. Collaboration between WH2 and proline‐rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP‐family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.     

Interactions with Actin Monomers,Actin Filaments,and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks

Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.     

WHAMM is an Arp2/3 complex activator that binds microtubules and functions in ER to Golgi transport

The Arp2/3 complex is an actin nucleator that plays a critical role in many cellular processes. Its activities are regulated by nucleation-promoting factors (NPFs) that function primarily during plasma membrane dynamics. Here we identify a mammalian NPF called WHAMM (WASP homolog associated with actin, membranes, and microtubules) that localizes to the cis-Golgi apparatus and tubulo-vesicular membrane transport intermediates. The modular organization of WHAMM includes an N-terminal domain that mediates Golgi membrane association, a coiled-coil region that binds microtubules, and a WCA segment that stimulates Arp2/3-mediated actin polymerization. Overexpression and depletion studies indicate that WHAMM is important for maintaining Golgi structure and facilitating anterograde membrane transport. The ability of WHAMM to interact with microtubules plays a role in membrane tubulation, while its capacity to induce actin assembly promotes tubule elongation. Thus, WHAMM is an important regulator of membrane dynamics functioning at the interface of the microtubule and actin cytoskeletons.     

RhoD regulates cytoskeletal dynamics via the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules

The Rho GTPases have mainly been studied in association with their roles in the regulation of actin filament organization. These studies have shown that the Rho GTPases are essential for basic cellular processes, such as cell migration, contraction, and division. In this paper, we report that RhoD has a role in the organization of actin dynamics that is distinct from the roles of the better-studied Rho members Cdc42, RhoA, and Rac1. We found that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM), as well as the related filamin A–binding protein FILIP1. Of these two RhoD-binding proteins, WHAMM was found to bind to the Arp2/3 complex, while FILIP1 bound filamin A. WHAMM was found to act downstream of RhoD in regulating cytoskeletal dynamics. In addition, cells treated with small interfering RNAs for RhoD and WHAMM showed increased cell attachment and decreased cell migration. These major effects on cytoskeletal dynamics indicate that RhoD and its effectors control vital cytoskeleton-driven cellular processes. In agreement with this notion, our data suggest that RhoD coordinates Arp2/3-dependent and FLNa-dependent mechanisms to control the actin filament system, cell adhesion, and cell migration.     

Functional Characterization of Wiskott-Aldrich Syndrome Protein and Scar Homolog (WASH), a Bi-modular Nucleation-promoting Factor Able to Interact with Biogenesis of Lysosome-related Organelle Subunit 2 (BLOS2) and γ-Tubulin

The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting. We found that overexpression of the WASH-WCA domain induced disruption of the actin cytoskeleton, whereas overexpression of full-length WASH in mammalian cells did not affect stress fiber organization. Furthermore, our analysis has revealed that nerve growth factor treatment of PC12 cells overexpressing full-length WASH leads to disruption of the actin cytoskeleton. We have also found that WASH interacts through its N-terminal region with BLOS2, a centrosomal protein belonging to the BLOC-1 complex that functions as a scaffolding factor in the biogenesis of lysosome-related organelles. In addition to BLOS2, WASH also interacts with centrosomal γ-tubulin and with pallidin, an additional component of the BLOC-1 complex. Collectively, our data propose that WASH is a bimodular protein in which the C terminus is involved in Arp2/3-mediated actin nucleation, whereas the N-terminal portion is required for its regulation and localization in the cells. Moreover, our data suggest that WASH is also a component of the BLOC-1 complex that is associated with the centrosomes.     

Regulation of actin dynamics by WASP family proteins

Rapid reorganization of the actin cytoskeleton underlies morphological changes and motility of cells. WASP family proteins have received a great deal of attention as the signal-regulated molecular switches that initiate actin polymerization. The first member, WASP, was identified as the product of a gene of which dysfunction causes the human hereditary disease Wiskott-Aldrich syndrome. There are now five members in this protein family, namely WASP, N-WASP, WAVE/Scar1, 2, and 3. WASP and N-WASP have functional and physical associations with Cdc42, a Rho family small GTPase involved in filopodium formation. In contrast, there is evidence that links the WAVE/Scar proteins with another Rho family protein, Rac, which is a regulator of membrane ruffling. All WASP family members have a VCA domain at the C-terminus through which Arp2/3 complex is activated to nucleate actin polymerization. Analyses of model organisms have just begun to reveal unexpected functions of WASP family proteins in multicellular organisms.     

The actin nucleation factor JMY is a negative regulator of neuritogenesis

Junction-mediating and regulatory protein (JMY) is a p53 cofactor that was recently shown to nucleate actin assembly by a hybrid mechanism involving tandem actin monomer binding and Arp2/3 complex activation. However, the regulation and function of JMY remain largely uncharacterized. We examined the activity of JMY in vitro and in cells, its subcellular distribution, and its function in fibroblast and neuronal cell lines. We demonstrated that recombinant full-length JMY and its isolated WASP homology 2 domain, connector, and acidic region (WWWCA) have potent actin-nucleating and Arp2/3-activating abilities in vitro. In contrast, the activity of full-length JMY, but not the isolated WWWCA domain, is suppressed in cells. The WWWCA domain is sufficient to promote actin-based bead motility in cytoplasmic extracts, and this activity depends on its ability to activate the Arp2/3 complex. JMY is expressed at high levels in brain tissue, and in various cell lines JMY is predominantly cytoplasmic, with a minor fraction in the nucleus. Of interest, silencing JMY expression in neuronal cells results in a significant enhancement of the ability of these cells to form neurites, suggesting that JMY functions to suppress neurite formation. This function of JMY requires its actin-nucleating activity. These findings highlight a previously unrecognized function for JMY as a modulator of neuritogenesis.     

Differential regulation of WASP and N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2

The Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP) are key players in regulating actin cytoskeleton via the Arp2/3 complex. It has been widely reported that the WASP proteins are activated by Rho family small GTPase Cdc42 and that Rac1 acts through SCAR/WAVE proteins. However, a systematic study of the specificity of different GTPases for different Arp2/3 activators has not been conducted. In this study, we have expressed, purified, and characterized completely soluble, highly active, and autoinhibited full-length human WASP and N-WASP from mammalian cells. We show a novel N-WASP activation by Rho family small GTPase Rac1. This GTPase exclusively stimulates N-WASP and has no effects on WASP. Rac1 is a significantly more potent N-WASP activator than Cdc42. In contrast, Cdc42 is a more effective activator of WASP than N-WASP. Lipid vesicles containing PIP2 significantly improve actin nucleation by the Arp2/3 complex and N-WASP in the presence of Rac1 or Cdc42. PIP2 vesicles have no effect on WASP activity alone. Moreover, the inhibition of WASP-stimulated actin nucleation in the presence of Cdc42 and PIP2 vesicles has been observed. We found that adaptor proteins Nck1 or Nck2 are the most potent WASP and N-WASP activators with distinct effects on the WASP family members. Our in vitro data demonstrates differential regulation of full-length WASP and N-WASP by cellular activators that highlights fundamental differences of response at the protein-protein level.     

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.