The novel Rho-family GTPase rif regulates coordinated actin-based membrane rearrangements

Small GTPases of the Rho family have a critical role in controlling cell morphology, motility and adhesion through dynamic regulation of the actin cytoskeleton [1,2]. Individual Rho GTPases have been shown to regulate distinct components of the cytoskeletal architecture; RhoA stimulates the bundling of actin filaments into stress fibres [3], Rac reorganises actin to produce membrane sheets or lamellipodia [4] and Cdc42 causes the formation of thin, actin-rich surface projections called filopodia [5]. We have isolated a new Rho-family GTPase, Rif (Rho in filopodia), and shown that it represents an alternative signalling route to the generation of filopodial structures. Coordinated regulation of Rho-family GTPases can be used to generate more complicated actin rearrangements, such as those underlying cell migration [6]. In addition to inducing filopodia, Rif functions cooperatively with Cdc42 and Rac to generate additional structures, increasing the diversity of actin-based morphology.     

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation

BACKGROUND: Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. RESULTS: We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. CONCLUSIONS: This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.     

Cdc42-induced actin filaments are protected from capping protein.

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.     

Rif-mDia1 interaction is involved in filopodium formation independent of Cdc42 and Rac effectors

Filopodia are cellular protrusions important for axon guidance, embryonic development, and wound healing. The Rho GTPase Cdc42 is the best studied inducer of filopodium formation, and several of its effectors and their interacting partners have been linked to the process. These include IRSp53, N-WASP, Mena, and Eps8. The Rho GTPase, Rif, also drives filopodium formation. The signaling pathway by which Rif induces filopodia is poorly understood, with mDia2 being the only protein implicated to date. It is thus not clear how distinct the Rif-driven pathway for filopodium formation is from the one mediated by Cdc42. In this study, we characterize the dynamics of Rif-induced filopodia by time lapse imaging of live neuronal cells and show that Rif drives filopodium formation via an independent pathway that does not involve the Cdc42 effectors N-WASP and IRSp53, the IRSp53 binding partner Mena, or the Rac effectors WAVE1 and WAVE2. Rif formed filopodia in the absence of N-WASP or Mena and when IRSp53, WAVE1, or WAVE2 was knocked down by RNAi. Rif-mediated filopodial protrusion was instead reduced by silencing mDia1 expression or overexpressing a dominant negative mutant of mDia1. mDia1 on its own was able to form filopodia. Data from acceptor photobleaching FRET studies of protein-protein interaction demonstrate that Rif interacts directly with mDia1 in filopodia but not with mDia2. Taken together, these results suggest a novel pathway for filopodia formation via Rif and mDia1.     

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.     

Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex

Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo. However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro, enhancing IRSp53-dependent membrane extensions in vivo. Cdc42 binds to and controls the cellular distribution of the IRSp53-Eps8 complex, supporting the existence of a Cdc42-IRSp53-Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53-Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.     

Protein-tyrosine kinase and GTPase signals cooperate to phosphorylate and activate Wiskott-Aldrich syndrome protein (WASP)/neuronal WASP

Protein-tyrosine kinases and Rho GTPases regulate many cellular processes, including the reorganization and dynamics of the actin cytoskeleton. The Wiskott-Aldrich syndrome protein (WASP) and its homolog neuronal WASP (N-WASP) are effectors of the Rho GTPase Cdc42 and provide a direct link between activated membrane receptors and the actin cytoskeleton. WASP and N-WASP are also regulated by a large number of other activators, including protein-tyrosine kinases, phosphoinositides, and Src homology 3-containing adaptor proteins, and can therefore serve as signal integrators inside cells. Here we show that Cdc42 and the Src family kinase Lck cooperate at two levels to enhance WASP activation. First, autoinhibition in N-WASP decreases the efficiency (kcat/Km) of phosphorylation and dephosphorylation of the GTPase binding domain by 30- and 40-fold, respectively, and this effect is largely reversed by Cdc42. Second, Cdc42 and the Src homology 3-Src homology 2 module of Lck cooperatively stimulate the activity of phosphorylated WASP, with coupling energy of approximately 2.4 kcal/mol between the two activators. These combined effects provide mechanisms for high specificity in WASP activation by coincident GTPase and kinase signals.     

Dynamin1 Is a Novel Target for IRSp53 Protein and Works with Mammalian Enabled (Mena) Protein and Eps8 to Regulate Filopodial Dynamics

Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-of-function actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics.     

The mechanisms used by enteropathogenic Escherichia coli to control filopodia dynamics

Enteropathogenic Escherichia coli (EPEC) subverts actin dynamics in eukaryotic cells by injecting effector proteins via a type III secretion system. First, WxxxE effector Map triggers transient formation of filopodia. Then, following recovery from the filopodial signals, EPEC triggers robust actin polymerization via a signalling complex comprising Tir and the adaptor proteins Nck. In this paper we show that Map triggers filopodia formation by activating Cdc42; expression of dominant-negative Cdc42 or knock-down of Cdc42 by siRNA impaired filopodia formation. In addition, Map binds PDZ1 of NHERF1. We show that Map–NHERF1 interaction is needed for filopodia stabilization in a process involving ezrin and the RhoA/ROCK cascade; expression of dominant-negative ezrin and RhoA or siRNA knock-down of RhoA lead to rapid elimination of filopodia. Moreover, we show that formation of the Tir-Nck signalling complex leads to filopodia withdrawal. Recovery from the filopodial signals requires phosphorylation of a Tir tyrosine (Y474) residue and actin polymerization pathway as both infection of cells with EPEC expressing TirY474S or infection of Nck knockout cells with wild-type EPEC resulted in persistence of filopodia. These results show that EPEC effectors modulate actin dynamics by temporal subverting the Rho GTPases and other actin polymerization pathways for the benefit of the adherent pathogen.     

Cdc42 participates in the regulation of ADF/cofilin and retinal growth cone filopodia by brain derived neurotrophic factor

Rho family GTPases have important roles in mediating the effects of guidance cues and growth factors on the motility of neuronal growth cones. We previously showed that the neurotrophin BDNF regulates filopodial dynamics on growth cones of retinal ganglion cell axons through activation of the actin regulatory proteins ADF and cofilin by inhibiting a RhoA-dependent pathway that phosphorylates (inactivates) ADF/cofilin. The GTPase Cdc42 has also been implicated in mediating the effects of positive guidance cues. In this article we investigated whether Cdc42 is involved in the effects of BDNF on filopodial dynamics. BDNF treatment increases Cdc42 activity in retinal neurons, and neuronal incorporation of constitutively active Cdc42 mimics the increases in filopodial number and length. Furthermore, constitutively active and dominant negative Cdc42 decreased and increased, respectively, the activity of RhoA in retinal growth cones, indicating crosstalk between these GTPases in retinal growth cones. Constitutively active Cdc42 mimicked the activation of ADF/cofilin that resulted from BDNF treatment, while dominant negative Cdc42 blocked the effects of BDNF on filopodia and ADF/cofilin. The inability of dominant negative Cdc42 to block ADF/cofilin activation and stimulation of filopodial dynamics by the ROCK inhibitor Y-27632 indicate interaction between Cdc42 and RhoA occurs upstream of ROCK. Our results demonstrate crosstalk occurs between GTPases in mediating the effects of BDNF on growth cone motility, and Cdc42 activity can promote actin dynamics via activation of ADF/cofilin.     

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.     

Control of actin assembly and disassembly at filament ends

The most important discovery in the field is that the Arp2/3 complex nucleates assembly of actin filaments with free barbed ends. Arp2/3 also binds the sides of actin filaments to create a branched network. Arp2/3's nucleation activity is stimulated by WASP family proteins, some of which mediate signaling from small G-proteins. Listeria movement caused by actin polymerization can be reconstituted in vitro using purified proteins: Arp2/3 complex, capping protein, actin depolymerizing factor/cofilin, and actin. actin depolymerizing factor/cofilin increases the rate at which actin subunits leave pointed ends, and capping protein caps barbed ends.     

Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking

Rho GTPases are well known to regulate actin dynamics. They activate two types of actin nucleators, WASP/WAVE proteins and Diaphanous-related formins (DRFs), which induce different types of actin organization. Their ability to interact with membranes allows them to target actin polymerization to discrete sites on the plasma membrane and to intracellular membrane compartments and thereby induce membrane protrusions or regulate vesicle movement. Most studies have concentrated on just three of the 22 mammalian Rho proteins, RhoA, Rac1 and Cdc42. However, recent research indicates that several other members of the Rho family, including Rif, RhoD, TC10 and Wrch1, and also related Rho-of-plants proteins (ROPs) in plants, stimulate actin polymerization and affect plasma membrane protrusion and/or vesicular traffic.     

Waltzing with WASP

Wiskott-Aldrich syndrome (WAS) is an inherited immune deficiency that is marked by eczema, bleeding and recurrent infections. The lymphocytes and platelets of WAS patients display cytoskeletal abnormalities, and their T lymphocytes show a diminished proliferative response to stimulation through the T-cell receptor-CD3 complex (TCR-CD3). The product of the WAS gene, WAS protein (WASP), binds to the small GTPase Cdc42. Small GTPases of the Rho family are crucial for the regulation of the actin-based cytoskeleton. WASP and its relative NWASP might play an important role in regulating the actin cytoskeleton. Since both WASP and NWASP have the potential to bind to multiple proteins, they might serve as a hub to coordinate the redistribution of many cellular signals to the actin cytoskeleton. In this review, the authors discuss the possible role of WASP/NWASP and of the newly described protein WIP, which interacts with WASP and NWASP, in coupling signals from the T-cell receptor to the actin-based cytoskeleton.     

Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange.

The Cdc42 GTPase, a member of the Rho subfamily of Ras proteins, can signal to the cytoskeleton through its effector, the Wiskott-Aldrich syndrome protein (WASP), activation of which results in localized polymerization of new actin filaments. NMR structures of WASP peptide models in the Cdc42-bound and free states suggest that GTPase binding weakens autoinhibitory contacts between the GTPase binding domain (GBD) and the C-terminal actin regulatory (VCA) region of the protein. In the study presented here, amide hydrogen exchange has been used with NMR spectroscopy to directly examine destabilization of the autoinhibited GBD-VCA conformation caused by GTPase binding. A truncated protein, GBD-C, which models autoinhibited WASP, folds into a highly stable conformation with amide exchange protection factors of up to 3 x 10(6). A novel hydrogen exchange labeling-quench strategy, employing a high-affinity ligand to displace Cdc42 from WASP, was used to examine the amide exchange from the Cdc42-bound state of GBD-C. The GTPase increases exchange rates of the most protected amides by 50-500-fold, with destabilization reducing the differences in the protection of segments in the free state. The results confirm that Cdc42 facilitates the physical separation of the GBD from the VCA in a tethered molecule, indicating this process likely plays an important role in activation of full-length WASP by the GTPase. However, destabilization of GBD-C is not complete in the Cdc42 complex. The data indicate that partitioning of free energy between binding and activation may limit the extent to which GTPases can cause conformational change in effectors. This notion is consistent with the requirement of multiple input signals in order to achieve maximal activation in many effector molecules.     

The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.     

IQGAP1 is a component of Cdc42 signaling to the cytoskeleton

The Ras-GAP related protein IQGAP1 binds several proteins, including actin, calmodulin, E-cadherin and the Rho family GTPase Cdc42. To gain insight into its in vivo function, IQGAP1 was overexpressed in mammalian cells. Transfection of IQGAP1 significantly increased the levels of active, GTP-bound Cdc42, resulting in the formation of peripheral actin microspikes. By contrast, transfection of an IQGAP1 mutant lacking part of the GAP-related domain (IQGAP1deltaGRD) substantially decreased the amount of GTP-bound Cdc42 in cell lysates. Consistent with these findings, IQGAP1DeltaGRD blocked Cdc42 function in cells that stably overexpress constitutively active Cdc42 and abrogated the effect of bradykinin on Cdc42. In cells transfected with IQGAP1deltaGRD, bradykinin was unable to activate Cdc42, translocate Cdc42 to the membrane fraction, or induce filopodia production. IQGAP1deltaGRD transfection altered cellular morphology, producing small, round cells that closely resemble Cdc42-/- cells. Some insight into the mechanism was provided by in vitro analysis, which revealed that IQGAP1deltaGRD increased the intrinsic GTPase activity of Cdc42, thereby increasing the amount of inactive, GDP-bound Cdc42. These data imply that IQGAP1 has a crucial role in transducing Cdc42 signaling to the cytoskeleton.     

Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex.

BACKGROUND: The Rho GTPases Rho, Rac, and Cdc42 regulate the organization of the actin cytoskeleton by interacting with multiple, distinct downstream effector proteins. Cdc42 controls the formation of actin bundle-containing filopodia at the cellular periphery. The molecular mechanism for this remains as yet unclear. RESULTS: We report here that Cdc42 interacts with IRSp53/BAP2 alpha, an SH3 domain-containing scaffold protein, at a partial CRIB motif and that an N-terminal fragment of IRSp53 binds, via an intramolecular interaction, to the CRIB motif-containing central region. Overexpression of IRSp53 in fibroblasts leads to the formation of filopodia, and both this and Cdc42-induced filopodia are inhibited by expression of the N-terminal IRSp53 fragment. Using affinity chromatography, we have identified Mena, an Ena/VASP family member, as interacting with the SH3 domain of IRSp53. Mena and IRSp53 act synergistically to promote filopodia formation. CONCLUSION: We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia.     

The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia

Formins have important roles in the nucleation of actin and the formation of linear actin filaments, but their role in filopodium formation has remained elusive. Dictyostelium discoideum Diaphanous-related formin dDia2 is enriched at the tips of filopodia and interacts with profilin II and Rac1. An FH1FH2 fragment of dDia2 nucleated actin polymerization and removed capping protein from capped filament ends. Genetic studies showed that dDia2 is important for cell migration as well as the formation, elongation and maintenance of filopodia. Here we provide evidence that dDia2 specifically controls filopodial dynamics by regulating actin turnover at the barbed ends of actin filaments.     

Wsp1, a GBD/CRIB domain-containing WASP homolog, is required for growth, morphogenesis, and virulence of Cryptococcus neoformans

Human endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogen Cryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins than Saccharomyces cerevisiae Las17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, the wsp1 mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. The wsp1 mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. An in vitro phagocytosis assay showed a higher phagocytic index for the wsp1 mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal virulence.