Actin related protein 2/actin related protein 3 (Arp2/3) complex nucleates new actin filaments in eukaryotic cells in response to signals from proteins in the Wiskott–Aldrich syndrome protein (WASP) ...family. The conserved VCA domain of WASP proteins activates Arp2/3 complex by inducing conformational changes and delivering the first actin monomer of the daughter filament. Previous models of activation have invoked a single VCA acting at a single site on Arp2/3 complex. Here we show that activation most likely involves engagement of two distinct sites on Arp2/3 complex by two VCA molecules, each delivering an actin monomer. One site is on Arp3 and the second is on ARPC1 and Arp2. The VCAs at these sites have distinct roles in activation. Our findings reconcile apparently conflicting literature on VCA activation of Arp2/3 complex and lead to a new model for this process.
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Directional cell movements during morphogenesis require the coordinated interplay between membrane receptors and the actin cytoskeleton. The WAVE regulatory complex (WRC) is a conserved actin ...regulator. Here, we found that the atypical cadherin Fat2 recruits the WRC to basal membranes of tricellular contacts where a new type of planar-polarized whip-like actin protrusion is formed. Loss of either Fat2 function or its interaction with the WRC disrupts tricellular protrusions and results in the formation of nonpolarized filopodia. We provide further evidence for a molecular network in which the receptor tyrosine phosphatase Dlar interacts with the WRC to couple the extracellular matrix, the membrane, and the actin cytoskeleton during egg elongation. Our data uncover a mechanism by which polarity information can be transduced from a membrane receptor to a key actin regulator to control collective follicle cell migration during egg elongation. 4D-live imaging of rotating MCF10A mammary acini further suggests an evolutionary conserved mechanism driving rotational motions in epithelial morphogenesis.
Retromer is a membrane coat complex that is recruited to endosomes by the small GTPase Rab7 and sorting nexin 3. The timing of this interaction and consequent endosomal dynamics are thought to be ...regulated by the guanine nucleotide cycle of Rab7. Here we demonstrate that TBC1d5, a GTPase-activating protein (GAP) for Rab7, is a high-affinity ligand of the retromer cargo selective complex VPS26/VPS29/VPS35. The crystal structure of the TBC1d5 GAP domain bound to VPS29 and complementary biochemical and cellular data show that a loop from TBC1d5 binds to a conserved hydrophobic pocket on VPS29 opposite the VPS29-VPS35 interface. Additional data suggest that a distinct loop of the GAP domain may contact VPS35. Loss of TBC1d5 causes defective retromer-dependent trafficking of receptors. Our findings illustrate how retromer recruits a GAP, which is likely to be involved in the timing of Rab7 inactivation leading to membrane uncoating, with important consequences for receptor trafficking.
Ena/VASP proteins and the WAVE regulatory complex (WRC) regulate cell motility by virtue of their ability to independently promote actin polymerization. We demonstrate that Ena/VASP and the WRC ...control actin polymerization in a cooperative manner through the interaction of the Ena/VASP EVH1 domain with an extended proline rich motif in Abi. This interaction increases cell migration and enables VASP to cooperatively enhance WRC stimulation of Arp2/3 complex-mediated actin assembly in vitro in the presence of Rac. Loss of this interaction in Drosophila macrophages results in defects in lamellipodia formation, cell spreading, and redistribution of Ena to the tips of filopodia-like extensions. Rescue experiments of abi mutants also reveals a physiological requirement for the Abi:Ena interaction in photoreceptor axon targeting and oogenesis. Our data demonstrate that the activities of Ena/VASP and the WRC are intimately linked to ensure optimal control of actin polymerization during cell migration and development.
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•Ena/VASP EVH1 interacts with the Abi subunit of the WRC•VASP enhances the ability of WRC to promote Arp2/3 actin assembly in presence of Rac•Ena/VASP and WRC interactions enhance cell migration•Drosophila photoreceptor axon targeting and oogenesis depends on Ena binding Abi
Ena/VASP proteins and the WAVE regulatory complex independently promote actin polymerization. Chen et al. demonstrate that the Ena/VASP EVH1 domain binds Abi in the WRC. This interaction enhances cell migration and Arp2/3-mediated actin polymerization. Loss of this interaction in Drosophila induces changes in macrophage morphology and defects in photoreceptor axon targeting and oogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. ...Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.
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The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large ...N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in ...diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid-liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.
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IJS, NUK, SBMB, UL, UM, UPUK
General methods to engineer genetically encoded, reversible, light-mediated control over protein function would be useful in many areas of biomedical research and technology. We describe a system ...that yields such photo-control over actin assembly. We fused the Rho family GTPase Cdc42 in its GDP-bound form to the photosensory domain of phytochrome B (PhyB) and fused the Cdc42 effector, the Wiskott-Aldrich Syndrome Protein (WASP), to the light-dependent PhyB-binding domain of phytochrome interacting factor 3 (Pif3). Upon red light illumination, the fusion proteins bind each other, activating WASP, and consequently stimulating actin assembly by the WASP target, the Arp2/3 complex. Binding and WASP activation are reversed by far-red illumination. Our approach, in which the biochemical specificity of the nucleotide switch in Cdc42 is overridden by the light-dependent PhyB-Pif3 interaction, should be generally applicable to other GTPase-effector pairs.
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The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain ...in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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