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Infection of insect cells with the baculovirus AcMNPV causes the formation of dynamic actin-rich invadosome structures. The viral protein ARIF-1 is necessary and sufficient for the formation of invadosomes, the host protein cortactin is present in these structures, and the host Arp2/3 complex is required for their formation.

Trypanosoma brucei, the causative agent of African sleeping sickness, uses its flagellum for movement, cell division, and signaling. The flagellum is anchored to the cell body membrane viathe flagellum attachment zone (FAZ), a complex of proteins, filaments, and microtubules that spans two membranes with elements on both flagellum and cell body sides. How FAZ components are carried into place to form this complex is poorly understood. Here, we show that the trypanosome-specific kinesin KIN-E is required for building the FAZ in bloodstream-form parasites. KIN-E is localized along the flagellum with a concentration at its distal tip. Depletion of KIN-E by RNAi rapidly inhibits flagellum attachment and leads to cell death. A detailed analysis reveals that KIN-E depletion phenotypes include failure in cytokinesis completion, kinetoplast DNA mis-segregation, and transport vesicle accumulation. Together with previously published results in procyclic form parasites, these data suggest KIN-E plays a critical role in FAZ assembly in T. brucei.Movie S1Movie S1Flagellum beating in a 90‐13 cell. Frames acquired every 20 ms, plays at 25 frames/sec.Movie S2Movie S2Flagella beating in a dividing 90‐13 cell. Frames acquired every 20 ms, plays at 25 frames/sec.Movie S3Movie S3Detached flagellum beating in a KIN‐ERNAicell at 24 hpi. Frames acquired every 20 ms, plays at 25 frames/sec.Movie S4Movie S4Detached flagella beating in a dividing KIN‐ERNAicell at 24 hpi. Frames acquired every 20 ms, plays at 25 frames/sec.

Small G-proteins regulate the recruitment and activation of WASP-family actin nucleation factors at the plasma membrane. The G-protein Rab1 interacts with the nucleation factor WHAMM to remodel internal membranes into tubules. Unlike other G-proteins that recruit nucleation factors, Rab1 inhibits actin assembly.

Live-cell imaging reveals the cellular-level pathway for B. thailandensis-induced cell–cell fusion and resolves how bacterial motility, plasma membrane protrusions, and the type VI secretion system-5 components VgrG5 and TagD5 work together to induce cell–cell fusion.

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.

Exposure of neutrophils to chemoattractant induces cell polarization and migration. These behaviors require the asymmetric activation of distinct signaling pathways and cytoskeletal elements in the protruding pseudopod at the front of cells and the retracting uropod at the rear. An important outstanding question is, how does the organization of the plasma membrane participate in establishing asymmetry during polarization and migration? To answer this question, we investigated the function of cholesterol, a lipid known to influence membrane organization. Using controlled cholesterol depletion, we found that a cholesterol-dependent membrane organization enabled cell polarization and migration by promoting uropod function and suppressing ectopic pseudopod formation. At a mechanistic level, we showed that cholesterol was directly required for suppressing inappropriate activation of the pseudopod-promoting Gi/PI3-kinase signaling pathway. Furthermore, cholesterol was required for dampening Gi-dependent negative feedback on the RhoA signaling pathway, thus enabling RhoA activation and uropod function. Our findings suggest a model in which a cholesterol-dependent membrane organization plays an essential role in the establishment of cellular asymmetry by balancing the activation and segregating the localization of competing pseudopod- and uropod-inducing signaling pathways during neutrophil polarization and migration.

In response to upstream signals, proteins in the Wiskott-Aldrich Syndrome protein (WASP) family regulate actin nucleation via the Arp2/3 complex. Despite intensive study of the function of WASP family proteins in nucleation, it is not yet understood how their distinct structural organization contributes to actin-based motility. Herein, we analyzed the activities of WASP and Scar1 truncation derivatives by using a bead-based motility assay. The minimal region of WASP sufficient to direct movement was the C-terminal WCA fragment, whereas the corresponding region of Scar1 was insufficient. In addition, the proline-rich regions of WASP and Scar1 and the Ena/VASP homology 1 (EVH1) domain of WASP independently enhanced motility rates. The contributions of these regions to motility could not be accounted for by their direct effects on actin nucleation with the Arp2/3 complex, suggesting that they stimulate motility by recruiting additional factors. We have identified profilin as one such factor. WASP- and Scar1-coated bead motility rates were significantly reduced by depletion of profilin and VASP and could be more efficiently rescued by a combination of VASP and wild-type profilin than by VASP and a mutant profilin that cannot bind proline-rich sequences. Moreover, motility of WASP WCA beads was not affected by the depletion or addback of VASP and profilin. Our results suggest that recruitment of factors, including profilin, by the proline-rich regions of WASP and Scar1 and the EVH1 domain of WASP stimulates cellular actin-based motility.

The cellular functions of the product of the Saccharomyces cerevisiae ANC1 (actin non-complementing) gene were investigated. ANC1 was previously identified in a screen for mutations that enhance the defect caused by a mutation in the actin gene. Here, we show that anc1-1 and anc1 delta 1::HIS3 (gene deletion) mutants exhibit a novel combination of defects in the organization of the actin cytoskeleton and the localization of Spa2p, a protein implicated in polarity development and cytokinesis. Morphological abnormalities exhibited by anc1 mutants include failure to form a mating projection in response to alpha-factor and development of swollen or elongated cell shapes during proliferation. These morphological aberrations correlate with cytoskeletal defects that were also observed. These phenotypes demonstrate that Anc1p is important for actin function and for the functions of other proteins involved in morphogenesis. In further support of these roles for Anc1p, the anc1 delta 1::HIS3 mutation was found to be synthetically lethal in combination with a null mutation in SLA1, a gene that is important for membrane cytoskeleton function. Surprisingly, Anc1p was found to be a nuclear protein and to have sequence similarity to the human proteins ENL and AF-9. These human proteins are implicated in the development of a subset of acute lymphoblastic leukemias, acute myeloid leukemias, and lymphomas. Our findings suggest that changes in the functions or organization of actin filaments might contribute to the establishment of the neoplastic state for these leukemias and lymphomas.