WHIMP is a WASP-family Actin Nucleation-promoting Factor that Functions in Cell Motility

Actin filament assembly is regulated by many actin-binding proteins that function to maintain cell shape and structure, enable vesicle trafficking, control motility, and support DNA repair. Identifying these regulatory proteins is critical for a comprehensive understanding of the dynamic organization of actin in various cellular processes. In the last decade, the discovery of new Wiskott-Aldrich Syndrome Protein (WASP) family actin nucleation factors such as WASH, WHAMM, and JMY has significantly contributed to our understanding of actin cytoskeleton functions, and opened new avenues of research into the physiological roles of these proteins in membrane trafficking. I have identified a new WASP-family protein, WAVE Homology In Membrane Protrusions (WHIMP), which displays characteristic actin nucleation-promoting features. This study was aimed at determining the role of WHIMP in cells. My work shows that WHIMP is a weak activator of the Arp2/3 complex compared to other WASP-family members. Upon overexpression, WHIMP induces peripheral and dorsal membrane protrusions and co-localizes extensively with F-actin and the Arp2/3 complex at the edge of the cell. Moreover, migration assays show that WHIMP expression enhances cell motility by directly activating the Arp2/3 complex and inducing tyrosine phosphorylation and Src tyrosine kinase activation at the membrane ruffles. Rapid cell motility is abolished following WHIMP depletion, or upon expression of a WHIMP truncation mutant lacking its actin and the Arp2/3 binding region. My current findings provide insight into the actin assembly properties, expression, localization, and function of WHIMP as a new motility factor within the WASP family. Collectively, these findings identify a role for WHIMP in cell migration, as well as expand our understanding of proteins that regulate the Arp2/3 complex

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility.

Cell motility is governed by cooperation between the Arp2/3 complex and nucleation-promoting factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X chromosome of a subset of mammals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation-promoting factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-associated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family members

Genetic control of conventional and pheromone-stimulated biofilm formation in Candida albicans.

Candida albicans can stochastically switch between two phenotypes, white and opaque. Opaque cells are the sexually competent form of C. albicans and therefore undergo efficient polarized growth and mating in the presence of pheromone. In contrast, white cells cannot mate, but are induced - under a specialized set of conditions - to form biofilms in response to pheromone. In this work, we compare the genetic regulation of such "pheromone-stimulated" biofilms with that of "conventional" C. albicans biofilms. In particular, we examined a network of six transcriptional regulators (Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1) that mediate conventional biofilm formation for their potential roles in pheromone-stimulated biofilm formation. We show that four of the six transcription factors (Bcr1, Brg1, Rob1, and Tec1) promote formation of both conventional and pheromone-stimulated biofilms, indicating they play general roles in cell cohesion and biofilm development. In addition, we identify the master transcriptional regulator of pheromone-stimulated biofilms as C. albicans Cph1, ortholog of Saccharomyces cerevisiae Ste12. Cph1 regulates mating in C. albicans opaque cells, and here we show that Cph1 is also essential for pheromone-stimulated biofilm formation in white cells. In contrast, Cph1 is dispensable for the formation of conventional biofilms. The regulation of pheromone- stimulated biofilm formation was further investigated by transcriptional profiling and genetic analyses. These studies identified 196 genes that are induced by pheromone signaling during biofilm formation. One of these genes, HGC1, is shown to be required for both conventional and pheromone-stimulated biofilm formation. Taken together, these observations compare and contrast the regulation of conventional and pheromone-stimulated biofilm formation in C. albicans, and demonstrate that Cph1 is required for the latter, but not the former

Examination of six downstream targets of Cph1 and Tec1 reveals a general role for Hgc1 in biofilm formation.

<p>(A) Heatmap showing expression of six target genes that are regulated by Cph1 and Tec1 during pheromone treatment of white cells. (B) Pheromone-stimulated biofilm formation in mutants lacking each of the six candidate genes. Top panel, images of white cells adhering to plastic. Bottom panel, quantification of the number of adherent cells. Light blue bars on graphs, no pheromone added. Dark blue bars, MFα pheromone present. (C) Analysis of the six candidate genes in a conventional biofilm assay on silicone squares. Hgc1 and Tec1 are both necessary for conventional biofilm formation. Values are the mean ± SD from two independent experiments with at least three replicates. “#” represents <i>P</i><0.01 and “*” represents <i>P</i><0.001 for the difference with the wildtype strain. The complemented <i>HGC1</i> strain (HGC AB) showed a significant increase in biofilm formation compared to the <i>hgc1</i> mutant. (WT: CAY716; <i>Δste2/Δste2</i>: CAY1234; <i>Δorf19.7167/Δorf19.7167</i>: CAY3445; <i>Δorf19.7170/Δorf19.7170</i>: CAY3447; <i>Δorf19.7305/Δorf19.7305</i>: CAY3693; <i>Δpbr1/Δpbr1</i>: CAY3689; <i>Δcfl11/Δcfl11</i>: CAY3687; <i>Δhgc1/Δhgc1</i>: CAY3465; <i>HGC1 AB</i>: CAY3702).</p

The <i>CPH1</i> gene is up-regulated in <i>C. albicans</i> white cells responding to pheromone.

<p>Northern blotting reveals that (A) <i>CPH1</i> is highly induced upon 10 µM α-factor treatment of P37005 white cells for 4 h in Spider medium. (B) <i>PBR1</i> is also highly induced in white cells responding to pheromone. (C) Quantitative RT-PCR indicated that expression of the <i>TEC1</i> gene did not change significantly following pheromone treatment under a variety of culture conditions. Each data is the mean ± SD from two independent experiments with at least three replicates. Light blue bars on graphs, no pheromone added. Dark blue bars, MFα pheromone present. (WT P37005: CAY716; <i>Δcph1/Δcph1</i>: CAY2899; <i>Δtec1/Δtec1</i>: CAY2506; <i>Δste2/Δste2</i>: CAY1234; WT SC5314: RBY717). P37 indicates strain derived from P37005, SC indicates derived from SC5314.</p

Transcriptional profiling of <i>C. albicans</i> white and opaque cells in response to pheromone.

<p><i>C. albicans</i> P37005 cells were grown in Lee's medium at 25°C as planktonic cells or under biofilm conditions. Cells were treated with 10 µM α pheromone or a DMSO mock control, and collected after incubation for 4 h or 24 h. cDNA was prepared and hybridized against a <i>C. albicans</i> Agilent microarray (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003305#s4" target="_blank">Materials and Methods</a>). For each array, pheromone-treated samples were hybridized against the mock-treated control. (A) Left panel: gene expression of <i>C. albicans</i> white (lanes 1–16) and opaque (lane 17) cells in planktonic (lane 1–6) or biofilm (lane 7–17) culture conditions. Pheromone up-regulated genes are shown in red, and down-regulated genes are shown in green. Profiling reveals that Cph1 (lanes 5–6 and lane 12) is essential for pheromone signaling in <i>C. albicans</i> white cells under both planktonic and biofilm conditions. Fold changes in gene expression are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003305#ppat.1003305.s007" target="_blank">Table S3</a>. (B) Comparison of numbers of genes up- or down-regulated between different planktonic and biofilm culture conditions (lanes 1–2 vs. lanes 7–9). (C) Difference in gene expression between WT, <i>cph1</i> and <i>tec1</i> strains under biofilm conditions (lanes 7–9 vs. lanes 10–11 vs. lane 12). (D) Comparison between gene expression in WT and <i>wor1</i> strains (lanes 7–9 vs. lanes 13–14) in biofilm conditions at 4 h. (E) Changes of gene expressions between the WT and <i>wor1</i> mutant after pheromone treatment for 24 h (lane 15 vs. lane 16). (F) Comparison of pheromone-stimulated white (Wh, 24 h induction) and opaque (O, 4 h induction) gene expression under biofilm culture conditions (lane 15 vs. 17). (B–F) In all venn diagrams where there is overlap, the overlap is significant by a chi squared test (p<5×10⁻²⁵⁴).</p

Genetic Control of Conventional and Pheromone-Stimulated Biofilm Formation in <i>Candida albicans</i>

<div><p><i>Candida albicans</i> can stochastically switch between two phenotypes, white and opaque. Opaque cells are the sexually competent form of <i>C. albicans</i> and therefore undergo efficient polarized growth and mating in the presence of pheromone. In contrast, white cells cannot mate, but are induced – under a specialized set of conditions – to form biofilms in response to pheromone. In this work, we compare the genetic regulation of such “pheromone-stimulated” biofilms with that of “conventional” <i>C. albicans</i> biofilms. In particular, we examined a network of six transcriptional regulators (Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1) that mediate conventional biofilm formation for their potential roles in pheromone-stimulated biofilm formation. We show that four of the six transcription factors (Bcr1, Brg1, Rob1, and Tec1) promote formation of both conventional and pheromone-stimulated biofilms, indicating they play general roles in cell cohesion and biofilm development. In addition, we identify the master transcriptional regulator of pheromone-stimulated biofilms as <i>C. albicans</i> Cph1, ortholog of <i>Saccharomyces cerevisiae</i> Ste12. Cph1 regulates mating in <i>C. albicans</i> opaque cells, and here we show that Cph1 is also essential for pheromone-stimulated biofilm formation in white cells. In contrast, Cph1 is dispensable for the formation of conventional biofilms. The regulation of pheromone- stimulated biofilm formation was further investigated by transcriptional profiling and genetic analyses. These studies identified 196 genes that are induced by pheromone signaling during biofilm formation. One of these genes, <i>HGC1</i>, is shown to be required for both conventional and pheromone-stimulated biofilm formation. Taken together, these observations compare and contrast the regulation of conventional and pheromone-stimulated biofilm formation in <i>C. albicans</i>, and demonstrate that Cph1 is required for the latter, but not the former.</p> </div

Role of Cph1 and Tec1 in the <i>C. albicans</i> response to pheromone by white cells.

<p>Pheromone-stimulated biofilm formation was measured in an adherence to plastic assay. P37005 cells (or mutant derivatives) were inoculated into 6-well cluster plates and treated with 10 µM α pheromone (MFα) for 24 h at room temperature. Wells were washed to remove non-adherent cells and photographed, or cells in the biofilm resuspended and quantified. (A) Cph1 is essential for pheromone-mediated biofilm formation. (B) Pheromone-stimulated biofilm formation is reduced, but not abolished, in the absence of Tec1. (C) Confocal scanning laser microscopy of biofilm formation also indicates that Cph1, but not Tec1, is necessary for biofilm formation in response to pheromone. For each image, the top panel shows the top view and the bottom panel shows the reconstructed side view, with the plastic substrate at the bottom of the image. Scale bars are 50 µm. AB indicates strains in which the target gene has been reintegrated into the mutant background. Values are the mean ± SD from two independent experiments with at least three replicates. “#” represents <i>P</i><0.05 and “*” represents <i>P</i><0.001 for WT v. mutant. Light blue bars on graphs, no pheromone added. Dark blue bars on graphs, pheromone present. (WT P37005: CAY716; <i>Δste2/Δste2</i>: CAY1234; <i>Δcph1/Δcph1</i>: CAY2899; <i>CPH1 AB</i>: CAY3028; <i>Δtec1/Δtec1</i>: CAY2506; <i>TEC1 AB</i>: CAY2750).</p