. 2017 Jun 5;216(6):1673-1688.
Epub 2017 May 4.
WASP and SCAR Are Evolutionarily Conserved in Actin-Filled Pseudopod-Based Motility
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WASP and SCAR Are Evolutionarily Conserved in Actin-Filled Pseudopod-Based Motility
J Cell Biol
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Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call "α-motility." Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE-activators of branched actin assembly-make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP's role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin.
© 2017 Fritz-Laylin et al.
WASP colocalizes with the SCAR complex at the leading edge of neutrophils. Microscopy of HL-60 cells expressing TagRFP-WASP and Hem-1–YFP, a component of the SCAR regulatory complex. (A) TIRF images of live HL-60 cells. Top: WASP localization in three sequential time points, overlaid on the far right (0 s in red, 20 s in green, and 40 s in blue). Middle: same sequence of images but for Hem-1. Bottom: overlay of WASP and Hem-1 at each time point. The plot shows line scans of normalized fluorescence intensity of WASP (red) and Hem-1 (green). The location for generating the line scans is shown in the adjacent image (white line). (B) Two additional examples of live HL-60 cells in TIRF and corresponding line scans (indicated by white lines in the images). (C) Spinning-disk confocal images of two fixed HL-60 cells showing an axial slice through the middle of thick pseudopods. The slices shown were taken 3 µm above the coverslip. See also Fig. S1 (for additional images and kymographs) and Video 1. For all cells, the direction of migration is to the left.
WASP is crucial for pseudopod formation of neutrophils. (A) Western blots showing WASP and WAVE2 expression in control HL-60 cells, cells expressing shRNA to WASP, and exogenous WASP with three silent mutations in the region corresponding to the shRNA (KD + rescue) or cells expressing anti-WASP shRNA and empty vector rescue control (KD + control). Approximately equal amounts of total protein were loaded in each lane, which was confirmed by using actin as a loading control. (B) Brightfield images of control and WASP-KD cells. (C) KD of WASP using shRNA reduces the percentage of cells that have pseudopods. (D) Immunofluorescence of control and WASP-KD HL-60 cells showing microtubules (green, antibody stained), actin filaments (red, phalloidin stained), and DNA (blue, DAPI). Note the signature rhino phenotype in the WASP-KD cell. See also Fig. S2. (E) Rescue of rhino protrusion phenotype by expression of shRNA-insensitive WASP as described in A. Note: no wild-type (WT) cells were observed to exhibit the rhino phenotype. (C and E) Means and SD (bars) of three biological replicates (dots) with >130 cells total; p-values were obtained with two-tailed paired t tests. (F) Maximum projections of spinning-disk confocal stacks of living HL-60 cells with fluorescent probes specific for polymerized actin (mCherry fused to the calponin homology domain of Utrophin; Utr261). Insets are cross sections through the rhino horns at positions indicated by yellow lines, confirming that they are hollow with a shell of actin. See also Fig. S2 for a time lapse of the right-hand cell showing the dynamics of the rhino horn protrusion.
WASP is crucial for explosive actin polymerization during pseudopod formation in neutrophils. (A) Spinning-disk confocal images of polymerized actin of control and WASP-KD HL-60 cells after stimulation for the indicated time with chemoattractant (20 nM fMLP) stained with fluorescent phalloidin. (B) FACS quantification of actin polymerization in control (gray circles) and WASP-KD HL-60 cells (green triangles) after stimulation for the indicated time with chemoattractant stained with fluorescent phalloidin. 10,000 cells were counted for each sample, and means were normalized to time 0 for control cells within each experiment to control for detector variability. AU, arbitrary unit. (C) Example spinning-disk confocal images of pseudopod-forming WASP-KD and control cells with polymerized actin stained with phalloidin (red) and DNA stained with DAPI (blue). (D) Quantification of phalloidin staining shown in C. Only cells with pseudopods were analyzed. Mean pixel values for z projections of image stacks was measured for each cell, and then the mean background pixel value was subtracted. Means and SD (bars) from three biological replicates (dots) are shown, with >150 cells total; the p-value was obtained from a one-tailed paired t test.
WASP is crucial for neutrophil motility. (A) Worm plots showing the tracks of cells migrating up an fMLP chemoattractant gradient. Control cells are on the left, and WASP-KD cells are on the right, with cells exhibiting the rhino phenotype in red. Cells were imaged for 20 min and migration paths were overlaid, with time 0 at (0,0). The endpoint of each cell’s path is shown with a dot. See also Video 2. (B) Depletion of WASP protein leads to reduced cell speed. The mean instantaneous speed for each cell in A is plotted as a dot color-coded by biological replicate to highlight the consistency from experiment to experiment. (C) Reduction of WASP protein leads to no significant change in directional persistence (the ratio of the Euclidean distance to the accumulated distance) of cells tracked in A. Means of the three replicates are displayed as horizontal lines; p-values were obtained from two-tailed paired t tests.
Genomic retention of both WASP and SCAR correctly predicts pseudopod formation by the infectious chytrid fungus Bd. (A) Time lapse showing examples of dynamic pseudopods from chytrid cells with (top) and without a flagellum (middle) or one cell of each (bottom). See also Video 3. (B) Percentage of cells with pseudopods within the first 6 h after release from zoosporangia. The mean and SD (bars) of four biological replicates (dots) is shown, with 3,782 cells total. (C) Pseudopod extension rates. The means and SD (bars) of the individual values (dots) combined from three biological replicates is shown. (D) Scanning electron micrographs of fixed chytrid zoospores. Brackets denote pseudopods and arrowheads denote flagella. See also Fig. S4 B for more examples.
Chytrid pseudopods are actin filled and require both actin polymerization and Arp2/3 activity. (A) Fixed chytrid cells with and without a flagellum (arrow). Staining with fluorescent phalloidin reveals a thin shell of cortical actin surrounding the cell body and a dense network of polymerized actin filling the pseudopods (brackets). DIC, differential interference contrast. (B) Two examples of chytrid cells with pseudopods that lose them when treated with 10 nm latrunculin B, an inhibitor of actin polymerization. Dynamic pseudopods (brackets) return after drug washout. (C) Quantification of reversible inhibition of pseudopods by latrunculin B (Lat B). Only cells that were making pseudopods before treatment and that were not washed away during the experiment were counted. (D) Two examples of chytrid cells with pseudopods that lose them when treated with 10 µm CK-666, an inhibitor of Arp2/3 activity. Pseudopods return after drug washout. (E) Quantification of reversible inhibition of pseudopods by CK-666. Only cells that were making pseudopods before treatment and that were not washed away during the experiment were counted. Symbols are means from three biological replicates, each with at least 29 (C) or 17 (E) cells. P-values were obtained from two-tailed paired t tests.
Genomic retention of both WASP and SCAR correctly predicts α-motility in the infectious chytrid fungus Bd. (A) Example chytrid zoospores with and without a flagellum migrating when confined between 1-µm spaced glass coverslips. See also Video 4. (B) Worm plots showing the tracks of 26 migrating chytrid zoospores with migration paths overlaid and with time 0 at (0,0). The endpoint of each cell’s path is shown with a dot. Only those cells obviously migrating were tracked, and cells were tracked for the duration of their movement. (C) Mean instantaneous speed of cells tracked in B. (D) Directional persistence (the ratio of the Euclidean distance to the accumulated distance) of cells tracked in B. Means and SD (bars) of the individual values (dots) combined from three biological replicates are shown.
Only organisms that make pseudopods retain both WASP and SCAR genes. Diagram showing the relationships of extant eukaryotes (based on a study by Fritz-Laylin et al., 2010) with the presence or absence of SCAR (blue) and WASP (green) genes from complete genome sequences as described previously (Kollmar et al., 2012). Each representative organism whose genome was used for the analysis is listed to the right. For groups with similar morphological and sequence patterns, a single species is used. For example, there is no known plant species that forms pseudopods or retains the WASP gene, so only a single species is shown ( Arabidopsis thaliana); similarly, Aspergillus nidulans represents all dikarya. See Kollmar et al. (2012) for additional sequence information. An amoeba glyph indicates organisms that build pseudopods. Outlined rectangles indicate a lack of an identifiable gene. See Table 1 for citations and full species names. *, Although we were not able to find a reference to pseudopod formation in A. macrogynus, a relative ( C. anguillulae) does assemble pseudopods used for motility (Deacon and Saxena, 1997; Gleason and Lilje, 2009). Because of this and the conservation of both WASP and SCAR in Bd (highlighted in bold), we correctly predicted this species is also capable of pseudopod formation. ++, These species form pseudopods for feeding rather than motility. The question mark indicates uncertainty regarding the structure of the protrusions for phagocytosis in E. histolytica (see the Evolutionary retention of both WASP and SCAR correlates with pseudopod formation section). The time of divergence of extant eukaryotic groups has been estimated to be 1.1–2.3 billion years ago (bya; Chernikova et al., 2011; Parfrey et al., 2011; Knoll, 2014) and has been predicted to have possessed both WASP and SCAR gene families (Kollmar et al., 2012) and therefore may have built pseudopods.
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Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Actin-Related Protein 2-3 Complex / metabolism
Chytridiomycota / genetics
Chytridiomycota / metabolism*
Fungal Proteins / genetics
Fungal Proteins / metabolism*
Neutrophils / metabolism*
Pseudopodia / metabolism*
Wiskott-Aldrich Syndrome Protein / genetics
Wiskott-Aldrich Syndrome Protein / metabolism*
Wiskott-Aldrich Syndrome Protein Family / genetics
Wiskott-Aldrich Syndrome Protein Family / metabolism*
Actin-Related Protein 2-3 Complex
Wiskott-Aldrich Syndrome Protein
Wiskott-Aldrich Syndrome Protein Family
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