VASP-mediated actin dynamics activate and recruit a filopodia myosin

Figures

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  Figure 1.. DdMyo7 has a distinct cortical localization from its tail domain. (A) (left) Schematic of DdMyo7 illustrating its motor domain (gray), 4 IQ domains (yellow) and tandem MyTH4-FERM domains (blue-MyTH4, red-FERM) in the tail, the tail fragment, and a motor forced dimer (motor-FD); (right) Dictyostelium control, or myo7 null cells visualized with DIC and the membrane dye FM4-64 showing DdMyo7 is critical for filopodia formation. (B) Confocal images showing two examples of wild-type cells co-expressing DdMyo7-mCherry and GFP-DdMyo7-tail. The localization of DdMyo7-mCherry is at the cortex and in filopodia tips, and GFP-tail fragment localized around cortex. (C) Line intensity profile along the line shown in panel B. (D) Cytofluorograms of a representative field of cells comparing the colocalization between DdMyo7-mCherry intensity (x-axis) and GFP-DdMyo7 or GFP-DdMyo7 tail intensity (y-axis). (E) Analysis strategy for measuring entire cell peripheral intensity. (F) Micrographs of cells expressing RFP-Lifeact, GFP-DdMyo7, GFP-Tail, or GFP-Motor-Forced Dimer (FD) asterisks (*) on GFP-tail indicates the cell analyzed in G. (A,B,E,F) Scale bars are 10 µm. (G) Peripheral line scan…

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  Figure 1—figure supplement 1.. DdMyo7 is localized to filopodia and required for their formation. (A) Micrographs of control (AX2), myo7 null or myo7 null with GFP-DdMyo7 rescue construct expressing RFP-Lifeact (actin, top) and GFP-DdMyo7 (bottom). (B) Micrographs of myo7 null cells co-expressing either GFP-DdMyo7 and DdMyo7-mCherry (left) or GFP-DdMyo7-tail and DdMyo7-mCherry (right). A-B. Scale bars: 10 µm.

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  Figure 1—figure supplement 2.. Analysis of protein expression. Whole cell lysates from each line used in this study were analyzed for expression of endogenous proteins or expressed fusion proteins. Approximately 3 × 105 cells were loaded per lane. The blot was also probed for the 125 kD MyoB heavy chain serving as the loading control. Antibodies used to probe each set of blots are indicated below and the molecular weights in kD marked on the side. (A) Control wild type (WT, Ax2), myo7 null or vasp null cell lines. Note that DdVASP runs at ~50 kD, higher than its calculated molecular weight of ~40 kD. (B) GFP-DdMyo7 expression in control (WT, Ax3) and vasp null cells, and GFP-VASP in control wild type (Ax2) and myo7 null cells. (C) Expression of wild type or mutant GFP-DdMyo7 in myo7 null cells. Note that GFP-DdVASP runs at ~75 kD, higher than its calculated molecular weight of ~65 kD (D) Expression of wild type VASP and VASP mutants (not fused to a fluorescent protein) in vasp null cells. (E) Numbers are molecular weight standard. Wild type, vasp null and vasp null cell line overexpressing GFP-dDia2 CA. (F) Western blot of GFP-V1 induced (+Dox) in control (Ax3) cells.

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  Figure 2.. Actin dynamics regulate DdMyo7 recruitment to the cortex. (A) Dictyostelium co-expressing GFP-DdMyo7 and RFP-Lifeact. (B) Line intensity profile from yellow dotted line in A (circle = beginning, arrowhead indicates end of scan). (C) Cytofluorogram from a representative field of cells showing the colocalization of actin and DdMyo7, r is correlation coefficient. (D) Confocal image series of an extending pseudopod. (E) Normalized linescan intensity profile of DdMyo7 and actin in extending pseudopod along the line from panel D. (F) Intensity correlation of GFP-DdMyo7 and RFP-Lifeact plotted as the average spline fit of 10 extending pseudopodia (SD - light green shading, SEM - dark green shading, see also Figure 2—source data 1). (G) Confocal micrographs of cells expressing GFP-DdMyo7 (top) or RFP-Lifeact (actin, bottom) treated with specified drug. (A,D,G) Scale bar is 10 µm. (H) Cortex:cytoplasm ratio (cortex is 0.8 µm band of cell periphery, cytoplasm is the rest of cell excluding the nucleus) of GFP-DdMyo7 of cells treated with anti-actin drugs, circles are experimental means (see also Table 1). One-way ANOVA with multiple comparison correction, shown to 1% DMSO control, **p<0.01,…

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  Figure 2—figure supplement 1.. Effects of microtubule and membrane phospholipid inhibitors on DdMyo7 targeting and filopodia formation. (A) Micrographs of cells expressing GFP-DdMyo7 or GFP-CRAC treated with LY294002 (LY), wortmannin (WM). (B) Quantification of GFP-DdMyo7 cortical recruitment. (C) Quantification of filopodia formation. (D) (top) Micrograph of representative cell expressing GFP-tubulin (GFP-tub) and DdMyo7-mCherry and the merged image. (bottom) Cytofluoragram from a representative field of cells of pixel-wise overlap of a cell expressing GFP-tubulin and DdMyo7-mCherry. (E) Micrographs of cells expressing mNeon-DdMyo7 or GFP-tubulin under DMSO (vehicle control) or noted concentration of nocodazole for 20–35 min. (F) Micrograph of cells expressing GFP-DdMyo7 tail fragment under control or 30 µM nocodazole for 30 min. (G) Quantification of DdMyo7 cortical recruitment. (H) Quantification of DdMyo7-tail cortical recruitment. (I) Quantification of filopodia formation. (B–I). Circles on graphs are experimental means. One-way ANOVA with multiple comparison correction to DMSO, ns, not significant, *p<0.05, **p<0.01, ***p<0.0001 (see also Table 1 and Table 1—source data 1–4). (A–E). Scale bars on…

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  Figure 3.. VASP is required for DdMyo7 cortical recruitment. (A) Confocal images of wild type, myo7 null, vasp null or dia2 null cells expressing RFP-Lifeact (actin). (B) Violin plot of number of filopodia per cell (see also Figure 3—source data 1). (C) Micrographs of cells expressing GFP-DdMyo7 (top) or GFP-VASP in myo7 null, vasp null or dDia2 null cells. (D) Quantification of the cortical band (0.8 µm of periphery) relative to the cytoplasmic intensity of either GFP-Myo7 or GFP-VASP. (A,C) Scale bar is 10 µm (see also Figure 3—source data 3). (B, D) One-way ANOVA with multiple comparison correction or student’s t-test to compare GFP-VASP, ns, not significant, p***<0.001, p****<0.0001, circles are experimental means (see also Figure 3—source data 2 and 4). Figure 3—source data 1.Number of filopodia per cell counted for each cell in strains in Figure 3B. Figure 3—source data 2.Statistical test results for Figure 3B. Figure 3—source data 3.Cortex: cell ratio values for each cell for lines analyzed in Figure 3D. Figure 3—source data 4.Statistical test results for Figure 3D.

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  Figure 3—figure supplement 1.. VASP is not present in DdMyo7 immunoprecipitates. (A) GFP-DdMyo7 or GFP-DdMyo7-KKAA (see Figure 5) were immunoprecipitated from a clarified lysate (post nuclear spin sup; PNS) using GFP nanobody beads. Western blot analysis of PNS and immunoprecipitate pellet (IP Pell) probed with antibodies for DdMyo7, actin, and DdVASP.

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  Figure 4.. Linear actin polymerization drives DdMyo7 to the cortex. (A) Image series showing DdMyo7 is absent from latrunculinA-induced actin waves in control (top) or vasp null (bottom) cells. (B) (top) Confocal images of GFP-DdMyo7 in vasp null cells treated with either DMSO or 50 nM jasp treatment. (bottom) Images of vasp null cells expressing GFP or mCherry DdMyo7 and different actin modulating proteins (GFP-V1, green; GFP-dia2WT, green; RFP-dia2CA, magenta). (C, D) Average actin intensity (phalloidin staining, top) of cells through the longest cell axis. The line is the mean and the shaded area is the SEM (graphs, bottom) (see also Figure 4—source data 1). (A–D), Scale bar is 10 µm. (E) Quantification of the cortical band intensity of DdMyo7 in vasp null cells, with no treatment, treated with jasp, or also overexpressing V-1, dia2, or dia2-CA (see also Figure 4—source data 2). (F) Violin plot of filopodia per cell (see also Figure 4—source data 4). (E-F). One-way ANOVA with multiple comparison correction, ns, not significant, **p<0.05, p****<0.0001 (see also Figure 4—source data 3 and 5). Figure 4—source data 1.Actin line scans for Figure 4C,D. Figure 4—source data 2.Cortex: cell ratio…

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  Figure 4—figure supplement 1.. Effects of actin modulating drugs and proteins. (A) Kymographs of cells expressing RFP-LimEΔcoil and GFP-DdMyo7 induced to make actin waves in either control (left) or vasp- cells. Scale bar is (x,y: 5 µm, 30 s). (B) (top) rotated kymograph of vasp- cell, scale bar is 5 µm. (bottom) Plot profile through center of kymograph above during time-lapse of the cell making actin waves. Arrows above point to peak actin intensity (magenta line) which corresponds to wave formation, green line shows DdMyo7 intensity, which is not well correlated with actin waves. (C) Phalloidin staining of vasp null cells treated with either DMSO or 50 nM jasplakinolide (jasp). (D) Quantification of induction of filopodia formation by control cells (no V1 OE) or cells that overexpress GFP-V1 (see also Figure 4—figure supplement 1—source data 1). Student's t-test, ****p<0.001 (see also Figure 4—figure supplement 1—source data 2). (E) Clustal Omega alignment of the DAD region of diaphanous related formins, the conserved basic residues are highlighted in yellow. Figure 4—figure supplement 1—source data 1.Filopodia per cell values for lines shown in Figure 4—figure supplement 1D. Figure 4—figure supplement…

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  Figure 5.. Reduced cortical recruitment of DdMyo7 by VASP mutants. (A) Schematic of domains of DdVASP (top) and proposed interaction of DdVASP wildtype, monomeric (∆tet), and F-actin binding (FAB K-E) mutant with actin filaments. (B) Quantification of the cortical recruitment of GFP-DdMyo7 co-expressed in the vasp null with wildtype or mutant DdVASP (non-fluorescent) rescue constructs (see also Figure 5—source data 1). (C) Quantification of GFP-DdMyo7 positive filopodia per cell of vasp null cells with wildtype or mutant DdVASP rescue constructs (see also Figure 5—source data 3). (B–C) Circles represent experimental means. One-way ANOVA with multiple comparison correction, p****<0.0001, ns not significant (see also Figure 5—source data 2 and 4). (D) Clustal Omega alignment of Dictyostelium and human VASP sequences with conserved domains highlighted and mutated residues starred. (E) Micrographs of GFP-DdMyo7 in vasp nulls, or vasp nulls expressing wildtype DdVASP or mutant DdVASP rescue constructs. Scale bar is 10 µm. Figure 5—source data 1.Cortex: cell ratio values for each cell for lines analyzed in Figure 5B. Figure 5—source data 2.Statistical test results for Figure 5B. Figure 5—source…

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  Figure 6.. VASP-mediated actin assembly relieves DdMyo7 head-tail autoinhibition to promote targeting and filopodia formation. (A) (top) Diagrams depicting mutants analyzed. (bottom) Micrographs of GFP-DdMyo7 fusion proteins in control and vasp null cells, scale bar is 10 μm. Arrows indicate cortical enrichment of DdMyo7. (B) Quantification of cortical recruitment of GFP-DdMyo7 and variants in vasp null cells (see also Table 2 and Figure 6—source data 1). The line represents the mean GFP-DdMyo7 recruitment in wild type cells. (C) Comparison of cortical targeting of activated DdMyo7-KKAA or tail in vasp null versus control cells (see also Figure 6—source data 1 and 2). (D) Quantification of number of filopodia per cell in control or vasp null cells (see also Table 2; Figure 6—source data 3). (B–D) Circles represent experimental means. One way ANOVA with multiple comparison test, ns not significant, p***<0.001, p****<0.0001, ns, not significant (see also Figure 6—source data 2 and 4). (E) Quantification of the cortical band intensity variation of DdMyo7-KKAA in control versus vasp null cells (see also Figure 6—source data 5). Students t-test ****p<0.0001. Figure 6—source data 1.Cortex: cell…

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  Figure 7.. DdMyo7 motor activity is required to release autoinhibition. (A) Schematic of proposed effect of mutations on DdMyo7 function (see alignment in Figure 7—figure supplement 1). (B) Confocal images of myo7 null cells expressing GFP-DdMyo7 fusion proteins, scale bar is 10 µm. (C) Quantification of the cortical band intensity variation. Mean lines from Figure 1I data on graph for comparison. DdMyo7 versus I426A uncoupler, p=0.07, not significant (see also Figure 7—source data 1 and 2). (D) Fraction of DdMyo7 cosedimenting with the cytoskeleton, symbols with the same shape are technical replicates, students t-test ****p<0.0001 (see also Figure 7—figure supplement 1, Figure 7—source data 3). (E) Quantification of cortical recruitment of DdMyo7 and mutants (see also Figure 7—source data 4). (F) Filopodia number per cell of wildtype and DdMyo7 mutants (see also Figure 7—source data 6). (E–F) Data for KKAA is taken from Figure 6, experimental means shown as circles. (C, E, and F) One-way ANOVA with multiple comparison correction, p*<0.05, p***<0.001, p****<0.0001, ns not significant (see also Figure 7—source data 5 and 7). Figure 7—source data 1.Values of the cortical standard deviation…