The filopodial myosin DdMyo7 is a slow, calcium-regulated motor

Figures

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  Figure 1. DdMyo7 motors used in study. Illustrations of full-length Dictyostelium. Myosin 7 (FL-DdMyo7) and motors of varying lengths used in this study. These include long HMM (lnHMM) and forced-dimers of long HMM (lnHMM-FD), short HMM (shHMM-FD), long S1 (lnS1-FD), and S1 (S1-FD). The domains depicted are the motor (gray oval), IQ motifs (yellow ovals), single alpha-helix (SAH), candidate dimerization domain (cDD), proline-rich regions (Pro), SH3 domain (purple rectangle) and MF domains (blue rods–MyTH4; red circles indicating three lobes of the FERM domain). The GFP (green square), dimerization sequence (CC-Lzip purple helix) and epitope tags (FT – FLAG Tag; ST – Strep-tag) are also shown. The drawing on the right illustrates a forced-dimer with bound LCs (purple lines/dots). The previously mapped TalA binding region (amino acids 902–1156) (20) is highlighted in red above FL-DdMyo7. HMM, heavy meromyosin; LC, light chain; S1, subfragment 1.

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  Figure 2. CalA and CalB are the DdMyo7 light chains and binding is calcium dependent.A, DdMyo7 heavy chain (HC) pull downs revealing CalA and CalB as the DdMyo7 light chains (LCs). Following densitometry of the gel lanes, the molar ratios were calculated by normalizing HC, CalA, and CalB signal to their MWs (HC: 36.1, CalA: 83.0, CalB: 83.5, arbitrary units) then determining a ratio of CalA/HC and CalB/HC. CalA/HC: 2.08 ± 0.12, CalB/HC: 2.01 ± 0.16; N = 3. Average LC/HC ratio and S.E.M. reported. B, immunoblot of a shHMM-FD pull down probed for shHMM-FD-GFP (HC), CalA, and Flag (FL)-CalB. C, DdMyo7 shHMM-FD pull downs in the presence or absence of Ca2+. Immunoblots were probed for shHMM-FD-GFP (HC), CalA, and FL-CalB. Immunoblot signal in the presence or absence of Ca2+ was normalized to correct for gel load and heavy chain abundance. CalA reduction + Ca2+: 99.7% ± 0.15, CalB reduction + Ca2+: 75.1% ± 3.46; N = 3. Average reduction percentage and S.E.M. reported. FD, forced-dimer.

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  Figure 3. Characterization of DdMyo7 motor localization.Left: quantification of cortex-to-body intensity ratio of each dimerized motor in WT or talA null cells. lnHMM-FD WT (N: 3, n: 178), lnHMM-FD TalA- (N: 5, n: 91), shHMM-FD WT (N: 3, n: 520), shHMM-FD TalA- (N: 4, n: 66), lnS1-FD WT (N: 3, n: 270), lnS1-FD TalA- (N: 3, n: 39), S1-FD WT (N: 4, n: 32), S1-FD TalA- (N: 2, n: 25). N = number of imaging sessions, n = number of cells (used for statistical analysis). One-way ANOVA with pairwise comparison (∗∗∗ p value: <0.001, NS: not significant). Mean and standard deviation reported. Right: representative WT (left column) and talA null (right column) cells expressing the motor fragments. Yellow arrows indicate shHMM-FD and lnS1-FD anchored by TalA at the cortex. The scale bars represent 10 μm. TalA, Talin A; FD, forced-dimer; lnHMM, long heavy meromyosin.

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  Figure 4. TIRF-based in vitro motility assay.A, illustration of the in vitro motility assay. B, Left: kymograph of two representative motility events with velocities shown to the left. Right: sequential images of the upper event over 40 s (every 10 s) with yellow arrows tracking the individual shHMM-FD motor over time. FD, forced-dimer.

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  Figure 5. Single molecule characterization of the DdMyo7 shHMM-FD motor. Distribution of DdMyo7 shHMM-FD (A) velocity, (B) run length, and (C) run duration. N (motor purification): 3, N (chambers): 10, n (motility events): 414, mean ± S.E.M. reported. Data fit (teal) with either a log-normal (A) or exponential function (B–C). FD, forced-dimer.

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  Figure 6. DdMyo7 motility in the presence of calcium and on fascin-bundled actin.A, shHMM-FD motility minus (blue) and plus (green) calcium. B, total observed motility events minus (blue) and plus (green) calcium. N = motor purification and chamber number, n = individual motility events (used for statistical analysis) tracked over at least three frames. C, shHMM-FD velocity minus (N: 3, n: 120, blue) and plus (N: 3, n: 14, green) calcium. Two-sided Mann-Whitney U test, ∗ p value: 0.02, mean and S.E.M. reported. D, shHMM-FD motility on filamentous (orange) and FSCN-bundled (red) actin. E, shHMM-FD velocity on filamentous (N (motor purification): 2, N (chambers): 2, n (motility events): 74, orange) and FSCN-bundled actin (N (motor purification): 2, N (chambers): 3, n (motility events): 166, red). Two-sided student t test, p value: 0.02, mean and S.E.M. reported. F, shHMM-FD run length on filamentous (N: 2, n: 74, orange) and FSCN-bundled actin (N: 3, n: 166, red). Two-sided student t test, ∗ p value: 0.09, mean and S.E.M. reported. FD, forced-dimer; FSCN, fascin.