Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

Figures

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  Figure 1. Ca2+enhances the actin filament severing activity of gelsolin. For the depolymerization assays, actin was polymerized and diluted in polymerization buffer to a final concentration of 20 nM to induce spontaneous depolymerization. Gelsolin (GSN) was used at 5 nM final concentration at pCa 4.0 or pCa > 9 (1 mM EGTA added). A, spontaneous actin disassembly in the presence of either 100 μM CaCl2 (pCa 4.0) or 1 mM EGTA (pCa > 9) but in the absence of GSN. Data are presented as the average of two independent measurements at each condition. B, actin disassembly in the presence of gelsolin (5 nM) (unlabeled; UL or Alexa-647 labeled; A647) and in the presence of either 100 μM CaCl2 (pCa 4.0) or 1 mM EGTA (pCa > 9). Data are presented as the average of 2 (A647 GSN) and 4 (UL GSN) independent measurements. C, depolymerization rate (negative) derived from linear fitting of the pyrenyl transients. Mean ± SD, n = 2 to 4. Note negligible effects of gelsolin on depolymerization rate in the absence of Ca2+ but a substantial accelerating effect with added Ca2+ (pCa 4.0).

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  Figure 2. Number of actin filaments in the in vitro motility assay at different time points after addition of MgATP-containing assay solution (to start motility) including Ca2+(pCa 5.7) without or with gelsolin (GSN; ∼1 nM) and different surface incubation concentrations of HMM (171.4 or 342.8 nM). Concentration ratio of added actin and gelsolin, [Actin(monomer)]/[gelsolin]: 5. Data shown as mean ± 95% CI. Temperature: 25 to 27 °C. Data from individual experiments superimposed on bars representing mean values. Nonoverlapping 95% CIs between bars indicate (approximate) statistical significance for differences corresponding to p < 0.05. HMM, heavy meromyosin.

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  Figure 3. Number of actin filaments in the in vitro motility assay at different time points after addition of MgATP (to start HMM propelled actin sliding) under conditions with preincubation (2 min) of the motility assay flow cell with HMM at different concentrations; 171.4 to 342.8 nM to give increasing HMM surface density. Motility assays conditions with Ca2+(pCa 8.2) with or without gelsolin (GSN; 5 nM). [Actin(monomer)]/[gelsolin]: ∼1. Data shown as mean ± 95% CI. Nonoverlapping 95% CIs indicate statistically significant differences corresponding to p < 0.05. Temperature: 25 to 27 °C. Data from individual experiments superimposed on bars representing mean values. Note, we attribute the decrease in the number of filaments at late time points to detachment of filaments from the surface and difficulty to observe short filaments because of weak total fluorescence (see further text). Inset: number of actin filaments in the in vitro motility assay 60 s after MgATP addition as a function of the HMM concentration used for preincubation of the flow cell. CI, confidence interval; HMM, heavy meromyosin.

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  Figure 4. Effects of gelsolin (5–10 nM) in the presence of low [Ca2+] (pCa 8.2) on HMM-induced F-actin sliding velocity versus filament length in the in vitro motility assay. [Actin(monomer)]/[gelsolin]: 0.5 to 1. A, HMM incubation concentration 342.8 nM in the absence (black dots) and presence (red dots) of gelsolin. Full lines (with 95% confidence band; dotted lines) represent Equation 1 fitted to the data obtained in the control solution (orange; v∞ =1, [0.986, 1.014]; f= 0.118, [0.042, ∞]; mean, [95% CI]) and in the presence of gelsolin (blue; v∞ =0.759, [0.744, 0.775]; f = 0.0260 [0.0202, 0.042]; mean, [95% CI]). Data from six experiments. To be able to appropriately compare different experiments, the data in each individual experiment (30–80 filaments) were normalized to the average absolute velocity in the absence of gelsolin (8.42 [7.29, 9.06]; mean, [range]). Plots of data from individual experiments are presented in Fig. S2. B, HMM-induced F-actin sliding velocity with HMM added at 171.4 nM in the absence (black dots) and presence (red dots) of gelsolin. Full lines (with 95% confidence bands) represent Equation 1 fitted to the data obtained without gelsolin (orange; v∞=1.000,…

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  Figure 5. TIRF microscopy images showing F-actin severing activity in the presence of 5 nM gelsolin either with or without calcium. A, unlabeled gelsolin without calcium. B, Alexa-647-labeled gelsolin without calcium. C, unlabeled gelsolin, pCa 5.7. D, Alexa-647-labeled gelsolin, pCa 5.7. Preincubation with HMM at 34.2 nM before addition of actin filaments (rhodamine phalloidin labeled). Assay solution was added without MgATP, resulting in rigor actomyosin state keeping filaments stationary. Note, each image is an average of up to 100 subsequent image frames in a sequence. Image processing was done using ImageJ (Fiji, Ver. 1.53a). Time points are represented as min:sec. Scale bars, 10 μm. HMM, heavy meromyosin; TIRF, total internal reflection fluorescence. Temperature, 23 °C.

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  Figure 6. TIRF microscopy images showing gelsolin binding and F-actin severing activity in the presence of 5 nM Alexa-647 gelsolin. A, no calcium, same experiment and filaments as in Figure 5B. B, pCa 6.3, C., pCa 5.7, same experiment, and filaments as in Figure 5D. Preincubation with HMM at 34.2 nM before addition of actin filaments (rhodamine phalloidin labeled). Assay solution was added without MgATP resulting in rigor actomyosin state. Note, images for the time point 00:00 are single channel images (rhodamine phalloidin; pseudocolored), whereas those in 01:00 and 06:00 are merged images of Alexa-647 and rhodamine phalloidin channels. Each image is an average of up to 100 subsequent images. Image processing was done using ImageJ (Fiji, Ver. 1.53a). Time points are represented in min:sec. Scale bars, 5 μm. Note, the 30 s frame in C shows the same filaments as in the 0 s frame, but the filaments have moved probably because some of the filament segments formed after cleavage were initially not directly linked via HMM to the surface, consistent with use of 10 times lower HMM incubation concentration (34.2 nM) than in the in vitro motility assay (342.8 nM). Temperature, 23 °C. HMM, heavy…

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  Figure 7. Motility of actin filaments (green, Alexa-488 phalloidin labeled) with attached gelsolin (red, Alexa-647 labeled). Conditions used in the assay, Ca2+(pCa 8.2), gelsolin (GSN; 5 nM), and HMM (342.8 nM). A, time projection image of background-subtracted video (summary of 57 subsequent frames acquired at rate of 20 s−1) showing actin filament fluorescence. B, time projection image of background-subtracted video (sum of sequence of 90 frames acquired at rate of 20 s−1) showing gelsolin fluorescence. Note that gelsolin can be bound to the actin filaments (motile trajectories observed) and nonspecifically to the surface (static spots; in majority). C, merged image of actin and gelsolin from A and B with motile actin filaments having bound gelsolin indicated by yellow rectangles. Note, visible double-colored filament trajectories. D, zoom in of one double-colored filament trajectory in C as indicated by dashed lines. E, Time-lapse sequence of the filament trajectory shown in D with filament moving from bottom to top. Video (see Video S1) was acquired as follows: first FITC filter cube was used to observe Alexa-488 phalloidin-labeled actin filament motility, followed by switch to Cy5…