Fig. 5: Actin density-dependent myosin penetration and flows.

a Representative actin networks (upper panels) and SspB-mScarlet-I-VCA distribution (middle panels) in the absence (left column) or presence (middle and right columns) of 200 nM Alexa546-SMM. Dense condition: 5 μM actin, 15 μM profilin, 100 nM Arp2/3 complex, 25 nM capping protein (CP), and 150 nM SspB-mScarlet-I-VCA. Sparse condition: 2 μM actin, 6 μM profilin, 100 nM Arp2/3, 10 nM CP, and 150 nM SspB-mScarlet-I-VCA. Images were acquired after 5 min illumination. Blue squares indicate illuminated regions. b Box plots of actin fluorescence intensities 2 μm above the SLB after 5 min illumination. n = 10 for each condition. Statistical significance was assessed by unpaired two-sided t-test; ***p < 0.001. c Line profiles of actin and myosin intensity along the orange lines in (a). d Dynamics of actin and myosin puncta after 5 min illumination. Elapsed times are indicated in Alexa647-actin images. Orange arrows indicate direction of puncta movements. e Illumination pattern to induce actin flow (top), and resultant SspB-mScarlet-I-VCA recruitment (bottom). Maximum intensity projections of actin networks formed by the illumination described in (e), in the absence (f) or presence (g) of Alexa546-SMM. The SMM signal is shown in middle panels of (g). Protein concentrations are same as the sparse condition in (a). Blue light was illuminated for 2 min (t = 0–2 min). Elapsed times are indicated in Alexa647-actin images. h Temporal color-coded images of actin in (f, g) after stopping illumination. i Fluorescence intensity of Alexa546-SMM in the three regions in (e) after stopping illumination. The bold lines indicate the mean values of three independent experiments (thin lines). j Illumination pattern to induce actin flow from both sides (top) and resultant SspB-mScarlet-I-VCA recruitment (bottom). k Maximum intensity projections of actin and SMM formed by the illumination described in (j). Orange arrowheads indicate disconnected actin structures within sparse regions. f, g, k Blue dashed boxes indicate the illuminated regions. Scale bars, 20 μm (a, e, f, g, h, j, k), 10 μm (d). Source data are provided as a Source Data file.