Figure 3. Optically controlled localization of myosin motors in live cells.

a, Simplified schematic of a cell with different actin architectures at various locations in the cell. b, Block diagram and cartoon of the molecular construct imaged in live cells. c, Maximum intensity projection of a 3D confocal stack of fluorescence images (excitation at 639 nm) of a sheet of mouse fibroblasts in culture immediately after 150 s stimulation with blue light, displaying fluorescent puncta concentrated around the periphery of the cell. Insets show a region of the sheet before and after the blue light stimulation. Images are representative of a set of 7 independent experiments with comparable blue light stimulation protocols (Supplementary Figure 7). d-f, Fluorescence intensity contained in puncta$I_{puncta}$, normalized to the total intensity in the image and plotted after further normalization to the value at the start of the series,$I_{puncta\text{,}{t}_0}$. The blue bands indicate the period of blue light stimulation using a blue laser line in the scanning confocal microscope. d, Intensity in puncta calculated for the image series on the cells in panel c. e, Intensity in puncta over 7 stimulation cycles of 2 minutes each, separated by 40-minute wait times. In each stimulation cycle, cells were imaged for 5 frames, with one frame without blue light preceding 4 frames with blue light on. Grey dashed lines are visualization guides. f, Time traces of averaged responses (black datapoints) upon stimulation with blue light, from 7 independent sample preparations (grey datapoints). Black datapoints, mean of the 7 measurements. Error bands, standard deviation of the measurements. White datapoints, response in absence of blue light. Top, fluorescence intensity contained in puncta; the maximum level of $I_{puncta}/I_{puncta\text{,}{t}_0}$ after 300 s of stimulation was 11 ± 4. Bottom, fluorescence of cytoplasm. g-h, Characterization of MyLOVChar4~1R~TET~mRuby3 in hippocampal neurons in culture. g, Block diagram of molecular construct and illustration of actin architecture in neurons. h, Wide-field fluorescent images of MyLOVChar4~1R~TET~mRuby3 before, during and after stimulation with blue light (see also Supplementary Video 13). Arrows highlight puncta as expected for motor accumulation in dendritic spines. Data are representative of an experiment replicated twice on different days, with 13 cells.