Extended Data Figure 3-1
Photostimulation of LOV2-JBD in dendritic spines rapidly immobilizes spine-head actin in the peripheral domain. A, Time-lapse sequences from 16-d hippocampal neurons expressing mCherry-actin and LOV2-JBD variants as indicated. Cells were stimulated for 1 s with 0.4 mW of 458-nm light. B, The effect on spine motility of LOV2-JBD photoactivation in mushroom spines (head diameter:neck length ratio >3) is shown. Spine motility is measured from arithmetic difference projection ratios (motility after light/motility before light). Optical stimulation of mushroom spines did not alter mCherry-actin motility in spines even when laser power was increased to compensate for larger spine-head volume. At least six spines from four experimental repeats were measured for each condition. Mean data ± SEM are shown. C, High-resolution maximum projections show temporally color-coded dendritic spines from 10-s time lapse from 3D recordings using Airyscan mode, at 1-s intervals. D, Footprints of time lapse sequences are shown for color-coded time projections from cells expressing mCherry-actin with or without LOV2-JBD for spines shown in C. E, Movies generated from 3D Airyscan recordings (1-s interval) of dendritic spines of a cell expressing mCherry-actin with LOV2-JBD as shown in Movie 1: time lapse movie 0-movie 120 s, blue circle depicts ROI of 458-nm illumination. Movie 2, volumetric temporal color coding of a dendritic spine generated from 48 to 58 s with 1-s 458-nm illumination at 48 s. Download Figure 3-1, TIF file.