Optogenetically induced cross-linking decreases MT comet velocities and increases the area void of MT plus ends in an F-actin–dependent manner. (A) Representative images of EGFP-SKIP-LZ-iLID^Lit coexpressed with CH-CH-tgRFP-SspB or tgRFP-SspB in S2 cells treated with DMSO as a control, or LatA (2 nM or 2 µM) for 1 h before imaging. Top and middle: EGFP and RFP channels, respectively. Bottom: Maximum projections of the EGFP channel (60 frames collected over 3 min). Bar, 5 µm. (B) EGFP comet velocity/cell (μm/min) for control cells (SKIP-LZ-iLID^Lit + SspB) and constitutively cross-linked cells (SKIP-LZ-iLID^Lit + CH-CH-SspB) treated with DMSO or LatA (2 nM or 2 µM). P-values were determined by two-way unpaired Student’s t test. (C) MT-void area as a percentage of the total cell area for constitutively cross-linked cells and control cells treated with DMSO, 2 nM LatA, or 2 µM LatA. Maximum projection images of 60 frames (collected over 3 min) were used to determine the area of the cell void of growing MT plus ends (representative image used for quantification shown in A). MT-void area decreased in constitutively cross-linked cells with the addition of LatA as compared with control DMSO treatment. P-values were determined using a two-tailed nonparametric Mann–Whitney U test. For plots in B and C, numbers in parentheses indicate (number of experiments, total number of cells quantified). Central lines represent the mean, and error bars indicate SD. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001.