CH-CH MT plus end recruitment is required to decrease MT comet velocities and generate a peripheral MT exclusion zone. (A) Representative still images of SKIP-LZ-iLID or SKIP-LZ-iLID^Lit coexpressed with CH-CH-tgRFP or tgRFP-SspB in S2 cells, showing EGFP and RFP channels. Right: Maximum projections of 60 frames (collected over 3 min) showing the area traversed by MT plus ends. Bar, 5 µm. (B) EGFP comet velocity/cell (μm/min) for cross-linked control cells (SKIP-LZ-iLID + SspB activated with blue light and SKIP-LZ-iLID^Lit + SspB) and cells transfected with a CH-CH-tgRFP construct that binds F-actin, but lacks the SspB domain that mediates cross-linking to MT plus ends (SKIP-LZ-iLID + CH-CH and SKIP-LZ-iLID^Lit + CH-CH). P-values were determined by two-way unpaired Student’s t test. (C) Area void of MT plus ends as a percentage of the total cell area for control cells (SKIP-LZ-iLID + SspB activated with blue light and SKIP-LZ-iLID^Lit + SspB) and cells in which the CH-CH construct engages the actin network, but is not cross-linked to MT plus ends (SKIP-LZ-iLID + CH-CH and SKIP-LZ-iLID^Lit + CH-CH). Maximum projection images of 60 frames (collected over 3 min) were used to determine the area of the cell void of MT plus ends (representative image used for quantification shown in A). 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 < 0.0005; ****, P < 0.0001.