Photoactivated SxIP-iLID constructs rapidly recruit tgRFP-SspB to MT plus ends without altering MT comet velocities. (A) Diagram of SKIP-LZ-iLID and tgRFP-SspB constructs. (B) Cartoon diagram of tgRFP-SspB binding to SKIP-LZ-iLID upon blue light activation (light: hν). SKIP-LZ-iLID localizes to MT plus ends via association with EB dimers. (C) Representative images of S2 cells cotransfected with either a tandem SxIP-iLID construct or a dimeric SKIP-LZ-iLID construct and a tgRFP-SspB control construct, repeatedly pulsed with blue light (700 ms) every 3 s at 488 nm. Images show tgRFP-SspB localization before and after photoactivation (see accompanying Video 2). The SKIP-LZ-iLID^Lit construct is constitutively competent to bind SspB in the absence of blue light. Bars: (left) 5 µm; (inset) 1 µm. Kymograph bars: 2 µm, 25 s. (D) The postactivation/preactivation ratio of mean tgRFP comet intensity demonstrating effective tgRFP-SspB recruitment. tgRFP comet intensity per area on a MT plus end was calculated relative to the mean intensity per area in an adjacent cytoplasmic region. (E) SxIP-iLID-based recruitment of tgRFP-SspB to MT plus ends does not significantly alter mean MT plus end comet velocities compared with control cells cotransfected with EB1-GFP and tgRFP-SspB. Error bars indicate SD. Numbers in parentheses indicate (number of experiments, total number of cells quantified). P-values were determined by two-way unpaired Student’s t test. **, P < 0.005; ***, P < 0.0005.