Dynamics of blue light–dependent tgRFP-SspB MT plus end recruitment and dissociation. (A) Montage of tgRFP-SspB before and after activation in a S2 cell cotransfected with EGFP-SKIP-LZ-iLID. Time in seconds is relative to t = 0, at which time the cell was activated with a single 200-ms pulse of 0.15 mW 488-nm light. The montage shows blue light–dependent tgRFP-SspB recruitment to MT plus ends as well as subsequent dissociation over time (see accompanying Video 3). Bar, 5 µm. (B) Plot displaying the apparent cellular kinetics of tgRFP-SspB MT plus end association and dissociation over time. The system was activated at t = 0 with a single pulse of 0.15 mW 488-nm light. n = 5 cells. In cells cotransfected with SKIP-LZ-iLID, tgRFP-SspB immediately associates with MT plus ends, reaches maximal recruitment within 30 s after activation, and dissociates within 90 s. Inset shows the general kinetic steps that enable tgRFP-SspB MT plus end association, the convolution of which yields the apparent kinetics observed. The apparent half life of tgRFP-SspB on the MT plus end after activation is 25.1 ± 6.7 s. (C) Multiple rounds of activation of cells cotransfected with SKIP-LZ-iLID and tgRFP-SspB show the ability of tgRFP-SspB to be recruited to MT plus ends multiple times. Cells were activated at t = 0, 150, and 300 s with a single pulse of 0.10 mW 488-nm light (see accompanying Video 4). n = 7 cells. For B and C, black points and gray area represent the mean and SEM, respectively.