Figure 4.
Figure 4. Optogenetically induced cytoskeletal cross-linking decreases MT comet velocities and increases the MT-void area. (A) Schematic of Drosophila (D.m.) Shot. Shot contains tandem N-terminal CH domains that bind F-actin, a C-terminal GAR domain that binds MTs, and a SxIP motif that confers MT plus end localization. We functionally parsed Shot’s F-actin– and MT-binding activity into the SxIP-iLID system, fusing Shot’s CH domains to tgRFP-SspB and using the SKIP-LZ-iLID construct, which contains a SxIP motif from the mammalian spectraplakin MACF2. (A’) Image of CH-CH-tgRFP-SspB in a transfected S2 cell (left). Bar, 10 µm. See accompanying Video 5. The kymograph at right represents a time-lapse of the red line scan at left, and shows retrograde CH-CH-tgRFP-SspB movement in the lamellar region. Kymograph bars: 1 µm, 25 s. (B) Representative images of S2 cells cotransfected with EGFP-SKIP-LZ-iLID and either a tgRFP-SspB control, or the F-actin–binding CH-CH-tgRFP-SspB construct, and repeatedly pulsed with blue light (250 ms every 3 s at 488 nm). Top and middle: Single images from the EGFP and RFP channels. Bar, 5 µm. Right: Kymographs show representative EGFP-SKIP-LZ-iLID plus end comets. Kymograph bars: 2 µm, 25 s. Bottom: Maximal projections of 60 frames (total 3 min) are shown, revealing the area of the cell traversed by EGFP-SKIP-LZ-iLID–containing MT plus ends. Areas void of MT plus ends can be seen in the cross-linked cell (arrowhead). Blue boxes: Blue light recruitment of tgRFP-SspB constructs. (C) Representative images of S2 cells cotransfected with EGFP-SKIP-LZ-iLIDLit and either a tgRFP-SspB control or the F-actin–binding CH-CH-tgRFP-SspB construct. Top and middle: single images from the EGFP and RFP channels. Bar, 5 µm. Right: Kymographs show representative EGFP-SKIP-LZ-iLIDLit plus end comets. Kymograph bars: 2 µm, 25 s. Bottom: Maximal projections of 60 frames (total 3 min) are shown, revealing the area of the cell traversed by EGFP-SKIP-LZ-iLIDLit–containing MT plus ends. Areas void of MT plus ends can be seen in the constitutively cross-linked cell (see accompanying Video 6). (D) EGFP comet velocity/cell (μm/min) for control cells (iLID +SspB and iLIDLit+SspB), light-activated cross-linked cells (iLID + CH-CH-SspB), and constitutively cross-linked cells (iLIDLit + CH-CH-SspB). P-values were determined by two-way unpaired Student’s t test. (E) MT-void area as a percentage of the total cell area for control and cross-linked cells. Maximum projection images of 60 frames (spanning 3 min) were used to determine the area of the cell void of MTs (representative images used for quantification are shown in B and C and Video 6). P-values were determined by two-tailed nonparametric Mann–Whitney U test. For plots in D and E, numbers in parentheses indicate (number of experiments, total number of cells quantified). Line represents the mean, and error bars indicate SD. *, P < 0.05; **, P < 0.005; ****, P < 0.0001.

Optogenetically induced cytoskeletal cross-linking decreases MT comet velocities and increases the MT-void area. (A) Schematic of Drosophila (D.m.) Shot. Shot contains tandem N-terminal CH domains that bind F-actin, a C-terminal GAR domain that binds MTs, and a SxIP motif that confers MT plus end localization. We functionally parsed Shot’s F-actin– and MT-binding activity into the SxIP-iLID system, fusing Shot’s CH domains to tgRFP-SspB and using the SKIP-LZ-iLID construct, which contains a SxIP motif from the mammalian spectraplakin MACF2. (A’) Image of CH-CH-tgRFP-SspB in a transfected S2 cell (left). Bar, 10 µm. See accompanying Video 5. The kymograph at right represents a time-lapse of the red line scan at left, and shows retrograde CH-CH-tgRFP-SspB movement in the lamellar region. Kymograph bars: 1 µm, 25 s. (B) Representative images of S2 cells cotransfected with EGFP-SKIP-LZ-iLID and either a tgRFP-SspB control, or the F-actin–binding CH-CH-tgRFP-SspB construct, and repeatedly pulsed with blue light (250 ms every 3 s at 488 nm). Top and middle: Single images from the EGFP and RFP channels. Bar, 5 µm. Right: Kymographs show representative EGFP-SKIP-LZ-iLID plus end comets. Kymograph bars: 2 µm, 25 s. Bottom: Maximal projections of 60 frames (total 3 min) are shown, revealing the area of the cell traversed by EGFP-SKIP-LZ-iLID–containing MT plus ends. Areas void of MT plus ends can be seen in the cross-linked cell (arrowhead). Blue boxes: Blue light recruitment of tgRFP-SspB constructs. (C) Representative images of S2 cells cotransfected with EGFP-SKIP-LZ-iLIDLit and either a tgRFP-SspB control or the F-actin–binding CH-CH-tgRFP-SspB construct. Top and middle: single images from the EGFP and RFP channels. Bar, 5 µm. Right: Kymographs show representative EGFP-SKIP-LZ-iLIDLit plus end comets. Kymograph bars: 2 µm, 25 s. Bottom: Maximal projections of 60 frames (total 3 min) are shown, revealing the area of the cell traversed by EGFP-SKIP-LZ-iLIDLit–containing MT plus ends. Areas void of MT plus ends can be seen in the constitutively cross-linked cell (see accompanying Video 6). (D) EGFP comet velocity/cell (μm/min) for control cells (iLID +SspB and iLIDLit+SspB), light-activated cross-linked cells (iLID + CH-CH-SspB), and constitutively cross-linked cells (iLIDLit + CH-CH-SspB). P-values were determined by two-way unpaired Student’s t test. (E) MT-void area as a percentage of the total cell area for control and cross-linked cells. Maximum projection images of 60 frames (spanning 3 min) were used to determine the area of the cell void of MTs (representative images used for quantification are shown in B and C and Video 6). P-values were determined by two-tailed nonparametric Mann–Whitney U test. For plots in D and E, numbers in parentheses indicate (number of experiments, total number of cells quantified). Line represents the mean, and error bars indicate SD. *, P < 0.05; **, P < 0.005; ****, P < 0.0001.

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