FIGURE 6.

PRC1 crosslinks do not substantially resist the relative sliding of two microtubules by kinesin-5. (A) Schematic illustrating the assay used to examine the effect of PRC1 (green) on kinesin-5 (cyan)-mediated relative sliding of two microtubules (orange), when extent of overlap between microtubules is unchanged. Near-simultaneous dual-mode microscopy was used to image microtubules (via wide-field fluorescent speckle microscopy) and GFP-PRC1-FL (via Total Internal Reflection Fluorescence (TIRF) microscopy). (B) Frames from a time-lapse sequence (1 min interval) show GFP-PRC1-FL (green) enriched at regions where two crosslinked microtubules (red) overlap. (C) Corresponding kymograph shows the surface-attached static microtubule (vertical streaks) and a moving microtubule (diagonal streaks, 16.5 nm/s). (D) Kymograph shows that the GFP-PRC1-FL decorated region moves at the velocity of the moving microtubule. (E) Schematic illustrating the assay when overlap between microtubules decreases during relative microtubule sliding. (F) Frames from a time-lapse sequence (2 min interval) and corresponding kymographs showing microtubule movement (7 nm/s) (G) and GFP-PRC1-FL localization to overlap region that reduces due to relative sliding of filaments (H). (I) Velocity distributions for kinesin-5 driven microtubule sliding at 1.8 nM kinesin-5 and 0.034 (V = 23.1 ± 6.3 nm/s, N = 43), 0.14 nM (V = 17.7 ± 3.5 nm/s, N = 39), and 0.54 nM GFP-PRC1-FL (V = 9.7 ± 3.1 nm/s, N = 41). Scale bars: 1.5 μm, 100 s. See also Figure S5