Fig. 4.

Cin8-induced antiparallel MT sliding of stable and dynamic MTs. (A,B) Cin8, clustered at the minus ends of the MTs, drives plus-end-directed antiparallel MT sliding (Movie 6). (A) Kymographs of the mobile MTs and of Cin8 motility (MTs 2 and 3 in Movie 6). Merged images of the MTs and Cin8 are shown at the top. Minus and plus ends of the MTs are indicated based on the directionality of the mobile (yellow) MT. In all cases, part of the Cin8 cluster moved along the leading minus end of the mobile MT, and some of the cluster became dispersed in the overlap region. (B) MT 3 (Movie 6) switched between two MTs while sliding. A montage of images at selected time points is shown, indicated at the bottom left of each frame (seconds). Cin8 leads the switch between the two red MTs. Arrows indicate the cluster of Cin8 that leads the switch of the yellow MT on top of the two red MTs. (C) Cin8 promotes crosslink and sliding apart of dynamic MTs (Movie 9). Kymographs of Cin8-mediated sliding of MTs in a dynamics-permitting experiment, as described in Fig. 2. In the left-hand kymographs, sliding of one GMPCPP-stabilized seed on top of the other is observed. Both seeds promote MT dynamics, second kymograph from the left. White arrow points to a gap between the two stable seeds, indicating that they are held together by Cin8-mediated crosslinking of at least one dynamic MT. Yellow arrows point to Cin8 tracking along the minus ends of the dynamic MTs. Yellow asterisks indicate the Cin8 particles that are likely to crosslink the dynamic MTs (Movie 9).