Figure 2. The kinesin-5 C-terminal tail domain is required for microtubule sliding at physiological ionic strength.

(A) Schematic of the microtubule sliding assay. Rhodamine-labeled microtubules from solution were crosslinked to surface-immobilized microtubules by kinesin-5, which drives relative sliding. (B–D) Kymographs show the motion of microtubules (left, red) and Kin5-GFP constructs (center, green). Bar, 3 µm. Microtubule sliding in the presence of (B) Kin5-GFP (1 nM) in high salt buffer, (C) unlabeled kinesin-5 (2 nM) and Kin5-Δtail-GFP (3 nM) in high salt buffer, and (D) Kin5-Δtail-GFP (3 nM) in low salt buffer. Dashed red lines in center kymographs indicate position of moving microtubule. Green arrows indicate Kin5-Δtail-GFP accumulated at microtubule tips. (E) Microtubule binding curves for Kin5-GFP (), Kin5-Δtail-GFP (), and Kin5-Δtail-GFP (). Microtubule affinity was determined by cosedimentation of the kinesin-5 constructs with microtubules in low salt buffer with 2 mM ADP. The fraction bound was calculated from the relative fraction remaining in the pellet following cosedimentation, and binding constants were determined by fitting to a hyperbola. Error bars = sem. See also Figure S2 and Movies S3–S5.