The Tail of XCTK2 Contains Two Distinct Microtubule Binding Domains

Abstract

Molecular motors organize the microtubules (MTs) that make up the mitotic spindle, and defects in motor function can cause improper spindle assembly that leads to chromosome mis-segregation. The Kinesin-14 family of molecular motors facilitates spindle pole focusing by cross-linking and sliding anti-parallel and parallel MTs through their motor and tail domains. How the tail contributes to these functions is unclear. Kinesin-14 tail MT binding is inhibited by importin α/β association through a nuclear localization signal (NLS). To understand how importin α/β association regulates the cross-linking and sliding activity of Kinesin-14s, we visualized Xenopus XCTK2 cross-linking and sliding in vitro. Surprisingly, importin α/β preferentially inhibited anti-parallel cross-linking and sliding. These results suggest that the tail may contain two different MT binding domains (MBDs) that recognize the anti-parallel and parallel MT orientations. To test this hypothesis, we created a deletion series of proteins from the N-terminus and C-terminus of the tail and tested them for MT binding. We identified two MBDs, which we call MBD1 and MBD2, that had MT binding abilities similar to the full-length tail. YPet-tagged MBD1, which contains the NLS, displayed robust FRET with importin α-CyPet and importin β, indicating tight association; whereas, MBD2 did not. In the presence of importin α/β, MBD1 was also completely inhibited from binding to MTs. These results are consistent with the model that MBD1 recognizes the anti-parallel conformation, and MBD2 recognizes the parallel conformation. We are currently generating site-specific mutations within MBD1 and MBD2 to inhibit MT binding so that we can demonstrate separation of cross-linking activities in the full-length protein. Once we identify the mutations that disrupt anti-parallel or parallel MT cross-linking, we will test how these modes of cross-linking and sliding contribute to spindle assembly.