The motor domain of the kinesin Kip2 promotes microtubule polymerization at microtubule tips.

Kinesins are microtubule-dependent motor proteins, some of which moonlight as microtubule polymerases, such as the yeast protein Kip2. Here, we show that the CLIP-170 ortholog Bik1 stabilizes Kip2 at microtubule ends where the motor domain of Kip2 promotes microtubule polymerization. Live-cell imaging and mathematical estimation of Kip2 dynamics reveal that disrupting the Kip2-Bik1 interaction aborts Kip2 dwelling at microtubule ends and abrogates its microtubule polymerization activity. Structural modeling and biochemical experiments identify a patch of positively charged residues that enables the motor domain to bind free tubulin dimers alternatively to the microtubule shaft. Neutralizing this patch abolished the ability of Kip2 to promote microtubule growth both in vivo and in vitro without affecting its ability to walk along microtubules. Our studies suggest that Kip2 utilizes Bik1 as a cofactor to track microtubule tips, where its motor domain then recruits free tubulin and catalyzes microtubule assembly.

Author Correction: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Remote control of microtubule plus-end dynamics and function from the minus-end.

In eukaryotes, the organization and function of the microtubule cytoskeleton depend on the allocation of different roles to individual microtubules. For example, many asymmetrically dividing cells differentially specify microtubule behavior at old and new centrosomes. Here we show that yeast spindle pole bodies (SPBs, yeast centrosomes) differentially control the plus-end dynamics and cargoes of their astral microtubules, remotely from the minus-end. The old SPB recruits the kinesin motor protein Kip2, which then translocates to the plus-end of the emanating microtubules, promotes their extension and delivers dynein into the bud. Kip2 recruitment at the SPB depends on Bub2 and Bfa1, and phosphorylation of cytoplasmic Kip2 prevents random lattice binding. Releasing Kip2 of its control by SPBs equalizes its distribution, the length of microtubules and dynein distribution between the mother cell and its bud. These observations reveal that microtubule organizing centers use minus to plus-end directed remote control to individualize microtubule function.

Structure-Function Relationship of the Bik1-Bim1 Complex.

In budding yeast, the microtubule plus-end tracking proteins Bik1 (CLIP-170) and Bim1 (EB1) form a complex that interacts with partners involved in spindle positioning, including Stu2 and Kar9. Here, we show that the CAP-Gly and coiled-coil domains of Bik1 interact with the C-terminal ETF peptide of Bim1 and the C-terminal tail region of Stu2, respectively. The crystal structures of the CAP-Gly domain of Bik1 (Bik1CG) alone and in complex with an ETF peptide revealed unique, functionally relevant CAP-Gly elements, establishing Bik1CG as a specific C-terminal phenylalanine recognition domain. Unlike the mammalian CLIP-170-EB1 complex, Bik1-Bim1 forms ternary complexes with the EB1-binding motifs SxIP and LxxPTPh, which are present in diverse proteins, including Kar9. Perturbation of the Bik1-Bim1 interaction in vivo affected Bik1 localization and astral microtubule length. Our results provide insight into the role of the Bik1-Bim1 interaction for cell division, and demonstrate that the CLIP-170-EB1 module is evolutionarily flexible.

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.