Molecular requirements for transition from lateral to end-on microtubule binding and dynamic coupling

Abstract

Abbreviations: MAP -microtubule associated protein, MT -microtubule, MSD -mean squared displacement, TIRF -total internal reflection fluorescence. ABSTRACTAccurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are ill-understood. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plusend coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned forcedependent motility characteristics. S. Macedo-Ribeiro (Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal) for providing CLASP2 protein. We also thank Dr. A. Kiyatkin, P.-T. Chen and V. Mustyatsa for help with protein purification, and Grishchuk lab members for discussions.

Higher-order assembly is a regulatory switch that promotes outer kinetochore recruitment

“…When incubated with stabilized microtubules in vitro, the oligomers interacted with the microtubule walls (Figure 3C, D). Using immunofluorescence, we confirmed that the microtubule-bound oligomers had copurified with the NDC80 complex (Supplementary Figure 2C), which likely mediated their interaction with microtubules 2,39,40 . By contrast, GFP oligomers isolated from mitotic HeLa cells did not bind microtubules (Figure 3C, D), indicating that microtubulebinding is specific to CENP-T 1-242 oligomers.…”