Kinetochore Attachments Require an Interaction between Unstructured Tails on Microtubules and Ndc80Hec1

Abstract

Summary Kinetochores bind and regulate the plus-ends of microtubules during mitosis. These attachments must be tight enough to move chromosomes, while the microtubule end both remains dynamic and repositions within the attachment pocket so that connections are maintained during depolymerization. Hec1/Ndc80 (Hec1) is a subunit of the four-protein Ndc80 complex, which is an important structural component of the kinetochore [1-3]. Kinetochores are unable to bind microtubules after Hec1 knockdown [2,4]; however, because the Ndc80 complex has structural roles, it is unclear if Hec1 directly mediates kinetochore microtubule attachments. Hec1 has a microtubule-binding site composed of both an unstructured N-terminal tail and a calponin homology domain [5-7]. Here we show that, surprisingly, the N-terminal tail is sufficient for microtubule binding affinity in vitro. The interaction is salt sensitive and the positively charged Hec1 tail is unable to bind microtubules lacking negatively charged tails. We have replaced the endogenous Hec1 subunit with a mutant lacking the N-terminal tail. These cells assemble kinetochores properly but are unable to congress chromosomes, generate tension across sister kinetochores, or establish cold-stable kinetochore-microtubule attachments. Our data argue that the highest affinity interactions between kinetochores and microtubules are ionic attractions between two unstructured domains. We discuss the importance of this finding for models of repositioning of microtubules in the kinetochore during depolymerization.