Incremental Phosphorylation of a Dynamic Lawn of NDC80 Complexes Provides Graded Control of Kinetochore-Microtubule Affinity

Abstract

The stability of kinetochore-microtubule (KMT) attachments is finely tuned to drive different mitotic processes. This regulation involves phosphorylation of NDC80 complex, a major component of the MT-binding interface at kinetochores. The fundamental question of how the number and stability of KMT attachments is regulated by NDC80 phosphorylation remains unanswered. The Hec1 subunit of the NDC80 complex has an unstructured “tail,” which is required for KMT attachment in vivo and contributes to the NDC80 complex-MT binding in vitro. This tail is an established target for Aurora kinases and has 9 mapped phosphorylation sites. To understand how phosphorylation of the Hec1 tail affects MT-binding characteristics of single NDC80 complexes we expressed and purified NDC80Bonsai complexes with different number of phospho-mimetic mutations in Hec1 tail. With TIRF microscopy we found that Hec1 tail phosphorylation leads to a graded increase in NDC80 diffusion and the shortening of its MT residency time, but cooperativity of NDC80-MT binding is only weakly affected by Hec1 tail phosphorylation. To understand physiological relevance of the phosphoregulation of NDC80 complexes we used computational approaches to model kinetochore-MT interface containing multiple NDC80 molecules. We show that the behavior of such interface strongly depends on the spatial organization of the NDC80 complexes. KMT interface that contained “repetitive sites” of NDC80 complexes greatly amplified the relatively small, phosphorylation-induced changes in the residency time of single NDC80. However, the KMT interface that contained a dynamic “lawn” of un-clustered and uncoordinated NDC80 complexes exhibited a graded response to phosphorylation and produced an excellent fit to our data with cells in prometaphase and metaphase. We conclude that incremental phosphorylation of NDC80 complexes drives the graded regulation of kinetochore-microtubule affinity during mitotic progression.