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.

Temporal changes in Hec1 phosphorylation control kinetochore–microtubule attachment stability during mitosis

“…This result provides a functional link between the observation that kinetochore-MT stability is low in early mitosis (Zhai et al, 1995) and the finding that Aurora B kinase is responsible for increasing kinetochore-MT turnover and inducing kinetochore-MT detachment Pinsky et al, 2006). We propose that in early mitosis, when attachment errors are most frequently made, phosphorylation of the Hec1 tail, which contributes to the linkage between kinetochores and MTs (Guimaraes et al, 2008;Miller et al, 2008), reduces the positive charge of the domain and thus decreases the affinity of kinetochores for negatively charged MTs. Conversely, dephosphorylation of the tail of Hec1 after chromosomes have bioriented ensures that kinetochore-MT attachments are stabilized in late mitosis so that forces can be generated for chromosome movements and to silence the mitotic spindle assembly checkpoint.…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…CH domains have diverse functions and have been identified in other microtubule binding proteins (Hayashi and Ikura 2003;Dougherty et al 2005). An unstructured N-terminal tail on Ndc80 enhances the microtubule binding activity of the complex (Wei et al 2005;DeLuca et al 2006;Wei et al 2007;Ciferri et al 2008;Miller et al 2008;Alushin et al 2010), although it is not essential for yeast viability due to redundancy with Dam1 (Kemmler et al 2009;Demirel et al 2012;Lampert et al 2013). The interaction between Ndc80 and microtubules is largely electrostatic and requires the C-terminal tails of tubulin ).…”

A Redundant Function for the N-Terminal Tail of Ndc80 in Kinetochore–Microtubule Interaction in Saccharomyces cerevisiae

“…MT-binding affinity of yeast and human Ndc80 complexes is decreased when the Ndc80 N-terminal tail is deleted (Wei et al 2005;Ciferri et al 2008). Reducing the positive charge by mutating some residues in the Ndc80 N-terminal tail abolishes MT attachments (Guimaraes et al 2008;Miller et al 2008;. KTs are structurally intact in engineered Hec1DN HeLa cells; however, the cells are unable to stably bind MTs, generate tension between sister KTs, or congress chromosomes (Miller et al 2008).…”

“…Reducing the positive charge by mutating some residues in the Ndc80 N-terminal tail abolishes MT attachments (Guimaraes et al 2008;Miller et al 2008;. KTs are structurally intact in engineered Hec1DN HeLa cells; however, the cells are unable to stably bind MTs, generate tension between sister KTs, or congress chromosomes (Miller et al 2008). Surprisingly, the N-terminal tail of the Ndc80 protein is dispensable for viability in Saccharomyces cerevisiae although a detailed phenotypic analysis has not been reported (Kemmler et al 2009;Lampert et al 2010).…”

“…The subtle phenotypes associated with ndc80-112 are surprising, given that the N-terminal tail of Ndc80 is essential in human cells and is required for yeast Ndc80 to bind MTs in vitro (Wei et al 2005;Ciferri et al 2008;Miller et al 2008;. One of the differences between kinetochores of yeast and human cells is the Dam1 complex that serves as a processivity factor for the Ndc80 complex, allowing Ndc80 to track the plus ends of microtubules and establish load-bearing connections (Lampert et al 2010; Figure 1 A mitotic delay in ndc80-112 cells.…”