Hec1 Tail Phosphorylation Differentially Regulates Mammalian Kinetochore Coupling to Polymerizing and Depolymerizing Microtubules

Abstract

Summary The kinetochore links chromosomes to dynamic spindle microtubules and drives both chromosome congression and segregation. To do so, the kinetochore must hold on to depolymerizing and polymerizing microtubules. At metaphase, one sister kinetochore couples to depolymerizing microtubules, pulling its sister along polymerizing microtubules [1,2]. Distinct kinetochore-microtubule interfaces mediate these behaviors: active interfaces transduce microtubule depolymerization into mechanical work, and passive interfaces generate friction as the kinetochore moves along microtubules [3,4]. Despite a growing understanding of the molecular components that mediate kinetochore binding [5–7], we do not know how kinetochores physically interact with polymerizing versus depolymerizing microtubule bundles, and whether they use the same mechanisms and regulation to do so. To address this question, we focus on the mechanical role of the essential load-bearing protein Hec1 [8–11]. Hec1’s affinity for microtubules is regulated by Aurora B phosphorylation on its N-terminal tail [12–15], but its role at the interface with polymerizing versus depolymerizing microtubules remains unclear. Here, we use laser ablation to trigger cellular pulling on mutant kinetochores and decouple sisters in vivo, and thereby separately probe Hec1’s role on polymerizing versus depolymerizing microtubules. We show that Hec1 tail phosphorylation tunes friction along polymerizing microtubules and yet does not compromise the kinetochore’s ability to grip depolymerizing microtubules. Together, the data suggest that kinetochore regulation has differential effects on engagement with growing and shrinking microtubules. Through this mechanism, the kinetochore can modulate its grip on microtubules over mitosis, and yet retain its ability to couple to microtubules powering chromosome movement.

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

“…This is similar to the plus-end polymerization rate of neighboring unmanipulated k-fibers during natural growth: lengthening at 1.4 ± 0.1 µm/min ( Fig. 2 G), while depolymerizing at the minus end at ∼0.5 µm/min results in a polymerization rate of ∼1.9 µm/min at plus ends (Mann-Whitney U test, P = 0.55; Long et al, 2017). This indicates that the applied force does not increase mammalian k-fiber plus-end polymerization rates.…”

Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner

“…Determining whether microtubule occupancy regulates only the Mad1 loss trigger or also its rate can have implications for both the SAC's molecular underpinnings and cellular role. To test how lowering steady-state microtubule occupancy affects Mad1 loss rates, we expressed Hec1-9D-FusionRed in PtK2 cells where endogenous Hec1 was depleted by RNAi (Guimaraes et al, 2008;Long et al, 2017) and monitored EYFP-Mad1 loss dynamics during attachment formation (visualized using SiR-Tubulin; Lukinavičius et al, 2014; Fig. 3 B).…”

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

“…At one end of Ndc80, two closely interacting calponin-homology (CH) domains near the N-terminal ends of NDC80 and NUF2 form a globular structure that binds the microtubule. An ~80-residue basic tail preceding the NDC80 CH domain (Ndc80-tail) has also been implicated in microtubule binding, and phosphorylation by Aurora kinase activity has been proposed to modulate electrostatic interactions with the negatively charged MT lattice (Alushin et al, 2012;Cheerambathur et al, 2017;Cheeseman et al, 2002;Cheeseman et al, 2006;Ciferri et al, 2008;DeLuca et al, 2006;DeLuca et al, 2011;DeLuca et al, 2018;Guimaraes et al, 2008;Long et al, 2017;Miller et al, 2008;Shrestha et al, 2017;Tooley et al, 2011;Umbreit et al, 2012;Wei et al, 2007;Ye et al, 2015;Zaytsev et al, 2015;Zaytsev et al, 2014).…”