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

Abstract

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] in mammalian cells. 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.

Keywords: Hec1; Ndc80; force generation; friction; kinetochore; kinetochore-microtubule interface; mechanics; microtubule; mitosis; spindle.

Figure 1. Hec1 tail phosphorylation regulates the magnitude and timescale of the mammalian kinetochore-microtubule interface’s response to force

(A) Assay to sever a k-fiber using laser ablation (red X) to induce a dynein-based poleward pulling force on a specific kinetochore pair to probe the back kinetochore’s movement on polymerizing microtubules in response to force. (B–D) Timelapse showing representative response of PtK2 (B) Hec1-WT-EGFP, (C) Hec1-9A-EGFP and (D) Hec1-9D–EGFP (each in Hec1 RNAi background – see also Figure S1) kinetochore pairs to k-fiber laser ablation. First frame after ablation set to 0:00. (E–G) Mean positions of Hec1-WT, Hec1-9A, and Hec1-9D (E) back and (F) front kinetochores and (G) K–K distance before and after laser ablation. Kinetochore position is shown normalized to its pre-ablation position. Traces are mean±SEM and are offset vertically for clarity in (E,F). (H) Velocity of the front and back kinetochores (from E,F) relative to the ablation-proximal spindle pole after the directional switch to poleward motion in response to ablation, until each kinetochore returned to motion away from that pole (* for p<0.05, n.s. not significant, Student’s T-test, n= number of kinetochores). (I) Distance traveled by the back kinetochore over the first 30s of poleward motion after ablation (* for p<0.05, Student’s T-test). See also Figure S1, Movie S1–3.

Figure 2. Hec1 tail phosphorylation regulates friction on kinetochores bound to polymerizing microtubules

(A) Assay to measure kinetochore velocity relative to the microtubule lattice, tracking kinetochores and poleward k-fiber microtubule flux by photomarking. (B) Representative timelapses of Hec1-EGFP and PA-GFP-tubulin PtK2 cells in a Hec1 RNAi background and (C) kymograph of poleward microtubule flux (dotted line) measured by photoactivation. Time 0:00 corresponds to photoactivation. The distance between the photomark and the kinetochore (ruler) provides velocity relative to the microtubule lattice. (D) Microtubule flux rate (mean±SEM, * for p<0.05, Student’s T-test) in cells with Hec1-WT, Hec1-9A, or Hec1-9D kinetochores (n= number of k-fibers). (E) Histogram of kinetochore velocity relative to the microtubule lattice (** for p<0.01, Student’s T-test). Hec1-9D kinetochore oscillations were too variable to quantify (see Supplement). (F) Spindle length (mean±SEM, n.s. for not significant, p=0.76 one-way ANOVA) in cells with Hec1-WT, Hec1-9A, or Hec1-9D kinetochores (n= number of cells). See also Movie S4.

Figure 3. Hec1 tail phosphorylation does not disrupt the mammalian kinetochore’s ability to couple to depolymerizing microtubules

(A) Assay to decouple sister kinetochores using laser ablation (red X) of one sister kinetochore to probe the remaining sister’s ability to track depolymerizing microtubules. (B) Timelapse of Hec1-WT-EGFP, Hec1-9A-EGP, or Hec1-9D-EGFP and GFP-tubulin in PtK2 cells before and after kinetochore ablation. (C) Response of kinetochores to sister ablation, colored by pre-ablation direction (n= number of kinetochores). (D) Kinetochore velocity relative to pole before and after its direction switch following sister ablation. (*** for p<0.001, Student’s T-test, n.s. for not significant). (E) Responses of kinetochores to sister ablation (n = number of kinetochores). (F) Kinetochore velocity after switching to poleward motion (depolymerization) due to ablation of sister. Kinetochore velocities relative to the pole (left) or to the microtubule lattice (right, adjusted for differences in flux from Figure 2) (same dataset as (D), n.s. for not significant, Student’s T-test). (G) Example traces and (H) mean delay of kinetochores switching direction after sister ablation (n.s. for not significant, Student’s T-test). (I) Summary of the role of Hec1 phosphorylation in regulating kinetochore velocity under different mechanical states. Kinetochore speeds are replotted from the indicated figures (Figure 1H values are adjusted for differences in flux from Figure 2). (J) Cartoon summarizing the mechanical role of Hec1 tail phosphorylation: it regulates velocity in polymerization (top, cyan) but does not disrupt coupling in depolymerization (bottom, yellow). For simplicity, numbers of microtubules and Hec1 molecules are diagrammed as constant across conditions. See also Movie S5–7.