# Bayesian data driven modelling of kinetochore dynamics: Space-time organisation of the human metaphase plate

## TL;DR

The study uses detailed imaging and modeling to show how kinetochores dynamically organize chromosomes during cell division, revealing variability that may ensure accurate chromosome segregation.

## Contribution

The paper introduces a Bayesian modeling framework to reverse engineer kinetochore dynamics and reveals intrinsic spatial and temporal heterogeneity in metaphase plate organization.

## Key findings

- Kinetochore forces show substantial sister asymmetry, contributing to transverse metaphase plate organization.
- Kinetochore dynamic properties are spatially organized within the metaphase plate.
- K-fiber mechanical parameters adjust over time, leading to an anaphase-ready state.

## Abstract

Mitosis is a complex self-organising process that achieves high fidelity separation of duplicated chromosomes into two daughter cells through capture and alignment of chromosomes to the spindle mid-plane. Chromosome movements are driven by kinetochores (KTs), multi-protein machines that attach chromosomes to microtubules (MTs), and through those attachments both control and generate directional forces. Using lattice light sheet microscopy imaging and automated near-complete tracking of kinetochores at fine spatio-temporal resolution, we produce a detailed atlas of kinetochore metaphase-anaphase dynamics in untransformed human cells (RPE1). Such data allows dynamic models to be reverse engineered and biological hypotheses to be addressed. We determined the support from this dataset for 17 models of metaphase dynamics using Bayesian inference, demonstrating (1) substantial sister asymmetry that generates transverse organisation of the metaphase plate (MPP), (2) substantial spatial organisation of KT dynamic properties within the MPP, and (3) significant time dependence of the K-fiber mechanical parameters whereby K-fiber forces tune over the last 5 mins of metaphase towards a set point, referred to as the anaphase ready state. These spatio-temporal trends are robust to perturbation of the spindle assembly pathway (nocodazole washout treatment), suggesting that the underlying processes generating kinetochore heterogeneity are intrinsic to mitosis and possibly play a role in ensuring high-fidelity segregation.

Cell division segregates newly duplicated chromosomes into two daughter cells. This is a mechanical process orchestrated by the mitotic spindle, a self-assembling molecular machine comprising dynamic fibres called microtubules. Chromosomes are attached to microtubules at protein complexes called kinetochores, forces from these attachments driving chromosome movements. During cell division the chromosomes are aligned at the cell equator, where they undergo pseudo-periodic oscillations for ∼10 minutes before chromosome segregation begins. The purpose of this 10-minute ‘holding pattern’ is however unclear. By tracking kinetochores in 3D, we estimated the forces acting on individual kinetochores using reverse engineering techniques. We discovered that kinetochore dynamics is very variable within a cell, with substantial dependence of forces on spindle location and substantial changes in forces occurring in the lead up to anaphase and segregation. We speculate that this variability is a design feature, with forces adapting to the local spindle curvature, whilst random variability may be important to prevent oscillation synchrony. This work establishes a framework to quantitatively analyse how kinetochores work collectively at the cell level, and how individual variability may contribute to cell division robustness. Our work demonstrates that kinetochores are not equal, a fact that may be crucial in unravelling error detection and correction mechanisms.