Ensemble-level organization of human kinetochores and evidence for distinct tension and attachment sensors

Abstract

<jats:title>Summary</jats:title><jats:p>Kinetochores are multi-protein machines that form dynamic attachments to microtubules and generate the forces for chromosome segregation. High-fidelity is ensured because kinetochores can monitor attachment status and tension, using this information to activate checkpoints and error correction mechanisms. To explore how kinetochores achieve this we used two and three colour subpixel fluorescence localisation to define how six protein subunits from the major kinetochore complexes CCAN, MIS12, NDC80, KNL1, RZZ and the checkpoint proteins Bub1 and Mad2 are organised in the human kinetochore. This reveals how the kinetochore outer plate is a liquid crystal-like system with high nematic order and largely invariant to loss of attachment or tension except for two mechanical sensors. Firstly, Knl1 unravelling relays tension and secondly NDC80 jack-knifes under microtubule detachment, with only the latter wired up to the checkpoint signalling system. This provides insight into how kinetochores integrate mechanical signals to promote error-free chromosome segregation.</jats:p>

Kinetochore life histories reveal the origins of chromosome mis-segregation and correction mechanisms

“…1a; Movie S1). For this, we used a non-transformed near-diploid human hTERT-RPE1 cell line in which one allele of the NDC80 gene is tagged with eGFP at the carboxy-terminus 49 . By adapting our existing kinetochore tracking (KiT) algorithms 50 , we were able to capture an average of 34±9 long tracks of paired sister kinetochores per cell, compared to the total diploid number (46 pairs), representing 74 ± 20%, (Fig.…”

Bayesian inference of multi-point macromolecular architecture mixtures at nanometre resolution

“…We illustrate our algorithm on simulated data with (true) triangle lengths and measurement errors similar to those observed in biological complexes, [24]. We simulate data with J = 3 markers and N = 400 independent measurements, using the core model described in subsection Polygon parametrisation and inference, see supplementary section Simulated data for details.…”

“…We apply a two-state version of our algorithm to human kinetochores, macro-molecular complexes that play a vital role during cell division. Kinetochore attachment to microtubules is critical for the correct positioning of chromosomes in mitosis, microtubule attachment in fact leading to a conformational change within the kinetochore complex, [24]. By using our two-state model on triple-labelled kinetochores (re-analysing datasets from [24] thereby improving the resolution of those results), we demonstrate for the first time that there is conformational heterogeneity of the kinetochore complex during metaphase.…”

“…In 3D, however, typically PSFs are anisotropic, with the resolution along the optical axis usually reduced relative to that in the focal plane. For anisotropic measurement errors no analytical solution is known; however a Bayesian approach was developed for pair-wise distance correction [24]. Here we extend this methodology to multiple fluorophores/arbitrary polygons.…”

“…For the single-state method we demonstrate the algorithm’s advantages over existing pair-wise analyses for triangle (three fluorophore) datasets. In section Analysis of the human kinetochore: a structured macro-molecular complex we use our algorithm to analyse the architecture of the human kinetochore from experimental 3D fluorescence imaging data, [24]. In subsection Experimental data for two-state mixture model we present results for the analysis of heterogeneous experimental samples assumed to comprise two states.…”