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  • Ensemble-Level Organization of Human Kinetochores and Evidence for Distinct Tension and Attachment Sensors.

Ensemble-Level Organization of Human Kinetochores and Evidence for Distinct Tension and Attachment Sensors.

Cell reports (2020-04-30)
Emanuele Roscioli, Tsvetelina E Germanova, Christopher A Smith, Peter A Embacher, Muriel Erent, Amelia I Thompson, Nigel J Burroughs, Andrew D McAinsh
ABSTRACT

Kinetochores are multi-protein machines that form dynamic attachments to microtubules and control 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-color subpixel fluorescence localization to define how proteins from six major complexes (CCAN, MIS12, NDC80, KNL1, RZZ, and SKA) and the checkpoint proteins Bub1, Mad1, and Mad2 are organized in the human kinetochore. This reveals how the outer kinetochore has a high nematic order and is largely invariant to the loss of attachment or tension, except for two mechanical sensors. First, Knl1 unravels to relay tension, and second, NDC80 undergoes jackknifing and loss of nematic order under microtubule detachment, with only the latter wired up to the checkpoint signaling system. This provides insight into how kinetochores integrate mechanical signals to promote error-free chromosome segregation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-α-Tubulin antibody, Mouse monoclonal, clone B-5-1-2, purified from hybridoma cell culture
Sigma-Aldrich
Dimethyl sulfoxide, sterile-filtered, BioPerformance Certified, meets EP, USP testing specifications, suitable for hybridoma
Sigma-Aldrich
Nocodazole, ≥99% (TLC), powder
Taxol, phyproof® Reference Substance