Highlights d 3D mapping of kinetochore architecture in human RPE1 cells d Outer kinetochore (NDC80, Mad1, and RZZ) has high nematic order d NDC80 jackknives and KNL1 unravels upon loss of attachment and tension, respectively d Recruitment of Mad2 is only coupled to the occupancy sensor (NDC80)

SummaryKinetochores 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.

No abstract

Gaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales. This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores. Here we present a flexible Bayesian computational approach that infers the distances and angles between multiple fluorophores and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorophore individually and infers the correlations between polygon lengths. The latter is essential for determining the full multi-fluorophore 3D architecture. We further developed the algorithm to infer the mixture composition of a heterogeneous population of multiple polygon states. We use our algorithm to analyse the 3D architecture of the human kinetochore, a macro-molecular complex that is essential for high fidelity chromosome segregation during cell division. Using triple fluorophore image data we unravel the mixture of kinetochore states during human mitosis, inferring the conformation of microtubule attached and unattached kinetochores and their proportions across mitosis. We demonstrate that the attachment conformation correlates with intersister tension and sister alignment to the metaphase plate.

Gaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales (≲ 50nm). This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores. Here we present a flexible Bayesian computational approach that infers the distances and angles between multiple markers and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorescent marker individually and infers the correlations between polygon lengths. The latter is essential for determining the full multi-fluorophore 3D architecture. We further developed the algorithm to infer the mixture composition of a heterogeneous population of multiple polygon states. We use our algorithm to analyse the 3D architecture of the human kinetochore, a macro-molecular complex that is essential for high fidelity cell division. We examine the conformational change induced by microtubule attachment using triple fluorophore marked data and demonstrate for the first time that in metaphase kinetochore conformation is heterogeneous.