Adaptive changes in the kinetochore architecture facilitate proper spindle assembly

Abstract

Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that upon mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore-microtubule interactions precedes formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion-compaction in the context of lateral interactions correctly predict experimentally-observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.

Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture

“…The original search-and-capture model supposed that KC attachments to MTs are end-on, consistent with the results of Kalinina et al (24), while others observed primarily lateral attachments (14,19,20,23,25). Therefore, we studied how the probability of lateral or end-on attachment varied with MT length and the presence or absence of rotational diffusion in the model (Fig.…”

“…Simultaneously, a spherical KC of radius a ¼ 100 nm diffuses in the nucleus (24). We assumed the fission yeast KC size remained constant during mitosis, in contrast to recent work on human KCs (20). KC capture occurs when the KC contacts the MT, either at its end or along its lateral wall.…”

“…However, the simplest search-and-capture mechanism does not appear rapid enough to capture multiple chromosomes quickly enough to match measured time in mitosis. Extensions to the search-and-capture mechanism that can make KC capture more rapid in large cells or cell extracts include KC diffusion (12,15), MT growth that is spatially biased toward chromosomes (16)(17)(18), chromosome spatial arrangements and rotation (15,19,20), and KC-initiated MTs that can interact with searching MTs (15,21,22). KCs in human cells change size and shape during mitosis, which can both speed up capture and minimize errors (20).…”

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

“…Through gene editing and livecell imaging, we found that the human Rod-Zw10-Zwilch (RZZ) complex is dispensable for cell viability and initial recruitment of Mad1-Mad2 to kinetochores at NEBD, but then becomes necessary to tether Mad1-Mad2 at kinetochores and sustain SAC arrest in cells challenged with spindle poisons. We also show that RZZ forms the mesh-like fibrous corona, a structural expansion of the outer kinetochore important for timely chromosome congression [10][11][12][13] once Mps1 phosphorylates the N-terminus of Rod.Artificially tethering Mad1-Mad2 to kinetochores enabled long-term mitotic arrest in the absence of RZZ. Conversely, blocking early RZZ-independent recruitment of Mad1-Mad2 eliminated the transient SAC response in RZZ-null cells.…”

“…http://dx.doi.org/10.1101/297580 doi: bioRxiv preprint first posted online Apr. 9, 2018; 10 assembly downstream of Mad1-Mad2 [4]; however network-level compensation allows the SAC to adapt to chronic BUB1 loss, similar to other signaling pathways [48,49].RZZ is also required for microtubule attachment-sensitive expansion of the outer kinetochore [10,12,13]. Transgenic expression of LAP-Rod 2A and mCherry-Mis12-Mad1 rescued nocodazole-induced SAC arrest but not kinetochore expansion in KNTC1-null cells, implying that the latter is not rate limiting for SAC transduction.…”