Suchergebnisse

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end–directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics. ; Plasmids and protein purification protocols were generously provided by Drs. I. Cheeseman (Whitehead Institute, MIT), J. DeLuca (Colorado State Univ.), T. Surrey (Francis Crick Inst., UK) and D.W. Cleveland (Ludwig Cancer Research and Univ. of CA at San Diego). We are grateful to Drs. H. Maiato and S. Macedo-Ribeiro (Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal) for providing CLASP2 protein. We also thank Dr. A. Kiyatkin, P.-T. Chen and V. Mustyatsa for help with protein purification, and Grishchuk lab members for discussions. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM098389 to E.L.G., and by the American Cancer Society grant RSG-14-018-01-CCG to E.L.G. Theoretical modeling was supported by grant from Russian Science Foundation (16-14-00-224) to F. I.A. A.C.F. acknowledges support by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 681443) and FLAD Life Science 2020-award (to H. Maiato). F.I.A. acknowledges support from the Russian Foundation for Basic Research 17-00-00481 and 17-00-00480 to E.L.G.