CENP-F controls force generation and the dynein-dependent stripping of CENP-E at kinetochores

Accurate chromosome segregationdemands efficient capture of microtubulesby kinetochoresand their conversion to stable bi-oriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochorefollowing microtubuleattachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule- binding domainsand physically associates with dyneinmotor regulators. Here, we have combined CRISPRgene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochorefunction. We show here that the two microtubule- binding domainsmake distinct contributions to attachment stability and force generation that are required to minimise errors in anaphase, but are dispensable for congression. We further identify a specialised domain that functions to inhibit the dyneinmediated stripping of CENP-E motors. We show how this dynein-brake is crucial for ensuring kinetochorescontain the right number of CENP-E motors at the right time during mitosis, with loss of brake function delaying congression.