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

Abstract

Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and 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 kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show here that the two microtubulebinding domains make 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 dynein mediated stripping of CENP-E motors. We show how this "dynein-brake" is crucial for ensuring kinetochores contain the right number of CENP-E motors at the right time during mitosis, with loss of brake function delaying congression.

Leaving no-one behind: how CENP-E facilitates chromosome alignment

“…In addition to mechanical roles at the nuclear envelope and mitochondrial outer membrane in G2 [ 52 , 53 ], CENP-F is recruited to outer kinetochores through a direct interaction with Bub1 in mitosis, providing a potential Bub1-dependent pathway for CENP-E recruitment to kinetochores [ 33 , 35 , 50 , 52 ]. Individual depletion of CENP-E and CENP-F indicate they show interdependency in their kinetochore localization [ 50 , 54 , 55 ]. Yet, in nocodazole-treated cells CENP-E is retained strongly at the kinetochore in the absence of CENP-F, indicating that CENP-F is not essential for CENP-E targeting to unattached kinetochores [ 33 , 54 , 55 ].…”

“…Individual depletion of CENP-E and CENP-F indicate they show interdependency in their kinetochore localization [ 50 , 54 , 55 ]. Yet, in nocodazole-treated cells CENP-E is retained strongly at the kinetochore in the absence of CENP-F, indicating that CENP-F is not essential for CENP-E targeting to unattached kinetochores [ 33 , 54 , 55 ]. Whether CENP-E and CENP-F interact at the kinetochore remains controversial and a direct interaction between CENP-E and CENP-F has yet to be reconstituted in vitro .…”

“…The outer corona is disassembled by Dynein upon kinetochore-microtubule attachment [ 16 ]. CENP-E is indeed removed from kinetochores in a Dynein-dependent manner [ 16 , 54 ], but the physical linkage between Dynein and CENP-E remains unknown.…”

“…CENP-E also tip-tracks depolymerizing microtubules and stabilizes kinetochore-microtubule attachments [ 71 , 97 , 103 , 104 ]. In contrast with the enrichment of CENP-E at unattached kinetochores, only a residual amount of CENP-E is maintained at the kinetochores of aligned chromosomes following Dynein-dependent stripping of the fibrous corona ( Figure 2 C) [ 16 , 32 , 54 ]. The remaining pool of CENP-E at bi-oriented kinetochores is essential for the maintenance of chromosome alignment: inhibition of CENP-E ATPase activity results in the poleward movement of chromosomes from the metaphase plate [ 71 ].…”

RZZ-SPINDLY-DYNEIN: you got to keep ‘em separated

“…However, premature RZZ shedding from KTs must be avoided to allow some degree of KT-MT attachment plasticity and prevent stabilization of erroneous endon attachments that might form during early mitosis [45,47]. Similarly to a recently proposed "DYNEIN brake" mechanism to maintain CENP-E at KTs [74], it has been suggested that cells must block untimely removal of RZZ from KTs. However, the molecular underpinnings of such mechanism had never been established [61].…”