The pseudo GTPase CENP-M drives human kinetochore assembly

Abstract

Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore–centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers.DOI: http://dx.doi.org/10.7554/eLife.02978.001

The distinct functions of CENP-C and CENP-T/W in centromere propagation and function inXenopusegg extracts

“…However, it may stabilize the incorporated CENP-T either directly or through the HIKM complex. 29 Unexpectedly, we observe little CENP-W in interphase chromatin. While we cannot discard the possibility that the antibody that we use is unable detect CENP-W at interphase centromeres by immunofluorescence, immunoblot analyses show a clear increase of CENP-W on chromatin in mitosis with respect to interphase.…”

“…Previous results in human and chicken somatic cells using siRNA and genetic conditional deletion, respectively, suggest a more independent behavior of CENP-C and CENP-T/W in some studies 24,35 but not in other. 29 It is important to note that in our experimental system the mitotic centromeric chromatin assembled in CENP-C depleted extracts does not contain any CENP-C while in other systems complete removal of chromatin-bound CENP-C is difficult to achieve. 8 As shown in other organisms, 22,23 CENP-C likely interacts with Mis12C and this, in turn, can recruit Ndc80C to centromeres even in the absence of CENP-T/W.…”

“…23,24,28 Other evidences, however, are more consistent with the existence of a single pathway with CENP-C at the top. 29 To investigate this issue in our experimental system, we performed immunofluorescent staining of mitotic chromosomes assembled Quantification of CENP-A loading (black bars) and CENP-C loading (gray bars) in CENP-A depleted extracts relative to the corresponding mock depleted extract using the loading assay described in the main text. At least 250 centromeres from more than 10 pairs as the one shown in (A) were measured for each condition in 2 independent experiments (exp1 and exp2).…”

“…[17][18][19] CENP-C and CENP-T serve as a platform for recruitment of the KMN network through pathways that are not yet well understood. [20][21][22][23][24][25][26][27][28][29] From the CCAN components, CENP-N and CENP-C recognize CENP-A. [30][31][32] Depletion of CENP-N or CENP-C in human cells by RNA interference leads to decreased incorporation of new CENP-A at centromeres.…”

Stepwise unfolding supports a subunit model for vertebrate kinetochores

“…In contrast, CENP-C has a web of interactions with other CCAN members, including CENP-A and CENP-H/-I/-K/-M (35,36,41) as well as outer kinetochore components (10,42). CENP-T/W/S/X also interacts both with CENP-H/-I/-K/-M (10) and the Ndc80 complex (13,43).…”