Dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis

Abstract

Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. The outer layer of kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts following microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD-Zwilch-ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ-Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.

Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion

“…Kinetochore expansion is thought to accelerate mitotic ‘search and capture’ by promoting lateral microtubule attachment [4] and to enhance SAC signaling [5]. RZZ is closely related to endomembrane coatomers that form oligomeric lattices [18, 19] and is likely a “building block” of the corona itself [13, 14, 20, 21]. Consistent with these proposals, two other corona-associated proteins (CENP-E [22] and CENP-F [23]) did not form crescents in KNTC1 –/– cells (Figure 2A-B and S2A).…”

“…To ensure that these results reflected loss of kinetochore expansion and not protein mislocalization, we performed correlative light-electron microscopy in cells expressing CENP-A-GFP as a centromere marker. Serial sectioning revealed circumferential expansion of trilaminar plates and fibrous material in wildtype cells (n=14 kinetochores), whereas the kinetochores of KNTC1 –/– cells appeared as compact discs (n=15; Figure S2B and [13]). We conclude that the RZZ complex is required for fibrous corona formation.…”

“…Kinetochore expansion depends on mitotic kinases [5, 13], but relevant phosphorylation events are not known. We identified two Mps1-regulated phosphosites just upstream of Rod’s β-propeller domain [24] that are required for kinetochore expansion but not SAC arrest.…”

“…Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4–6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7–12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells.…”

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

“…Recent studies indicate that Rod and Spindly not only interact via this domain [17,20] but also inhibit their own self-assembly into polymers [51,52]. In light of these findings and the fact that Mps1 maximally phosphorylates Rod at unattached kinetochores [23], we propose that this modification overcomes the barrier to Spindly-RZZ polymerization and kinetochore expansion.…”