Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Abstract

SUMMARYThe kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signalling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, while dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into µm-scale filaments, and we identify the ROD-1 β-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZ’s capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

Cyclin B1 scaffolds MAD 1 at the kinetochore corona to activate the mitotic checkpoint

“…Although MAD1 is well known to bind the corona (Buffin et al , ; Kops et al , ; Caldas et al , ; Silio et al , ; Wynne & Funabiki, ; Qian et al , ; Luo et al , ; Pereira et al , ; Rodriguez‐Rodriguez et al , ; Sacristan et al , ; Zhang et al , ), an interaction with a corona component has never been mapped in vitro . In fact, the only established way to remove MAD1 from the corona is to deplete RZZ subunits, which simply abolishes corona formation altogether (Pereira et al , ; Rodriguez‐Rodriguez et al , ; Sacristan et al , ). Therefore, we next sought to explore whether Cyclin B1 might be the receptor that directly recruits MAD1 to the corona.…”

“…As well as MPS1 localisation, CDK1 positively and negatively regulates a number of other key enzymes at the kinetochore (for review, see Saurin, ), and therefore, these substrates may change dramatically upon kinetochore–microtubule attachment when Cyclin B1/MAD1 is stripped away along microtubules. Furthermore, the removal of the corona itself may depend on localised CDK1 inactivation because acute inhibition of CDK1 is known to cause premature corona detachment (Pereira et al , ; Sacristan et al , ). To address the potential importance of localised kinetochore CDK1 regulation, however, we believe it will be crucial to first identify the other receptor(s) for Cyclin B1 at the corona so that this pool can then be fully removed from unattached kinetochores.…”

Mutations in the Drosophila rough deal gene affecting RZZ kinetochore function

“…The earliest observations of RZZ behaviour during mitosis led to the proposal that it was a major component of the fibrous corona [Basto et al, 2004;Williams et al, 1996], an expanded outer domain of unattached kinetochores that promotes microtubule capture. Features of the modelled structure of RZZ [Mosalaganti et al, 2017] display similarities to other proteins that can self-assemble into networks, such as COP1 and Clathrin, and recently several groups have reported direct cell biological and biochemical evidence for the participation of RZZ and its interacting partner Spindly in the assembly of the corona in vivo and filamentous structures in vitro ([Pereira et al, 2018;Rodriguez-Rodriguez et al, 2018;Sacristan et al, 2018], reviewed in [Suzuki and Varma, 2018]). These reports have focused particular attention on the N-terminal domain of Rod, which appears to be necessary for kinetochore expansion.…”

“…These reports have focused particular attention on the N-terminal domain of Rod, which appears to be necessary for kinetochore expansion. The β-propeller is phosphorylated by Mps1 [Rodriguez-Rodriguez et al, 2018] and, along with Zwilch, [Gama et al, 2017;Pereira et al, 2018] is necessary for Spindly binding [Mosalaganti et al, 2017]. Indeed, specific mutations in Zwilch, in Spindly, and in the Rod N-terminal domain all interfere with the ability of RZZ to form expanded kinetochores in vivo and to oligomerise in vitro.…”

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.…”