Structure of the RZZ complex and molecular basis of Spindly‐driven corona assembly at human kinetochores

Abstract

In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein–dynactin adaptor Spindly and the ROD–Zwilch–ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high‐resolution cryo‐EM structure that captures the essential features of the RZZ complex, including a farnesyl‐binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ–Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation‐dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long‐sought‐for molecular basis of corona assembly on metazoan kinetochores.

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin

“…CC2* shares these characteristics with Spindly ∆276–306 ( Fig. 3 E and Raisch et al, 2022 ). Collectively, these observations suggest that the region of CC2 containing these residues is at the same time implicated in kinetochore binding and filament formation, suggesting a possible mechanism of kinetochore regulation of corona expansion.…”

“…Spindly Chimera promoted Spindly-dependent oligomerization of the RZZ complex in filaments in vitro ( Fig. 5 G ), which requires MPS1 kinase and mimics kinetochore corona assembly ( Raisch et al, 2022 ; Rodriguez-Rodriguez et al, 2018 ; Sacristan et al, 2018 ). Thus, the Spindly segment in Spindly Chimera is sufficient for polymerization.…”

“…We asked therefore if Spindly F interacted with DD in the presence of the RZZ complex. As the RZZ complex and Spindly are both known to be phosphorylated, with phosphorylation being critical for corona function ( Raisch et al, 2022 ; Rodriguez-Rodriguez et al, 2018 ), we tested binding to DD after dephosphorylation ( D RZZ and D Spindly F , Fig. 2 F ), or after additional incubation with a mix of ATP and mitotic kinases, including CDK1/Cyclin B, MPS1, and Aurora B ( P RZZ and P Spindly F , Fig.…”

“…The RZZ complex, considered the Spindly cargo at kinetochores, binds directly to farnesylated Spindly ( Mosalaganti et al, 2017 ; Raisch et al, 2022 ). The observation that binding of RZZ to Spindly was insufficient to unleash a conformational change compatible with DD binding motivated our detailed investigation of the determinants of Spindly’s closed conformation.…”

“…At kinetochores, phosphorylation by the MPS1 kinase promotes the polymerization of the RZZ–Spindly (RZZS) complex into a mesh ( Fig. 1 B ), the kinetochore corona, which adopts a characteristic crescent shape and contributes to the initial phases of chromosome alignment that precede end-on microtubule attachment and chromosome bi-orientation ( Kops and Gassmann, 2020 ; Magidson et al, 2015 ; Pereira et al, 2018 ; Raisch et al, 2022 ; Rodriguez-Rodriguez et al, 2018 ; Sacristan et al, 2018 ). Besides DD, the corona also recruits the MAD1:MAD2 complex ( Barisic et al, 2010 ; Chan et al, 2009 ; Cheerambathur et al, 2013 ; Gassmann et al, 2008 ; Gassmann et al, 2010 ; Griffis et al, 2007 ; Rodriguez-Rodriguez et al, 2018 ; Starr et al, 1998 ; Yamamoto et al, 2008 ), a central component of the spindle assembly checkpoint (SAC), which synchronizes cell-cycle progression with the completion of chromosome alignment ( Kops and Gassmann, 2020 ; Musacchio, 2015 ).…”