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

Cláudia Pereira; Rita M. Reis; José B. Gama; Dhanya K. Cheerambathur; Ana X. Carvalho; Reto Gassmann

We also show that RZZ's direct binding partner Spindly makes an important contribution to kinetochore expansion. This function of Spindly is separable from its function as the kinetochore adaptor for dynein-dynactin [15] , because DCTN1 depletion shows that dynein-dynactin are dispensable for kinetochore expansion. Previous work indicates that Spindly is recruited to kinetochores through a direct interaction with the ROD b-propeller [15, 16] , and, consistently, deletion of the ROD b-propeller suppresses . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/282707 doi: bioRxiv preprint kinetochore expansion [15] . However, the ZWILCH(E422A/D426A) mutant shows that Spindly recruitment by RZZ per se is not sufficient for kinetochore expansion. Work in C. elegans suggested that SPDL-1 Spindly , in addition to binding the ROD-1 b-propeller, also makes contact with ZWL-1 Zwilch [15] . The interaction between SPDL-1 Spindly and ZWL-1 Zwilch involves the conserved acidic residues we mutated in human ZWILCH, raising the possibility that an interaction between Spindly and ZWILCH may play role in RZZ selfassembly. Mitotic kinases have been implicated in the regulation of kinetochore expansion in Xenopus egg extracts [21] . It is therefore likely that kinetochore-localized kinase activity acts on RZZ and/or Spindly to promote self-assembly of RZZ. Mps1 is a particularly attractive candidate given that a recent study identified multiple Mps1-dependent phospho-sites on both Spindly and the ROD b-propeller [38] .