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

Rod proteins share a common architecture with membrane coat precursors, which selfassemble into higher-order structures [16] . Self-assembly of ROD into filaments could underlie the central role of ROD in kinetochore expansion that we describe above, but there is currently no evidence that Rod proteins can form oligomeric assemblies. In our search for such evidence, we turned to the nematode C. elegans. In mature oocytes and meiosis I embryos, small filamentous assemblies, referred to as 'linear elements', are present in the cytoplasm and The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 S3). Depletion of ROD-1 prevented the formation of linear elements (Fig. S3A ), suggesting that they may represent oligomeric assemblies of ROD-1. Intriguingly, we found that after tagging endogenous ROD-1 with GFP using CRISPR/Cas9-mediated genome editing, GFP::ROD-1 filaments were not only present in meiosis but also appeared in the cytoplasm of early multicellular embryos (Fig. 4A) . Live imaging revealed that µm-scale GFP::ROD-1 filaments formed with highly reproducible kinetics during mitosis at the 8-cell stage, but not in earlier divisions ( Fig. 4A ; Movie S1). In addition to forming filaments, GFP::ROD-1 localized transiently to holocentric kinetochores and was enriched in nuclei in early embryos at all stages ( Fig. 4A ). At the 8-cell stage, filaments formed rapidly (within ~40 s) in nuclei about 3 min prior to NEBD, at about the same time when the GFP::ROD-1 signal first appeared on kinetochores ( Fig. 4B ; Movie S2). GFP::ROD-1 filaments remained distinct from mitotic chromosomes and segregated to daughter cells by clustering at spindle poles ( Fig. 4B; Fig. S4A ; Movie S2). At the end of mitosis, the filaments largely disassembled before forming again in nuclei prior to NEBD of the subsequent division ( Fig. S4B ; Movie S2). Interestingly, depletion of SPDL-1 Spindly had no effect on the formation of GFP::ROD-1 filaments but prevented clustering at spindle poles (Fig. S4B) , suggesting that the filaments become tethered to spindle poles via dynein.