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

The ROD-1 b-propeller suppresses ubiquitous and complete oligomerization of ROD-1 into filaments In human cells, deletion of the ROD b-propeller prevents kinetochore expansion, which may at least in part be a consequence of Spindly's inability to bind RZZ. Because oligomerization of C. elegans ROD-1 into filaments was independent of SPDL-1 Spindly , we wanted to address the role of the ROD-1 b-propeller. We therefore generated animals expressing mCherry::ROD-1 without b-propeller (D1-372) from an RNAi-resistant transgene integrated in single copy at a defined chromosomal locus (Fig. 5A ). We found that mCherry::ROD-1(D1-372), just like full-length mCherry::ROD-1, localized to early embryonic nuclei and mitotic kinetochores when endogenous ROD-1 was present (Fig. 5B ). By contrast, when we depleted endogenous ROD-1 by RNAi, mCherry::ROD-1(D1-372), but not full-length mCherry::ROD-1, oligomerized into filaments measuring up to 15 µm in length that were ubiquitously present throughout the cytoplasm of the oocyte-producing gonad and early embryo, irrespective of developmental and cell cycle stage ( Fig. 5B, D; Fig. S5A ). Oligomerization of mCherry::ROD-1(D1-372) into filaments appeared to be essentially complete judging by the lack of diffuse cytoplasmic signal (Fig. 5B) . Consequently, after depletion of endogenous ROD-1, mCherry::ROD-1(D1-372) was no longer detectable at mitotic kinetochores, and, as predicted by RZZ's essential role at kinetochores, dividing embryos exhibited chromosome bridges in anaphase and were inviable (Fig. 5B, C) . We conclude that ROD-1's propensity to oligomerize into filaments is antagonized by its N-terminal b-propeller. The behavior of C. elegans ROD-1(D1-372) was unexpected given that human ROD(D1-375) prevented kinetochore expansion. While the molecular basis for this difference is currently unclear, the experiments in human cells and C. elegans identify the Rod b-propeller as a key regulator of RZZ self-assembly. The ROD-1(D1-372) mutant also illustrates that cells must have tight regulatory mechanisms in place to control Rod's tendency to oligomerize.