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 sought to investigate why GFP::ROD-1 filaments formed at this specific time in development. Quantification of the GFP::ROD-1 signal revealed that nuclear levels of GFP::ROD-1 gradually increased with every embryonic division, as well as during the course of each cell cycle, reaching a peak before NEBD (Fig. 4C -E) . Consequently, nuclear GFP::ROD-1 levels before NEBD in 8-cell embryos were significantly higher than nuclear levels in all previous divisions ( Fig. 4C -E ). This suggested a straightforward explanation for the invariant timing of filament formation, namely that GFP::ROD-1 must become sufficiently concentrated before oligomerization is triggered. To directly test this idea, we used a mild RNAi regime to slightly reduce total GFP::ROD-1 levels in the embryo (Fig. 4E , F). In these RNAi conditions, nuclear GFP::ROD-1 levels in 8-cell embryos were comparable to those in control embryos at the 2-cell stage (Fig. 4E) . Strikingly, GFP::ROD-1 no longer formed filaments in 8-cell embryos or at later developmental stages, despite the still prominent enrichment in nuclei and normal localization to mitotic kinetochores (Fig. 4E, F) . These results demonstrate that ROD-1 has a propensity to oligomerize into filaments and suggest that oligomerization can be triggered locally by concentrating ROD-1 above a critical threshold.