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 next sought to exploit the unique behavior of the ROD-1(D1-372) mutant to dissect the molecular requirements for ROD-1 oligomerization. Depletion of CZW-1 Zw10 by RNAi completely suppressed filament formation of mCherry::ROD-1(D1-372) (Fig. 5H ), likely because CZW-1 Zw10 is essential for ROD-1 stability [15] . By contrast, mCherry::ROD-1(D1-372) filaments were not affected by depletion of ZWL-1 Zwilch or SPDL-1 Spindly , consistent with the observation that ZWL-1 Zwilch and SPDL-1 Spindly did not localize to filaments. Furthermore, mCherry::ROD-1(D1-372) filaments readily formed after depletion of KNL-1, BUB-1, the Mis12 complex subunit KNL-3, and NDC-80, as well as after depletion of the mitotic regulators ICP-1 INCENP , CDK-1, and PLK-1 (data not shown). This indicated that a complex of ROD-1(D1-372) and CZW-1 Zw10 may be both necessary and sufficient for efficient oligomerization into filaments. To directly test this idea, we expressed the full-length RZZ complex and the ROD-1(D1-372)-CZW-1 Zw10 complex in insect Sf21 cells using baculovirus (Fig. 6A ). For purification and visualization, ROD-1 was N-terminally tagged with 6xHis followed by GFP, and CZW-1 Zw10 contained an N-terminal StrepTagII. After a first affinity purification step with nickel resin, size exclusion chromatography (SEC) showed that GFP::RZZ fractionated as a complex with equal stoichiometry of its three subunits and without other obvious protein contaminants (Fig. 6A ). After SEC, we examined the peak fractions by fluorescence microscopy but found no evidence that GFP::ROD-1 was present in filaments ( Fig. 6B ). We then applied the same purification procedure to the GFP::ROD-1(D1-372)-CZW-1 Zw10 complex, which we obtained in similar amounts and purity as GFP::RZZ (Fig. 6A) . In contrast to GFP::RZZ, GFP::ROD-1(D1-372)-CZW-1 Zw10 fractionated in a broader peak in SEC. Subsequent examination of peak fractions using fluorescence and transmission electron microscopy revealed . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 that GFP::ROD-1(D1-372) was present in filaments that were typically several µm in length (up to ~15 µm) and had an invariant width of ~50 nm (Fig. 6B -G) . Filaments frequently associated laterally with each other to form loose bundles (Fig. 6C , F, G). Staining for the StrepTagII confirmed that CZW-1 Zw10 co-localized with GFP::ROD-1(D1-372) on filaments (Fig. 6D) . We also readily obtained filaments with a ROD-1(D1-372)-CZW-1 Zw10 complex, confirming that the GFP tag was dispensable for ROD-1 oligomerization (Fig. S6 ). Once formed, filaments remained static and were stable at 4 ºC for several weeks, but did not tolerate freezing or high salt (500 mM NaCl), suggesting oligomerization involves electrostatic interactions. If the SEC step was omitted and partially purified GFP::ROD-1(D1-372)-CZW-1 Zw10 was examined directly after nickel affinity chromatography, filament formation could occasionally be followed by live imaging, which revealed that filaments grow from both ends (Fig. 6E) . We conclude that a purified ROD-1(D1-372)-CZW-1 Zw10 complex oligomerizes efficiently into µm-scale filaments in vitro, and that these filaments resemble those observed with this ROD-1 mutant in vivo. Thus, ROD-1(D1-372)-CZW-1 Zw10 represents a minimal RZZ construct sufficient for se