ROD-1 and Zw10^CZW-1 can target to kinetochores independently of Zwilch^ZWL-1. (A) Cartoon depicting localization dependencies of kinetochore dynein module components. (B) Summary of interactions between C. elegans RZZ subunits, based on yeast two-hybrid mapping (Fig. S1, A–C). Note that the intact RZZ complex likely exists as a dimer of the ROD-1–Zw10^CZW-1–Zwilch^ZWL-1 trimer, based on hydrodynamic analysis of the human and D. melanogaster complexes (Williams et al., 2003; Çivril et al., 2010). (C) Cartoon of the one-cell embryo spindle region shown in subsequent panels. (D–F) Localization dependency analysis of mCherry-tagged RZZ subunits. Still images are from time-lapse sequences. (G) Quantification of mCherry::ROD-1 levels at kinetochores in the conditions shown in D–F. Circles correspond to measurements in individual one-cell embryos in metaphase. Error bars represent the SEM with a 95% confidence interval. The t test was used to determine statistical significance (***, P < 0.0001 compared with no RNAi control; ns, P > 0.05). (H) Immunoblots showing that depletion of Zw10^CZW-1 but not Zwilch^ZWL-1 lowers mCherry::ROD-1 levels. α-Tubulin served as the loading control. (I) Immunofluorescence images of metaphase kinetochores stained for ROD-1. Endogenous ROD-1 is missing from kinetochores in Zwilch^ZWL-1-depleted embryos, whereas mCherry::ROD-1 localizes under the same conditions. (J) Stills from a time-lapse sequence showing that mCherry::ROD-1 supports kinetochore localization of GFP::Zw10^CZW-1 in the absence of Zwilch^ZWL-1. Bars: (D–F and J) 5 µm; (I) 2µm. (K) Cartoon summary of the data shown in C–J.