RZZ interacts with Spindly. (A) Representative images of the localization of the recombinant GFP-RZZ complex injected in mitotic HeLa cells transiently expressing mCherry-CENPA (to visualize kinetochores). Cells were synchronized in G2 by treatment with the Cdk1 inhibitor RO3306 and released into mitosis in the presence of the indicated drugs. Mitotic cells were live imaged before (Pre) and after (Post) microinjection with recombinant GFP or GFP–RZZ. Bars: (main images) 2 µm; (insets) 0.5 µm. Numbers of cells injected: for GFP, n = 2; for GFP-RZZ n = 8; for GFP in Reversine-treated cells, n = 2 (not depicted); for GFP-RZZ in Reversine-treated cells, n = 8. (B and C) Forward (For) and reverse (Rev) SILAC experiments (see the SILAC immunoprecipitation experiments section in Materials and methods) were performed as indicated in the upper part of the figure. Spindly is the most prominent component of the “Interactors” quadrant, which identifies specific binders of the GFP-RZZ complex in comparison with GFP. The experiments were performed in the absence (B) or presence (C) of detergent in lysis buffer. Baits are shown in green, interaction partners in red, and all quantified background proteins in gray. (D) Coiled-coil content of human Spindly and BicD2 predictions obtained with the program COILS (Lupas et al., 1991). Sequence alignment of Spindly and BicD2 shows excellent conservation in Spindly of a highly conserved motif previously identified in BicD2 (Hoogenraad and Akhmanova, 2016). Residues shown in red indicate conservation of signatures within motifs, but sequence conservation is more extensive and extends to the entire dynein-binding domain (not depicted).