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

HeLa cells were maintained at 37 °C in a 5 % CO 2 atmosphere in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10 % fetal bovine serum (Gibco), 100 units/mL penicillin, 100 units/mL streptomycin, and 2 mM GlutaMAX (Gibco). For immunofluorescence, cells were seeded on 12-mm poly-L-lysine-coated coverslips in 12well plates 24 h prior to transfection with siRNAs. Cells were transfected with siRNAs (Dharmacon On-Target plus; Table S2 ) targeting ROD/KNTC1, DCTN1, KNL1/CASC5, ZWILCH, and SPDL1/CCDC99, as described previously [43] . An siRNA against luciferase was used as a control. Transgene expression (Fig. 1K -M; Fig. 2C; Fig. 3B -D; Fig. S2) was induced with 0.2 µg/mL tetracycline (Sigma-Aldrich) 24 h post-transfection and cells were fixed 20 -24 h later. To depolymerize microtubules prior to immunofluorescence, cells were treated with 1 µM nocodazole (Sigma-Aldrich) for 4 h. To detach kinetochore crescents from centromeres ( Fig. 2; Fig. S1 ), cells were incubated for 4 h with 1 µM nocodazole followed by a 30-min incubation with nocodazole and 10 µM CDK1 inhibitor RO-3306 (Sigma-Aldrich).