ISWI is required for the maintenance of spindle MTs in anaphase and chromosome segregation. (A) A CSF extract was supplemented with 0.37 mg/ml rabbit IgG or anti–full-length ISWI antibodies. Each extract was incubated with sperm nuclei, Cy3-tubulin, and calcium at 20°C for 90 min (interphase). The samples were cycled into mitosis by adding fresh CSF extract and incubating at 20°C for 60 min (metaphase). They were further cycled into anaphase and interphase by adding calcium and incubating at 20°C for 60 min. At each time point, small aliquots were fixed on coverslips by squashing. This experiment was reproduced six times. Bar, 20 µm. (B) MTs disappear at anaphase onset in the presence of ISWI antibody. Spindle assembly and anaphase reactions were performed as described in A, but the samples were then fixed and spun down onto coverslips. The MT intensity around the sperm was quantified using a macro. n > 40 structures; n = 2 experiments. Error bars represent SD. (C) Chromosomes do not segregate in the presence of ISWI antibody. 20 min after anaphase onset, the percentage of the spindles with segregated chromosomes was quantified over the total number of structures counted. n = 300 spindles; n = 3 experiments. Error bars represent SD. (D) Cell cycle progression is normal in the presence of ISWI antibody. Histone H1 kinase activity was assayed in metaphase extracts (m), and 10, 20, 30, 40, and 60 min after calcium addition.