FIGURE 4:

Bipolar spindle formation in extracts required ER membranes and IP₃R. (A) Simultaneous disruption of reticular ER structure and bipolar spindles: Triton X-100 was added to a cytoplasmic droplet containing bipolar spindles (00:00) at a final concentration of 0.1%. Confocal time-lapse imaging (3D projections of the entire confocal stacks) was carried out following the behavior of the same spindle (n = 5). Scale bar = 20 µm. Shown is a representative series obtained in four independent experiments. (B) Cell-free cytoplasmic extract spindle assembly in the absence (control) or presence of 0.1% Triton X-100. Scale bar = 20 µm. Shown are representations of 6 independent experiments. (C) Oocytes injected with mRNAs coding for RFP-tubulin and eGFP-H2B were further injected with Trim21 mRNA as indicated (Trim). At 24 h after Trim21 mRNA injection, oocytes were injected with anti-IP₃R antibodies or anti-GST antibodies as indicated. At 4 h after antibody injection, all oocytes were incubated with progesterone overnight. The oocytes were then subjected to cytoplasm aspiration and spindle assembly assays. The ability of each oocyte to assemble bipolar spindles was assessed by confocal imaging and classified as bipolar spindle, disorganized chromosomes, or others (nuclei or microtubule asters). Shown are typical images of bipolar spindles and disorganized chromosomes, and a summary of four independent experiments with the total number of oocytes (n) in each group indicated. Scale bar = 20 µm. (D) Time-lapse (hh:mm) imaging of cell-free extract spindles following infusion of calcium-free OR2 (vehicle control) or heparin (200 µg/ml, two examples). Scale bar = 20 µm. The bar graph summarizes spindle microtubule abundance 7.5–20 min after heparin infusion relative to that before heparin infusion. For control, microtubule abundance 20 min after calcium-free OR2 infusion was compared with that before calcium-free OR2 infusion. Means ± SD; ** indicates p < 0.01; n = 10.