Li R , Ren Y , Mo G , Swider Z , Mikoshiba K , Bement WM , Liu XJ .

PJT-168891 CIHR

             spindle
             spindle assembly

	FIGURE 1: TrimAway IP3R in Xenopus oocytes. (A) Control oocytes and oocytes injected with mCherry-Trim21 mRNA were incubated for 24 h followed by injection with anti-IP3R antibodies, as indicated. Oocytes were lysed 4 h after antibody injection for immunoblotting, first using anti-IP3R antibodies followed by and anti-β-tubulin antibodies. Molecular weight markers are shown. Arrow depicts IP3R. Representative of three independent experiments. (B) Control oocytes and oocytes injected with eGFP-Trim21 mRNA were incubated for 24 h followed by injection with anti-IP3R antibodies or anti-GST antibodies, as indicated. Four hours following antibody injection, oocytes were lysed for immunoblotting using anti-IP3R antibodies or anti-tubulin antibodies. Arrow depicts IP3R. Representative of three independent experiments.
	FIGURE 2: IP3R depletion inhibited first polar body formation. Oocytes injected with mRNAs coding for RFP-tubulin (red) and eGFP-H2B (green) were further injected with GFP-Trim21 mRNA as indicated (Trim). Twenty-four (24) hours after Trim21 mRNA injection, oocytes were injected with anti-IP3R antibodies or anti-GTS antibodies as indicated. Four hours after antibody injection, all oocytes were incubated with progesterone overnight before being examined by confocal microscopy. Oocytes were recorded as metaphase II (MII, with a first polar body and a bipolar metaphase II spindle; scale bar = 20 µm), abnormal spindle (no polar body; arrow depicts oocyte cortex), or undetectable (UD) (no chromosomes seen within the depth of the confocal system, not shown). The graph summaries five independent experiments depicting the proportions of oocytes exhibiting the three phenotypes. Percentage of MII oocytes in each group is indicated within the bar, with total oocyte number indicated above.
	FIGURE 3: A novel cell-free spindle assembly system. (A) MI extracts: oocytes expressing RFP-tubulin (red) and eGFP-H2B (green) are stimulated to undergo meiosis. Cytoplasm is aspirated from an MI oocyte (1hour after GVBD) and mixed with demembranated sperm nuclei. An aster was seen about 5 min after mixing (00:00; hh:mm), which rapidly enlarged (00:03) and became a monopolar (00:09) and then bipolar (00:15 and 00:21) spindle. Anaphase was observed where all chromosomes moved to the same pole (00:24) before microtubules disappeared (00:33) (9/9 or 100%). Metaphase II spindles appeared 1 h later (5/9 or 56%) and remained stable. Scale bar = 20 µm. (B) MII extracts: time series (hh:mm) of bipolar spindle formation from a single sperm in cytoplasm aspirated from a metaphase II arrested oocyte expressing RFP-tubulin (red). Hoechst dye (1 µg/ml) was added to sperm droplet to visualize DNA (green). The bipolar spindle is stable, usually for hours. Time 0 represents 5 min after mixing the cytoplasm with sperm. Scale bar = 20 µm. (C) Representative stages of extract spindle assembly in oocytes expressing sfGFP-ER3 (DNA injection) and RFP-tubulin. An aster formed in the microtubule-and ER tubules-rich oocyte extracts (00:00), which rapidly enlarged and was permeated by thinner ER tubules (00:09). In monopolar (00:25) and bipolar spindles, the thinner ER tubules were found concentrated at the poles (arrows). The bright ER blob (*) represented circular ER tubule stack. Scale bar = 20 µm. Shown are representative of >10 independent experiments. (D) High-resolution (AiryScan) single-plane confocal images through the center of a bipolar spindle assembled in cell-free cytoplasmic extract expressing sfGFP-ER3 and RFP-tubulin. Scale bar = 5 µm. Shown are representative of >10 independent experiments. (E) Enrichment of IP3R at spindle poles: Oocytes were injected with GFP-IP3R1 plasmid DNA and RFP-tubulin mRNA. Cytoplasmic extracts were aspirated from metaphase II-arrested oocytes and mixed with sperm droplets containing Hoechst dye (1 µg/ml). (Left) A microtubule aster was seen associated with a sperm 5 min after cytoplasm and sperm were mixed. (Right) A bipolar spindle depicting close association of spindle poles and IP3R-positive ER elements. Scale bar = 5 µm. Shown are representative images obtained in two independent experiments.
	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.
	FIGURE 5: DB-BAPTA, but not EGTA, disrupt bipolar spindles in cell-free extracts. (A) Images of hh:mm. Scale bar = 20 µm. (B) The bar plot summarizes the relative microtubule abundance at 10 min after injection. Means ± SD; ** indicates p < 0.01; n = 4 (EGTA), n = 8 (DB-BAPTA).

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