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. 2022 Nov 18;33(14):br27. doi: 10.1091/mbc.E22-06-0218

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© 2022 Li et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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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.