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. 2023 Aug 31;222(11):e202211029. doi: 10.1083/jcb.202211029

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© 2023 Bourdais et al.

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Figure 4. — Two distinct myosin II pools coexist in the cortex of activated oocytes. (A) Detection of Cdc42·GTP (green) and RhoA·GTP (red) in live oocytes at the MII (left) and anaphase II (AII, right) stages. Oocytes were injected at the MII stage with cRNAs encoding the MCB-EGFP and mCherry-AHPH biosensors. Images are z-compressions of three consecutive confocal frames. (B) Immunofluorescence detection of MRCKβ in fixed MII (left) and AII (right) oocytes. (C) Immunofluorescence detection of ROCK1 in an AII oocyte. (D) Immunofluorescence detection of activated myosin II (P-MRLC) and MRCKβ in an AII oocyte. (E) Immunofluorescence detection of activated myosin II (P-MRLC) in the cortex of activated oocytes undergoing AII. Oocytes were treated with DMSO (top row), BDP9066 (1 µM; middle row), or a combination of BDP9066 and Y-27632 (respectively 1 and 50 µM; bottom row). Open arrowheads point to the P-MRLC rings overlying the chromatid clusters in the control (DMSO) oocyte. White arrows point to cytokinetic P-MRLC in the control (DMSO) and BDP9066-treated oocytes. Images are z-compressions of 33 (DMSO), 15 (BDP9066), and 30 (BDP9066 + Y-27632) consecutive confocal frames. (F) Bar graph depicting the percentage of activated oocytes showing ring myosin II activation (ring P-MRLC, light green) and/or cytokinetic myosin II activation (cytokinetic P-MRLC, dark green). Oocytes were treated with DMSO, BDP9066, or a combination of BDP9066 and Y-27632, as shown in E. P values were calculated using Fisher’s exact test. NS: not significant. The number of oocytes scored is indicated in parentheses. F-actin was labeled with Alexa Fluor 568–phalloidin. Chromosomes were stained with SiR-DNA (A) or TO-PRO-3 (B–E). Fluorescence intensity profiles in A and D correspond to the cortical regions delineated by the gray arrowheads in the merge images. In D, the white arrow points to the ingressing furrow region that is enriched in P-MRLC but devoid of MRCKβ. PB1: first polar body. Scale bars are 10 µm. In A and B, scale bars apply to the entire panel.