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. 2019 May 22;10:2271. doi: 10.1038/s41467-019-10171-7

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© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Clearance of egg cortex during cortical granule exocytosis. a Zona-free ovastacin^mCherry eggs were stimulated to exocytose and fixed after 5 and 15 min followed by immunostaining with phalloidin to label F-actin. Representative iSIM maximum intensity projection of egg cortex showing cortical granules (red) and actin (gray) with magnified view of actin-free area. Arrows, cortical granule; white dashed line, actin-free area. Schematic (right) shows clearance of cortical actin during exocytosis and the appearance of cortical granules inside cleared actin sites. Scale bar, 5 μm. b Zona-free wild-type and Mater^Null eggs were stimulated to exocytose and fixed after 5 and 15 min followed by immunostaining with phalloidin (red) and antibody specific to myoIIA (cyan). Representative iSIM projection of the egg cortex with magnified views of regions of interest (white insets). Arrowhead, myoIIA puncta; white dashed lines, actin-free area. Scale bar, 5 μm. c Quantification of actin-free areas in the cortex of fixed stimulated wild-type eggs after 5 (n, 20) and 15 min (n = 78), and Mater^Null eggs (n = 55) acquired by iSIM. Box plot includes the mean (horizontal line) and data between the 25th and 75th percentile. Error bars indicate the 90th and 10th percentiles. ***p < 0.001 by two-tailed Student’s t-test. The experiment reflects three biologically independent replicates. d Immunoblot of egg (60) lysates isolated from wild-type or Mater^Null female mice and probed with antibodies specific to myoIIA and myosin V (MyoV). Molecular mass, right. e Zona-free wild-type eggs were stimulated to exocytose in the presence of myosin II inhibitors blebbistatin and ML-7 as well as actin-nucleation inhibitors CK-666 and SMIFH2. Eggs were fixed after 15 min followed by immunostaining with phalloidin (gray). White dashed lines, actin-free area. Scale bar, 5 μm. f Quantification of actin-free areas in the cortex of wild-type eggs (n = 78) and eggs treated with blebbistatin (n = 35), ML-7 (n = 52), CK-666 (n = 44), and SMIFH2 (n = 62) acquired by iSIM. Box plot includes the mean (horizontal line) and data between the 25th and 75th percentile. Error bars indicate the 90th and 10th percentiles. ***p < 0.001 by two-tailed Student’s t-test. The experiment reflects three biologically independent replicates. Source data are provided as a Source Data file