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. 2023 Jul 10;11:1195806. doi: 10.3389/fcell.2023.1195806

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Copyright © 2023 Pal, Lin, Zhan, Banerjee, Kuhn, Providence and Devreotes.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Establishment of an opto-RhoGEF system in epithelial cells. Time-lapse confocal images of MCF-10CA1h cell expressing 2XPDZ-mCherry-LARG (DH) (red; (A) and Lifeact-7-iRFP670 (cyan); (B), before or after 488 nm laser was turned on globally. Confocal slices are focused at the substrate-attached basal surface of the cell. 488 nm light was turned on at 03:44 (min:sec), and images were acquired every 8 s. Pink arrows denote F-actin rich protrusions in the cell, and its subsequent decrease after laser was switched on. Cell morphology and protrusions were visualized in the DIC channel (C). Disappearance of protrusions and appearance of blebbing after recruitment can be visualized in magnified view with pink arrows. Time in min:sec format. Scale bars represent 5 μm. (D) A linescan across the bottom surface of the cell in (A; shown with green line) denoting uniform increase in LARG (DH) intensity on the cell membrane after laser was switched on globally. Quantifications display reduction in cortical F-actin (E) and decrease in cell size (F) upon opto-RhoGEF recruitment to the cell membrane.