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Fig. 3 The role of actin brushes and Rac1 GTPase in the establishment of the self-organizing ligand-independent polarization of PGCs. (A) Polarization of PAK-1 GBD (green), marking locations where Rac1 is active. Dashed line indicates the PAK-1 GBD crescent (Left). Actin brushes (magenta) form at the newly established PAK-1 GBD marked front. (B, B′) Light induced Rac1 activation is sufficient for directing polarization in tumbling PGCs (B) and for reversing polarity in already polarized cells (B′). Lifeact is presented in magenta and Esyt2a and Ezrin fusion proteins are in green. Blue circles indicate the area where the light-activated Rac1 was activated. (C) Rac1 activity is required for polarity establishment as judged by lack of stable focused accumulation of Lifeact (Left, magenta), Ezrin (Middle, green), and MTs (Right, green, CLIP-170) in cells expressing dominant negative Rac1 (DN-Rac1). Graphs show the polarity index of Ezrin (Left graph) and the MTs (Right graph). (D) Invasion of ER into front and back blebs. n = 140 front blebs and 29 back blebs in 16 cells. (D′, D″) Wild-type PGCs labeled with Lifeact (blue), ER (green), and cytoplasm (red). The blebs are marked by the dashed line. The asterisk indicates the front bleb and the arrowhead points at a bleb formed at the back. (Scale bars, 10 µm.) Time is in minutes and seconds and white arrows indicate the direction of migration.