Fig. 6: Phafin2 localization coincides with restructuring of the actin cytoskeleton.

a Light sheet imaging of RPE1 cells expressing Phafin2-GFP and Lifeact-SNAP. Representative image of n = 5 cells. Scale bar: 10 µm, inset: 5 µm. b Sequential images showing Phafin2 and Lifeact on macropinosomes. Representative image for n = 21 macropinosomes. Scale bar: 1 µm. c Localization dynamics of Phafin2 and Lifeact at macropinosomes. Lifeact localization shows partial temporal overlap with Phafin2 localization. n = 21 macropinosomes, mean + 95% CI. d Sequential images showing Phafin2 and Myo1E dynamics on a macropinosome. Representative image for n = 6 macropinosomes. Scale bar: 1 µm. e Myo1E is recruited earlier than the initial Phafin2 bursts and shows partial temporal overlap during the first phase of Phafin2 recruitment. n = 6 macropinosomes, mean + 95% CI. f Sequential images of Phafin2 and FilaminA on a macropinosome. Representative images for n = 46 macropinosomes. Scale bar: 1 µm. g Localization dynamics of Phafin2 and FilaminA on macropinosomes. n = 46 macropinosomes, mean + 95% CI. h Sequential images showing that actin (visualized by Lifeact) forms a coat around Phafin2-positive macropinosomes. Gaps in the actin coat allow the passage of the Phafin2-labeled vesicle (arrow). Representative image for 18 cells. Scale bar: 1 µm. i Sequential images showing that FilaminA forms a coat around Phafin2-positive macropinosomes. Gaps in the FilaminA coat allow the passage of the Phafin2-labeled vesicle (arrow). Representative image for 20 cells. Scale bar: 1 µm. j SIM super-resolution image showing a Phafin2-positive macropinosome passing a gap in the actin cytoskeleton (arrow). Representative image for ten cells. Scale bar: 10 µm, inset 2 µm. k Schematic overview of the observed recruitment dynamics. Statistics source data for b, d, and g are provided in this paper.