Fig. 7: Phafin2 can bind to actin.

a Confocal images showing the effect of Phafin2 depletion on the actin cytoskeleton. Representative image of the data scored in Fig. 7b. Scale bar: 10 µm. b Depletion of Phafin2 results in an increased number of cells with membrane ruffles, which can be rescued by expression of siRNA-resistant Phafin2. Data derived from high content imaging (2038, 1581, 1831, 2055 cells for siSCR, siPhafin2, siSCR(Rescue) and siPhafin2(Rescue) in total. n = 3 experiments, mean + 95% CI, two-sided t-test, p = 0.0017 (Control), p = 0.017 (Rescue). c Western blot showing in vitro actin-binding assays of MBP-Phafin2 and Phafin2 mutants. Phafin2 and the isolated Phafin2 PH domain co-sediment with actin. MBP or the isolated Phafin2 FYVE domain does not co-sediment. d Quantification of the in vitro actin-binding assays. n = 3 experiments, mean + 95% CI, ANOVA with Tukey’s post-test, p = 0.0009 (MBP vs Phafin2), p = 0.0073 (MBP vs. Phafin2 PH), p = 0.91 (MBP vs Phafin2 FYVE). e Images showing the effect of GFP, Phafin2-GFP, and Phafin2ΔC-GFP expression in RPE1 cells expressing Lifeact-SNAP. Representative image of 30 cells each. Arrowheads indicate cell retractions/ membrane blebs; arrows indicate flat cell protrusions. Scale bar: 10 µm. f Time-lapse images of cells expressing Phafin2ΔC before and after incubation with the PI4K inhibitor GSK-A1. Scale bar: 10 µm. g Quantification of membrane blebbing after treatment of Phafin2ΔC-expressing cells with the inhibitor GSK-A1. n = 81 cells. Two-sided Fisher’s exact test, p < 0.0001. h Time-lapse images of cells expressing light-dimerizable Phafin2 (Phafin2-mCherry-SSBP and Venus-iLID-CAAX) and Lifeact before and after light-induced plasma membrane recruitment. Arrowheads indicate membrane blebs, arrows cell retraction. Representative image of 25 cells. Scale bar: 10 µm. i Quantification of cell retraction after light-induced membrane recruitment of Phafin2. n = 28 cells, two-sided Fisher’s exact test, p < 0.0001. j Quantification of membrane blebbing after light-induced membrane recruitment of Phafin2. n = 28 cells, two-sided Fisher’s exact test, p < 0.0001. k Time series showing a forming and retracting membrane bleb—visualized by membrane-recruited Phafin2-and Lifeact after Phafin2 membrane recruitment. Scale bar: 10 µm. l Line plots showing intensities of membrane-recruited Phafin2 and Lifeact over time. Statistics source data for b, d, g, i, j, and l are provided in this paper.