Fig. 4: Two independent PtdIns3P pools control the biphasic recruitment of Phafin2. | Nature Communications

Fig. 4: Two independent PtdIns3P pools control the biphasic recruitment of Phafin2.

From: The phosphoinositide coincidence detector Phafin2 promotes macropinocytosis by coordinating actin organisation at forming macropinosomes

Fig. 4

a An isolated Phafin2 PH domain does not show membrane localization. Representative image for n = 12 cells. Scale bar: 10 µm. b A dual Phafin2 PH domain (2xPH) localizes to the plasma membrane and to forming macropinosomes but not to later, endosomal stages. Representative image for n = 19 cells. Scale bar: 10 µm. c Sequential images showing Phafin2 and the PtdIns4P probe SidC-P4C on forming macropinosomes. Representative image for n = 25 macropinosomes. Scale bar: 1 µm. d Localization dynamics of Phafin2 in relation to the PtdIns4P probe SidC-P4C. n = 25 macropinosomes, mean + 95% CI. e Sequential images showing the synthetic coincidence sensor SidM-FYVE(Phafin2) and the PtdIns3P probe 2xFYVE on forming macropinosomes. Representative image for n = 25 macropinosomes. Scale bar: 1 µm. f Localization dynamics of SidM-FYVE(Phafin2) in relation to the PtdIns3P probe 2xFYVE. n = 25 macropinosomes, mean + 95% CI. g Sequential images showing the synthetic coincidence sensor SidM-FYVE(Phafin2)(R176A) and the PtdIns3P probe 2xFYVE on forming macropinosomes. Representative image for n = 25 macropinosomes. Scale bar: 1 µm. h Localization dynamics of SidM-FYVE(Phafin2) (R176A) in relation to the PtdIns3P probe 2xFYVE. n = 25 macropinosomes, mean + 95% CI. i Liposome flotation assays showing Phafin2, Phafin2(R53C) and Phafin2(R176A) binding to liposomes. Shown is the liposome fraction. Representative gel from n = 3 experiments. j Quantification of Phafin2, Phafin2(R53C) and Phafin2(R176A) binding to liposomes. n = 3 experiments, mean + 95% CI. One-way ANOVA with Tukey’s post-test. p = 0,0114 (WT), n.s. (R53C) and p = 0.0024 (R176A). *p < 0.05, **p < 0.01, n.s. not statistically significant. k Exemplary image showing the localization of Phafin2-GFP, Phafin2ΔC, Phafin2ΔC(R176A). Representative image for 20 cells each. Scale bar: 10 µm. l Treatment of cells expressing Phafin2ΔC before and after treatment with the PI4K inhibitor GSK-A1. Representative image for 81 cells. Scale bar: 10 µm. m Quantification of (l). Two-sided Fisher’s exact test, n = 81 cells, p = 0.0001. Statistics source data and detailed statistics for d, f, h, j and m are provided in this paper.

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