Fig. 3: Mutational analysis of Phafin2. | Nature Communications

Fig. 3: Mutational analysis of Phafin2.

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

Fig. 3

a Mutational analysis of Phafin2. GFP fusions of Phafin2 truncation and point mutants were tested for their subcellular localization. b Protein-lipid binding assay of Phafin2 PH and FYVE domains. c Sequential images of a macropinosome showing recruitment dynamics of Phafin2 (WT) and Phafin2 with a defective PH domain (Phafin2(R53C)). Representative image for n = 6 macropinosomes. Scale bar: 1 µm. d Localization dynamics of Phafin2 and Phafin2 with a defective PH domain (R53C). n = 6 macropinosomes, mean + 95% CI. Phafin2 R53C is unable to localize to newly formed macropinosomes, whereas the second, endosomal localization is unaffected. e Sequential images of a macropinosome showing recruitment dynamics of Phafin2 (WT) and Phafin2 with a defective FYVE domain (Phafin2 (R176A)). Representative image for n = 15 macropinosomes. Scale bar: 1 µm. f Localization dynamics of Phafin2 and Phafin2 with a defective FYVE domain (R176A). n = 15 macropinosomes, mean + 95% CI. Mutation of the FYVE domain abolishes all membrane localization. g Sequential images showing Phafin2 and the PtdIns3P probe mCherry-2xFYVE on a macropinosome. Representative image for n = 20 macropinosomes. Scale bar: 1 µm. h Localization dynamics of Phafin2 in relation to the PtdIns3P probe mCherry-2xFYVE. n = 20 macropinosomes, mean + 95% CI. i Sequential images showing Phafin2 and the PtdIns(3,4,5)P3 probe AKT-PH on a macropinosome. Representative image for n = 19 macropinosomes. Scale bar: 1 µm. j Localization dynamics of Phafin2 in relation to the PtdIns(3,4,5)P3 probe AKT-PH. n = 19 macropinosomes, mean + 95% CI. k Sequential images showing Phafin2 and the PtdIns(3,4)P2 probe TAPP-2xPH on a macropinosome. Representative image for n = 20 macropinosomes. Scale bar: 1 µm. l Localization dynamics of Phafin2 in relation to the PtdIns(3,4)P2 probe TAPP-2xPH. n = 20 macropinosomes, mean + 95% CI. m Treatment of Phafin2-GFP expressing cells with the VPS34 inhibitor SAR-405 does not affect the first localization, but abolishes the second localization phase (n = 51 macropinosomes each, mean + 95% CI). n Overexpression of the PI3 phosphatase MTM1, but not of the catalytically inactive MTM1 (C375S) displaces Phafin2 from macropinosomes (n = 75 (WT) and 69 (C375S) cells). Two-sided Fisher’s exact test, p < 0.0001. Statistics source data for c, e, i, k, and m are provided in this paper.

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