Fig. 3: Generation of PI(4,5)P2 asymmetric vesicles with plasma membrane-like lipid composition. | Nature Communications

Fig. 3: Generation of PI(4,5)P2 asymmetric vesicles with plasma membrane-like lipid composition.

From: Plasma membrane transbilayer asymmetry of PI(4,5)P2 drives unconventional secretion of Fibroblast Growth Factor 2

Fig. 3: Generation of PI(4,5)P2 asymmetric vesicles with plasma membrane-like lipid composition.The alternative text for this image may have been generated using AI.

A Large unilamellar vesicles (LUVs) were prepared with 5 mol% PI(4,5)P2 or 5 mol% PI(4)P in plasma membrane (PM) like background. These LUVs were subjected to kinase reaction or mock reaction (without PIP5K1C) or left untreated. After the reaction, vesicles were washed and incubated with FGF2-Halo for 3 h, and unbound protein was removed by centrifugation before analysis using a sedimentation assay. The bound fraction of FGF2-Halo was analyzed by SDS-PAGE and detected with Coomassie staining. The gel was imaged using EPSON 4870 scanner. Data were normalized to 5 mol% PI(4,5)P2 within each experiment. Mean values with standard deviation for 3 biological replicates are presented. For statistical tests, ordinary one-way ANOVA was performed in Prism. Not significant (ns) P > 0.5, * P < 0.05, *** P < 0.001, **** P < 0.0001. Data distribution was assumed to be normal. B Giant unilamellar vesicles (GUVs) were prepared with 5 mol% PI(4,5)P2 or C 5 mol% PI(4)P along with membrane marker Rhodamine-PE, in PM like background. GUVs were subjected to kinase reaction or left untreated. After the kinase reaction, vesicles were washed to remove unreacted components or damaged vesicles. They were incubated with FGF2-GFP for 1 h and imaged with confocal microscope to access the enzymatic conversion of PI(4)P to PI(4,5)P2. Representative confocal images of FGF2-GFP binding to GUVs and their binding intensity profile with and without kinase reaction are shown (n = 3). D To test the asymmetry in GUVs, vesicles with 5 mol% PI(4,5)P2 or E 5 mol% PI(4)P along with membrane marker Rhodamine-PE, in PM like background, were immobilized on glass bottom ibidi chambers. Vesicles were monitored after the addition of Mg2+ + ATP + PIP5K1C and small tracer dye Alexa-647 in real time for 15 min using confocal microscope. Each time frame is separated by 1 min time. First slice was recorded at 1.5 min and last time slice was recoded at 15 min. Representative confocal images of GUVs with kinase reaction remains intact during the entire course of kinase reaction. Source data are provided as a Source Data file.

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