Fig. 1
From: Membrane curvature induced by proximity of anionic phospholipids can initiate endocytosis
PtdSer is redistributed upon cholesterol extraction. a Distribution of GFP-LactC2 in HeLa cells that were untreated or treated with 10 mM mβCD for 30 min. Images in A are representative of at least 4 experiments of each type. b Quantitation of the ratio of PM to cytoplasmic GFP-LactC2 from A, n = 40 cells. c Quantitation of GST-LactC2 to liposomes containing egg-PtdCho, dansyl-PtdEtn, 0% or 20% PtdSer, with 0% or 20% cholesterol. The term (F ÷ Fb) − 1 refers to the observed FRET resulting from the proximity of dansyl-PtdEtn in the liposome to tryptophan residues in GST-LactC2. d Comparison of the total PtdSer content of control and mβCD-extracted cells. PtdSer extracted from cells was reacted with fluorescamine and quantified by measuring fluorescence intensity after separation by TLC, then normalized to total protein of the cells.Data are means ± s.e.m from three separate experiments. e HeLa cells expressing mRFP-LactC2 (red) were treated with either 10 mM mβCD (left) or 10 μM ionomycin (right) for 10 min and subsequently incubated with Alexa-488-labeled Annexin V (green) to visualize exofacial PtdSer. Insets: pre-treatment images for each. f Comparison of the PtdSer content of plasma membranes isolated from control and mβCD-treated cells using polycationic beads. Plasma membranes were isolated as described in Supplementary Fig.1, and PtdSer quantified as in D. Data are means ± s.e.m of 4 separate experiments. g Subcellular distribution of PtdSer in control (black bars) and mβCD-extracted cells (open bars), determined by the colocalization using Pearson’s correlation coefficient of the GFP-LactC2 probe with the following organellar markers: PH-PLCδ, (PM marker); Sec61, (endoplasmic reticulum marker); GalT, (Golgi complex); MitoTracker, (mitochondria); Rab5, Rab11 and LAMP1, used as markers of early endosomes, recycling endosomes and late endosomes/lysosomes, respectively. Data represent the means ± s.e.m of for 35 independent cells collected from three separate days. NS = not significantly different, *p < 0.05, **p < 0.01 and ***p < 0.005; as determined using a two-tailed t-test. The same test and designation of p-value is used throughout the remaining figures. Scale bar = 10 µm in all images
