Endocytic Crosstalk: Cavins, Caveolins, and Caveolae Regulate Clathrin-Independent Endocytosis
Figure 2
Caveolar proteins specifically down-regulate CLIC endocytosis.
(A) In CAV1−/− MEFs transiently expressing CAV1-YFP and Cavin-1-GFP respectively, internalization assay was performed with anti-CD44 mAb and Tfn-647 for 2 min at 37°C. Cells were acid washed prior to fixation, and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. Arrows indicate CD44-labeled puncta. (B,C) 40–50 cells of each transfection from (A) were quantified for normalized fluorescent intensity of endocytic markers. (D) Cavin-1−/− MEFs were transiently transfected with Cavin-1-GFP, and uptake and quantification of internalized anti-CD44 mAb and Tfn-647 were performed as mentioned in (A,B). (E,F) CAV1−/− MEFs were transiently transfected with Myc-CAV2 and RFP-CAV3 respectively. Uptake and quantification of internalized anti-CD44 mAb and Tfn-647 were performed as mentioned in (B). (G) In CAV1−/− MEFs transiently expressing GFP vector, CAV1-YFP, and Cavin-1-GFP respectively, constitutive internalization of HRP (10 µg/ml) was performed for 2 min at 37°C followed by DAB reaction on live cells. Cells were fixed, embedded, and sectioned. Quantification of HRP-labeled CLICs per cell, using 16-20 cells from each transfection, is shown. For more images representing experimental variation obtained for uptake analysis, performed in untransfected and CAV1-YFP/Cavin-1-GFP-expressing CAV1−/− MEFs, see supporting information Figure S12, Figure S13, and Figure S14. In (B–G) data represent mean ± SEM of three independent experiments. *p<0.05,***p<0.0005,****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.