Fig. 6: The Cavin1 DR domains are essential for caveola formation with CAV1.

a GFP-tagged Cavin1 and truncations (green) were expressed for 24 h in PC3 cells, fixed and immunolabelled for Caveolin1 (CAV1) (red). Full length Cavin1 forms typical caveola puncta, colocalising with CAV1 at the cell surface. Cavin1-ΔDR1 mutant expression leads to formation of tethered intracellular CAV1-positive clusters. Cavin1-ΔDR3 shows cytoplasmic and microtubule localisation. Images were collected using a Zeiss fast Airyscan microscope. Scale bar = 10 μm; enlarged boxes scale bar—5 μm. Fluorescence recovery after photobleaching (FRAP) analysis of GFP-Cavin1-ΔDR3 before (b) and after (c) nocodazole (10 µM) addition. Scale bar—5 μm. d In PC3 cells GFP-tagged Cavin1-ΔDR1 truncation shows colocalization with the early endosomal marker (EEA1) (red) and CAV1 (blue). Inset shows merge images of GFP-Cavin1-ΔDR1/EEA1 and GFP-Cavin1-ΔDR1/CAV1. Scale bar = 10 μm; enlarged boxes scale bar—5 μm. e GFP-tagged Cavin1 and Cavin1-ΔDR1 were visualised in PC3 cells by electron microscopy and labelling of GFP tagged proteins using APEX-GBP staining. Scale bar = 1 and 2 μm. Enlarged boxes scale bar—500 nm and 1 μm. f Live imaging of PC3 cells expressing Rab5a-mCherry with either GFP-Cavin1 or GFP-Cavin1-ΔDR1. Images were acquired one frame per 4 s and frame numbers are indicated in boxes. Arrows indicate mCherry/GFP signal co-localisation or separation event. Scale bar—2 μm.