Figure 4. Dissociation of the cavin coat upon cell swelling induced by hypo-osmotic treatment.

(A) Histogram of apparent single-molecule sizes for cavin1-GFP before (green) and after (grey) membrane stretching induced by hypo-osmotic treatment. The size of observed cavin1 oligomers decreases from 60 nm to 20 nm. (B) Histogram of single-molecule coincidence for cavin1-GFP and cavin1-Cherry after hypo-osmotic treatment. We observe that solubilized cavin1 remains oligomeric as all bursts contain both GFP and Cherry fluorophores. (C) Burst brightness distribution for cavin1-GFP (green) and cavin1-Cherry (red) in oligomers released from the membrane upon hypo-osmotic treatment. Oligomers contain GFP and Cherry in equal amounts; the total fluorescence indicates that the sub-oligomers are typically made of 8–10 cavin1 proteins and hence significantly reduced in size compared to the isotonic conditions (Figure 1). (D) Histogram of single-molecule coincidence between cavin2 and cavin1 after hypotonic treatment. Data show that all cavin2 are bound to cavin1 but approximately 15% of the oligomers contain cavin1 only. (E) Histogram of single-molecule coincidence between cavin3 and cavin1 after membrane stretching. Data show that all cavin3 are bound to cavin1 and approx. 20% of cavin1-only oligomers are observed. (F) Histogram of single-molecule coincidence between cavin2 and cavin3 after membrane stretching. The absence of coincidence suggests that while cavin2 and cavin3 can localize to the same caveolae, their release from the membrane during stretch causes them to separate into two different subcomplexes. (G) Histogram of apparent single-molecule sizes obtained after hypo-osmotic treatment for cavin1-GFP, cavin2-GFP and cavin3-GFP, in the presence of co-expressed cavin1-Cherry. The measurements indicate that the cavin1-cavin1 and cavin1-cavin3 subcomplexes are very similar in size (average of 20 nm), but the apparent size of the cavin1-cavin2 subcomplex is higher with an average of 30 nm. (H) Model of cavin1-cavin1, cavin1-cavin2 and cavin1-cavin3 subcomplexes dissociation from CAV1 domains upon membrane stretching mediated by cell swelling.

DOI: http://dx.doi.org/10.7554/eLife.01434.013

Figure 4—figure supplement 1. Pull-down analysis of the effect of membrane stretching on the association between cavin members.

Experiments are performed in MDCK cells, which have endogenous levels of cavin1 and CAV1. MDCK cells were transfected with equal amounts of Cavin 1-GFP (C1-GFP) and Cavin2-Cherry (C2-C), C1-GFP and Cavin 3-Cherry (C3-C), C2-GFP and C3-C, Cavin 3-GFP (C3-GFP) and C2-C and Cavin1-Flag with C3-GFP and C2-C. Cells were then treated with 100% DMEM (Iso-osmotic; Iso) or with DMEM diluted in 90% sterile water (Hypo-osmotic; Hypo) for 20 min at 37°C. Post nuclear supernatant (soluble cytoplasmic fraction) were prepared by extensive washing of cells in PBS. Cells were then scraped into PBS containing protease and phosphatase inhibitors followed by mechanic disruption by syringe lysis. Cells were then pelleted at 2000 rpm, 10 min at 4°C. 120th of the supernatant was retained as the starting material (B). The remaining supernatant was mixed with 20 μl of prewashed GFP Trap beads for 30 min at 4°C on a rotating wheel. The beads were pelleted and washed three times in PBS supplemented with protease and phosphatase inhibitors and were boiled in 4× Sample buffer for 75°C for 2.5 min to preserve the fluorescence. Sample were separated by SDS-PAGE and the fluorescence corresponding to each of the overexpressed GFP or Cherry tagged Cavin construct was detected and quantification in the gel using the BioRad ChemiDoc MP Imaging System.

DOI: http://dx.doi.org/10.7554/eLife.01434.014