The role of EHD proteins in caveolae, and the role of caveolae in adipocytes
Caveolae are 50-60 nm flask-shaped invaginations of the plasma membrane that protect the plasma membrane from damage under stretch forces. They are highly abundant in cells that experience high levels of stress forces such as adipocytes, endothelial cells and muscle cells. Caveolae are generated by the oligomerisation and association of caveolin and cavin proteins, which form the caveolar coat complex at the caveolar bulb and are progressively well characterised. However, less is known about the proteins that localise to the caveolar neck. Using the CRIPSR/Cas9 system to generate gene knock-in and knockout cell lines, the role of EHD proteins at caveolae was investigated. It was found that, in addition to EHD2 being at the neck, both EHD1 and EHD4 were also present. The recruitment of other EHD proteins was markedly increased in the absence of EHD2. This functional redundancy was confirmed by the generation of EHD1, 2 and 4 triple knockout cell lines, which displayed two striking sets of phenotypes. Firstly, the characteristic higher-order clusters of caveolae are lost in the absence of EHD proteins. And secondly, caveolae are destabilised and the plasma membrane is more likely to rupture when the EHD1,2,4 knockout cells are subjected to cycles of stretch forces. The data identify the first molecular components that cluster caveolae into a membrane ultrastructure that potentially extends stretch buffering capacity. A second series of experiments tested different ideas about the function of caveolae in adipocytes. The insulin receptor and CD36 were found to at most partially colocalise with caveolae, and the role of caveolae in regulating signalling processes remains unclear. In contrast, the plasma membrane of adipocytes without caveolae is clearly more prone to rupture, confirming a mechanoprotective function.
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