EHD Proteins Cooperate to Generate Caveolar Clusters and to Maintain Caveolae during Repeated Mechanical Stress

Caveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. Here we make extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae. We show that EHD1, EHD2, and EHD4 are recruited to caveolae. Recruitment of the other EHDs increases markedly when EHD2, which has been previously detected at caveolae, is absent. Construction of knockout cell lines lacking EHDs 1, 2, and 4 confirms this apparent functional redundancy. Two striking sets of phenotypes are observed in EHD1,2,4 knockout cells: (1) the characteristic clustering of caveolae into higher-order assemblies is absent; and (2) when the EHD1,2,4 knockout cells are subjected to prolonged cycles of stretch forces, caveolae are destabilized and the plasma membrane is prone to rupture. Our data identify the first molecular components that act to cluster caveolae into a membrane ultrastructure with the potential to extend stretch-buffering capacity and support a revised model for the function of EHDs at the caveolar neck. •EHD1, 2, and 4 are all recruited to caveolae•EHD proteins promote the formation of clustered arrays of caveolae•EHD proteins are required for morphogenesis of caveolae under repeated stretch forces•Loss of EHD proteins increases stretch-induced damage to the plasma membrane Yeow et al. apply gene-editing approaches to show that members of the EHD protein family are recruited to plasma membrane invaginations termed caveolae. The EHD proteins act to promote clustering of caveolae into higher-order assemblies and also to protect the plasma membrane from rupture under stretch forces.