Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.
Clathrin-dependent endocytosis is one of the mechanisms used by cells to internalize specific proteins (cargo) from their surface. First, the cargo interacts with adaptor proteins that help cluster them in the cell's outer membrane, called the plasma membrane. This causes the protein clathrin to assemble into a lattice at the cytosolic side of the plasma membrane and deform the membrane into a pit. The pit grows deeper over time as more clathrin molecules assemble, eventually resulting in a deeply invaginated clathrin-coated pit that encloses the cargo to be taken up by the cell. The clathrin-coated pit then pinches off inside the cell in a process called fission to form a bubble-like structure called a vesicle, which transports the molecule to its destination.
The deep invagination of clathrin-coated pits that leads to fission is assisted by actin, a protein that assembles into filaments that are suggested to generate the forces needed for this process. Many other factors are also involved. One of them is epsin, the collective name for a family of three very similar proteins in mammalian cells. Epsin binds to several other proteins implicated in clathrin-dependent endocytosis, including clathrin itself, and to plasma membrane proteins specifically ‘tagged’ for internalization. In addition, a portion of the epsin molecule can insert into the plasma membrane and help it to curve, which is important for forming the invaginated pit. However, due to the number of possible functions epsin could perform, its main role has remained elusive.
Messa et al. created mouse cells that lack all three epsin proteins. Although these cells can form clathrin-coated pits, they struggle to develop into vesicles. The normal linking of the actin filaments to the clathrin coat does not occur, and another protein called Hip1R that also participates in clathrin-mediated endocytosis and links clathrin to actin, no longer accumulates at the clathrin-coated pits. Messa et al. also find that epsins can bind directly to actin. Overall, these results suggest that a main role of epsin is to help actin interact with the clathrin-coated pits and generate the force required for a pit to develop into a vesicle. However, epsin also performs many other roles, including recruiting a membrane protein (a so-called SNARE) that directs the fate of the vesicle to the clathrin-coated pit.
Additionally, Messa et al. find that cells lacking all three epsins have problems dividing correctly. More research is required to establish whether this effect is also due to epsin's interaction with the cell's actin cytoskeleton.