Annexin A4 senses membrane curvature in a density-dependent manner

Annexins (ANXs) are a family of peripheral membrane binding proteins which play a vital role in the maintenance and function of cellular membranes. These proteins are known to be part of the plasma membrane repair machinery where they are known to bind to negatively charged lipids in the cell membrane in a calcium dependent manner. The shape of the plasma membrane is known to be a regulator of protein density and thereby affects several biological functions of the cell such as exo-and endocytosis, cell motility, immune responses and also membrane repair. Membrane deformation and curvature sensing by proteins is a well described phenomenon which can assist in recruitment of specific proteins to certain regions in the cell and facilitate membrane bending. Following ruptures in the plasma membrane, calcium influx assist in the association of ANXs with the membrane around the ruptured area. Due to the expected increase in curvature at the damaged membrane site, it has been suggested that both membrane curvature and Ca2+ participate in the recruitment of ANXs. We have investigated the curvature sensing of ANXA4 in giant unilamellar vesicles (GUVs) by pulling high curvature membrane tethers from the vesicle surface using optical tweezers showing that ANXA4 recruitment increases with higher membrane curvature. We also describe an assay for determining protein density on the plasma membrane by utilizing ANXA4s property as a calcium dependent membrane binding protein. This new assay allows us to investigate the effect of protein density on curvature sensing.