Stepwise membrane binding of extended synaptotagmins revealed by optical tweezers

Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca²⁺- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER–PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca²⁺- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides. [Figure not available: see fulltext.]