The role of actin dynamics in vesicle formation during clathrin mediated endocytosis.

Clathrin-mediated endocytosis (CME) is an important internalization route for macromolecules, lipids, and membrane receptors in eukaryotic cells. During CME, the plasma membrane invaginates and pinches off forming a clathrin coated vesicle. We previously identified heterogeneity in this process with clathrin coated vesicles forming though multiple routes including simultaneous clathrin accumulation and membrane invagination (constant curvature; CCM) as well as membrane bending after accumulation of flat clathrin (flat to curved; FTC). The architectural dynamics of vesicle formation could be influenced by osmotic or confining pressure, membrane stiffness, fluid force, or cytoskeletal arrangement. Whether these biophysical factors regulate the heterogeneity of vesicle formation dynamics is not well understood. To address this, we investigated the interconnected roles of actin and membrane tension in CME using simultaneous two-wavelength axial ratiometry (STAR) microscopy with nanometer-scale axial resolution. First, we treated Cos-7 cells with latrunculin A (LatA) to inhibit actin polymerization and found the total number of clathrin coated vesicles increased significantly, short-lifetime curved events especially. The proportion of vesicles formed following the FTC model was reduced, the membrane curved sooner after clathrin recruitment, and vesicles were less stable in the x-y plane compared to control. Next, we disrupted actin branching by inhibiting Arp2/3 with CK-869. We found an increased delay between membrane invagination and clathrin recruitment, reduced number of curved events, increased vesicle stability and an increase in the FTC model compared to control. As loss of actin filaments also reduces membrane tension, we treated Cos7 with high osmolality to decrease membrane tension and observed similar result with LatA treated group except vesicle stability stayed unchanged. This suggested the increased curved events in LatA groups may result from reduced membrane tension. We conclude actin polymerization promotes FTC while actin branching promotes vesicle formation though the CCM.