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 to form clathrin-coated vesicles (CCVs). This rapid, nanoscale process involves significant changes to plasma membrane shape. We previously found heterogeneity in CCV formation: some vesicles form with simultaneous membrane bending and clathrin assembly (constant curvature), whereas others form with membrane bending after the accumulation of flat clathrin lattices (flat to curved). These architectural dynamics could be influenced by osmotic pressure, membrane stiffness, 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 stably expressing CLCa-iRFP713-EGFP with latrunculin A (LatA) to inhibit actin polymerization and found the frequency of CCVs increased significantly, especially for short-lifetime CCVs. The proportion of vesicles following the flat-to-curved model was reduced, the membrane curved sooner after clathrin recruitment, and forming vesicles were less stable in x-y compared with control. Next, we disrupted actin branching with CK869 and found the frequency of CCVs decreased. There was increased delay between membrane invagination and clathrin recruitment, increased x-y plane stability of forming vesicles, and increased proportion of vesicles following the flat-to-curved model compared with control. To address these opposing results, we considered the role of membrane tension. When membrane tension was decreased with high osmolality media, CCV formation mirrored the LatA-treated group, except the x-y stability of forming vesicles was unchanged. This suggests the increased CCV frequency following actin filament disruption may be due to reduced membrane tension. We conclude actin polymerization promotes the bending of flat clathrin, whereas actin branching promotes constant curvature.