In situ mapping of late-stage biomechanical coordination during clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) requires precise coordination between membrane deformation and force-generating protein assemblies, yet how these forces are dynamically organized in living cells remains poorly defined. Using multi-dimensional single particle tracking (SPT), we indirectly visualize the mechanical motions of individual clathrin-coated pits during the late stages of CME. We uncover a temporally ordered sequence of rotational behaviors that reflect distinct modes of membrane remodeling preceding vesicle scission. While dynamin-dependent in-plane twisting is a common feature of productive CME events, an additional out-of-plane rotational deformation, hereafter referred to as a “swing” motion, is selectively observed at a subset of endocytic sites that recruit actin. This mechanical heterogeneity correlates with differences in membrane deformation and scission efficiency, rather than representing an obligatory step in CME. By capturing these force-generating transitions in situ and under physiological conditions, our work provides a dynamic, biophysical framework for understanding how endocytic protein machines remodel membranes in living cells. This study uses single particle tracking to map clathrin-mediated endocytosis in living cells, revealing actin-associated swing motion and dynamin-driven twisting as coordinated mechanical steps.