Integrating Lateral Super-Resolution and Axial Progression Reveals Distinct Clathrin Pit Formation Pathways

Clathrin-mediated endocytosis (CME) relies on the dynamic assembly and remodeling of clathrin coats to drive membrane curvature and vesicle formation at the plasma membrane. Although live-cell fluorescence microscopy has provided critical insights into the timing and molecular composition of endocytic events, directly linking the nanoscale lateral organization of clathrin coats to their three-dimensional progression in real time has remained challenging. Structural approaches such as electron microscopy provide detailed snapshots of clathrin architecture but are inherently static, whereas axial TIRF-based methods report membrane-proximal position with limited lateral resolution. Here, we introduce variable-angle total internal reflection fluorescence structured illumination microscopy (vaTIRF-SIM), a live-cell imaging strategy that integrates lateral super-resolution with dynamic axial sensitivity near the plasma membrane. By combining TIRF-SIM with controlled variation of the evanescent field penetration depth, vaTIRF-SIM enables simultaneous visualization of clathrin coat architecture and relative axial displacement with high spatial and temporal resolution. Applying this approach to de novo clathrin-coated pits reveals coordinated lateral growth and progressive axial advancement from early stages of pit formation through maturation, consistent with early curvature generation that intensifies over time. Extending this analysis to clathrin plaques uncovers two distinct plaque-associated endocytic behaviors: slowly maturing pits that originate at plaque peripheries and progress similarly to de novo pits and rapid plaque subdomain internalization events marked by accelerated axial progression. Together, these results establish vaTIRF-SIM as an approach that, for the first time, enables direct real-time coupling of nanoscale clathrin coat organization with axial progression during CME in living cells, demonstrated here in genome-edited SUM-159 cells expressing AP2-EGFP from the endogenous locus.