Close-up of vesicular ER exit sites by volume electron imaging using FIB-SEM

The architecture of ER exit sites (ERES), the first sites of membrane remodeling in protein secretion, remains unclear, with descriptions ranging from vesicular clusters to extended tubular structures. We addressed this divergence by visualizing ERES in cells not overexpressing secretory cargo using large-scale volume-focused ion beam scanning EM (FIB-SEM) after high-pressure freeze substitution. Automated segmentation in EM (ASEM), our 3D U-Net pipeline trained with sparsely labeled 50–70-nm COPI vesicles near the Golgi, accurately detected them in HeLa, SVG-A, and iPSC-derived neurons. Using the same model, we identified abundant clusters of ∼5–40 larger vesicles (∼65–85 nm) confined within ∼250 nm³ regions adjacent to flattened ER domains, consistent with vesicular ERES. Similar assemblies also appeared alongside tubular networks and varicosities extending from enlarged ER domains, previously described as the sole ERES in HeLa cells. These findings reveal that vesicular ERES are widespread and morphologically diverse, resolving longstanding contradictions in early secretory pathway organization.