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

Oct 31, 2025The Journal of cell biology

Detailed 3D imaging of small vesicle exit points in the cell’s protein transport system

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Regenerative Health on OpenScience ↗PubMed ↗DOI ↗

Abstract

Vesicular exit sites (ERES) are found to be abundant and morphologically diverse, with clusters of ∼5-40 larger vesicles located within ∼250 nm3 regions near flattened ER domains.

ER exit sites were visualized in cells without overexpressing secretory cargo using advanced imaging techniques.
Automated segmentation successfully detected 50-70-nm COPI vesicles in multiple cell types, including HeLa and iPSC-derived neurons.
Clusters of larger vesicles, measuring ∼65-85 nm, were identified near flattened ER regions, supporting the existence of vesicular ERES.
Similar structures were also observed alongside tubular networks and varicosities from enlarged ER domains, previously thought to be the only ERES in HeLa cells.
These observations indicate that vesicular ERES are not only common but also exhibit a range of morphologies, addressing previous inconsistencies in the understanding of the early secretory pathway.

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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.

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

Abstract

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