Spatial proteomics of ER tubules reveals CLMN, an ER-actin tether at focal adhesions that promotes cell migration

The endoplasmic reticulum (ER) is structurally and functionally diverse, yet how its functions are organized within morphological subdomains is incompletely understood. Utilizing TurboID-based proximity labeling and CRISPR knock-in technologies, here we map the proteomic landscape of the human ER and nuclear envelope. Spatial proteomics reveals enrichments of proteins into ER tubules, sheets, and nuclear envelope. We uncover an ER-enriched actin-binding protein, Calmin (CLMN), and define it as an ER-actin tether that localizes to focal adhesions adjacent to ER tubules. CLMN depletion perturbs focal adhesion disassembly, actin dynamics, and cell movement. Mechanistically, CLMN-depleted cells also exhibit defects in calcium signaling near ER-actin interfaces, suggesting CLMN promotes calcium signaling near adhesions to facilitate their disassembly. Collectively, we map the sub-organelle proteome landscape of the ER, identify CLMN as an ER-actin tether, and describe a non-canonical mechanism by which ER tubules engage actin to regulate cell migration.