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 knockin technologies, we map the proteomic landscape of the human ER network. Sub-organelle proteomics reveals enrichments of proteins into ER tubules, sheets, and the 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. Mechanistically, we find that CLMN depletion perturbs adhesion disassembly, actin dynamics, and cell movement. CLMN-depleted cells display decreased polarization of ER-plasma membrane contacts and calcium signaling factor STIM1 and altered calcium signaling near ER-actin interfaces, suggesting that CLMN influences calcium signaling to facilitate F-actin/adhesion dynamics. 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.