Abstract
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.