ABSTRACT Cell migration is critical for processes such as tissue development and repair. A key aspect of mesenchymal cell migration is the formation and turnover of cellular adhesions, multilayered protein complexes that link the actin cytoskeleton to the extracellular matrix. Adhesions must be turned over to enable cell migration, and defects in adhesion disassembly underlie developmental defects in some tissues and cancer cell movement in metastasis. Local intracellular calcium signaling by the endoplasmic reticulum (ER) precedes adhesion disassembly, but the signals that initiate calcium signaling, as well as the mechanisms for how the ER is spatiotemporally targeted to focal adhesions destined to turn over, are not known. This proposal presents preliminary data revealing a novel ER-to-actin tether, calmin/CLMN, that links the ER to the cytoskeleton to influence cellular adhesion dynamics and cell migration. Mechanistically, CLMN has actin-binding domains and localizes to ER tubules; in addition, CLMN localizes to a subpopulation of cellular adhesions. CLMN-depleted migrating cells exhibit a greater number of cellular adhesions and migrate more slowly than controls. The driving hypothesis of this proposal is that CLMN mediates tethering of the ER to actin and facilitates adhesion turnover by positioning the ER near actin to spatially localize calcium influx, in turn enabling cell migration. The research proposed in this fellowship will use molecular biology, biochemical, and advanced microscopy techniques to determine the mechanisms for localization and actin targeting by CLMN. Furthermore, the proposed research will use live cell imaging of CLMN, ER, adhesion markers, and intracellular calcium biosensors in migrating cells to determine a) the sequence of events in recruiting ER to adhesions to facilitate migration and b) how this sequence is perturbed in the absence of CLMN. This work will fill a critical knowledge gap of how the ER interacts with the actin cytoskeleton and spatially coordinates ER-actin crosstalk for cell migration. Taken together, this research will also further our understanding of the mechanisms of adhesion disassembly during cell migration and inform changes that occur during normal and diseased tissue development. The proposed research and training plan will also prepare the applicant for a productive scientific career as an independent investigator leading studies of how organelle-cytoskeleton interactions influence cell physiology.