Intravital single-molecule microscopy reveals that basolateral membrane domains in salivary gland acinar cells are built on deeply folded membrane infolds maintained by continuous NMII and Tropomyosin 3.1 turnover, and that β-adrenergic stimulation accelerates NMII molecular exchange to drive membrane unfolding and cell expansion through kinetic regulation rather than spatial reorganization of the cytoskeleton.

Understanding how cells regulate plasma membrane architecture inside intact living organs in a live animal has been limited by the inability to directly measure molecular dynamics in vivo. Here we introduce intravital single-molecule microscopy (iSiMM), an imaging approach that enables tracking of individual, endogenously expressed cytoskeletal components at the plasma membrane in live mice. Applying iSiMM to murine acinar secretory cells, we identify discrete basolateral membrane domains built on deeply folded membrane infolds that function as a pre-existing membrane reservoir. Single-molecule measurements reveal continuous, regulated molecular turnover within these domains. Physiological stimulation accelerates cytoskeletal exchange promoting rapid membrane unfolding and cell expansion. Together, these findings establish iSiMM as a general strategy for probing molecular kinetics underlying dynamic cellular behaviors in intact organs.

Intravital single-molecule microscopy enables direct measurement of molecular kinetics underlying dynamic cellular behaviors in intact living organs.