STIM1 (a Ca 2þ sensor in the endoplasmic reticulum (ER) membrane) and Orai1 (a pore-forming subunit of the Ca 2þ -release-activated calcium channel in the plasma membrane) diffuse in the ER membrane and plasma membrane, respectively. Upon depletion of Ca 2þ stores in the ER, STIM1 translocates to the ER-plasma membrane junction and binds Orai1 to trigger store-operated Ca 2þ entry. However, the motion of STIM1 and Orai1 during this process and its roles to Ca 2þ entry is poorly understood. Here, we report real-time tracking of single STIM1 and Orai1 particles in the ER membrane and plasma membrane in living cells before and after Ca 2þ store depletion. We found that the motion of single STIM1 and Orai1 particles exhibits anomalous diffusion both before and after store depletion, and their mobility-measured by the radius of gyration of the trajectories, mean-square displacement, and generalized diffusion coefficient-decreases drastically after store depletion. We also found that the measured displacement distribution is non-Gaussian, and the non-Gaussian parameter drastically increases after store depletion. Detailed analyses and simulations revealed that single STIM1 and Orai1 particles are confined in the compartmentalized membrane both before and after store depletion, and the changes in the motion after store depletion are explained by increased confinement and polydispersity of STIM1-Orai1 complexes formed at the ER-plasma membrane junctions. Further simulations showed that this increase in the confinement and polydispersity after store depletion localizes a rapid increase of Ca 2þ influx, which can facilitate the rapid activation of local Ca 2þ signaling pathways and the efficient replenishing of Ca 2þ store in the ER in store-operated Ca 2þ entry.

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Recently, we demonstrated the use of 31 P CODEX (Centerband-Only Detection of Exchange) NMR to measure lateral diffusion of phospholipids.Here we demonstrate the multiplexing capabilities of CODEX, i.e. simultaneous measurement of multiple phospholipid lateral diffusion coefficients, and compare the effects of polylysine and a model anti-microbial peptide, KL-14 (KKLLK KAKKL LKKL), on lateral diffusion of various anionic phospholipids.