Plasma Membrane ORAI1 and Septin Organization during Calcium Signaling

Abstract

The STIM-ORAI-mediated calcium release-activated calcium channel, or CRAC channel, is a key source of calcium influx that sustains intracellular Ca2+ signaling and effector function in T lymphocytes. STIM1 in the ER membrane senses depletion of ER calcium stores and moves to ER-plasma membrane junctions, then recruits plasma membrane ORAI channels to the junctions and directly gates the channels. We have shown previously that septins are necessary for efficient STIM1-ORAI1 cluster formation following store depletion. Septins are known to specify diffusive barriers in the plasma membrane and to serve as scaffolds to recruit signaling proteins, but their detailed role in calcium signaling remains to be characterized. Here we utilize live-cell super-resolution microscopy and single-molecule tracking to map ORAI1 relative to STIM1 and membrane-localized septins. We further investigate the trajectories and mobility of ORAI1 molecules in the context of septin-deficient cells or when the interaction between ORAI1 and STIM1 is perturbed by mutagenesis. These analyses at high spatial and temporal resolution are providing new insight into the modulation of STIM-ORAI signaling by septins and the local plasma membrane environment.