Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca 2+ store depletion

Abstract

Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca 2+ sensor that responds to ER Ca 2+ store depletion and activates Ca 2+ channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca 2+ store depletion. These oligomers rapidly dissociated when ER Ca 2+ stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER–PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca 2+ store depletion but failed to form puncta at ER–PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 μm to reach ER–PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca 2+ gradients. Together, our live-cell measurements dissect the STIM1 ER-to-PM signaling relay into four sequential steps: ( i ) dissociation of Ca 2+ , ( ii ) rapid oligomerization, ( iii ) spatially restricted translocation to nearby ER–PM junctions, and ( iv ) activation of PM Ca 2+ channels.