Michael Wm. Roe

Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca²⁺ signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca²⁺-binding protein, with Ca²⁺-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca²⁺ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca²⁺ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.

Resveratrol, a naturally occurring polyphenol found in some fruits and especially in grapes, has been reported to provide diverse health benefits. Resveratrol’s mechanism of action is the subject of many investigations, and some studies using the ratiometric calcium indicator Fura-2 suggest that it modulates cellular calcium responses. In the current study, contradictory cellular calcium responses to resveratrol applied at concentrations exceeding 10 μM were observed during in vitro imaging studies depending on the calcium indicator used, with Fura-2 indicating an increase in intracellular calcium while Fluo-4 and the calcium biosensor YC3.60 indicated no response. When cells loaded with Fura-2 were treated with 100 μM resveratrol, excitation at 340 nm resulted in a large intensity increase at 510 nm, but the expected concurrent decline with 380 nm excitation was not observed. Pre-treatment of cells with the calcium chelator BAPTA-AM did not prevent a rise in the 340/380 ratio when resveratrol was present, but it did prevent an increase in 340/380 when ATP was applied, suggesting that the resveratrol response was an artifact. Cautious data interpretation is recommended from imaging experiments using Fura-2 concurrently with resveratrol in calcium imaging experiments.