Proximity ligation assay visualizes in cultured neurons the interaction between endogenous ORAI1 and STIM proteins
Calcium sensors STIM1 and STIM2, located in ER, and calcium channel forming protein ORAI1 are involved in the store-operated calcium entry (SOCE). The process relies on extracellular calcium influx through the plasma membrane channels. In non-excitable cells STIM interaction with ORAI1 is a crucial element of SOCE, but in neurons its mechanism remains unclear. We showed earlier that STIM1 is likely involved in thapsigargin induced SOCE, while STIM2 is mostly active after EGTA-driven depletion of extracellular Ca2+ (Gruszczynska-Biegala et al. 2011). Depletion of calcium from ER increased number of puncta-like colocalization of YFP-STIM1 and ORAI1, but not of YFP-STIM2 and ORAI1. In contrast, reduction of extracellular calcium level triggered puncta formation for both YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1. In this work we aimed to determine whether after SOCE induction it is possible to detect complexes containing endogenous STIMs and ORAI1. We showed that in cultured rat cortical neurons STIM1 and STIM2 can interact with ORAI1 what can be observed by proximity ligation assay (PLA). Using PLA we were able to visualize fluorescent dots, which represent the site where two antibodies are bound: one against ORAI1 and another one against either STIM1 or STIM2. These dots identify likely the complexes between STIMs and ORAI1. The interaction between them was quantified and found to correlate well with the number of exogenous complexes formed under the same conditions. To confirm that observed PLA dots represent authentic STIMORAI1 complexes we use different pairs of anti-STIM and antiORAI1 antibodies. The positive findings will allow us to confirm that the interaction between endogenous STIMs and ORAI1 occurs in neurons during SOCE in vivo and to demonstrate the feasibility of the PLA technique with antibodies of low specificity.
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