RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells

• refSOFI maps protein-protein interactions in super-resolution in living cells
• BiFC-competent fragments of Dronpa were developed based on an enhanced variant
• Venus and corresponding BiFC fragments are suitable for SOFI/refSOFI
• Stimulating SOCE increases the number rather than size of STIM1/ORAI1 puncta

SummaryIt has become increasingly clear that protein-protein interactions (PPIs) are compartmentalized in nanoscale domains that define the biochemical architecture of the cell. Despite tremendous advances in super-resolution imaging, strategies to observe PPIs at sufficient resolution to discern their organization are just emerging. Here we describe a strategy in which PPIs induce reconstitution of fluorescent proteins (FPs) that are capable of exhibiting single-molecule fluctuations suitable for stochastic optical fluctuation imaging (SOFI). Subsequently, spatial maps of these interactions can be resolved in super-resolution in living cells. Using this strategy, termed reconstituted fluorescence-based SOFI (refSOFI), we investigated the interaction between the endoplasmic reticulum (ER) Ca2+ sensor STIM1 and the pore-forming channel subunit ORAI1, a crucial process in store-operated Ca2+ entry (SOCE). Stimulating SOCE does not appear to change the size of existing STIM1/ORAI1 interaction puncta at the ER-plasma membrane junctions, but results in an apparent increase in the number of interaction puncta.

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