Detecting In-Situ oligomerization of engineered STIM1 proteins by diffraction-limited optical imaging.

Several signaling proteins require self-association of individual monomer units to be activated for triggering downstream signaling cascades in cells. Methods that allow visualizing their underlying molecular mechanisms will immensely benefit cell biology. Using enhanced Green Fluorescent Protein (eGFP) complementation, here I present a functional imaging approach for visualizing the protein-protein interaction in cells. Activation mechanism of an ER (endoplasmic reticulum) resident Ca2+ sensor, STIM1 (Stromal Interaction Molecule 1) that regulates store-operated Ca2+ entry in cells is considered as a model system. Co-expression of engineered full-length human STIM1 (ehSTIM1) with N-terminal complementary split eGFP pairs in mammalian cells fluoresces to form 'puncta' upon a drop in ER lumen Ca2+ concentration. Quantization of discrete fluorescent intensities of ehSTIM1 molecules at a diffraction-limited resolution revealed a diverse set of intensity levels not exceeding six-fold. Detailed screening of the ehSTIM1 molecular entities characterized by one to six fluorescent emitters across various in-plane sections shows a greater probability of occurrence for entities with six emitters in the vicinity of the plasma membrane (PM) than at the interior sections. However, the number density of entities with six emitters was lesser than that of others localized close to the PM. This finding led to hypothesize that activated ehSTIM1 dimers perhaps oligomerize in bundles ranging from 1-6 with an increased propensity for the occurrence of hexamers of ehSTIM1 dimer units close to PM even when its partner protein, ORAI1 (PM resident Ca2+ channel) is not sufficiently over-expressed in cells. The experimental data presented here provide direct evidence for luminal domain association of ehSTIM1 monomer units to trigger activation and allow enumerating various oligomers of ehSTIM1 in cells.