Date Published: March 25, 2019
Publisher: Public Library of Science
Author(s): Prasanna Srinivasan, Laszlo Csernoch.
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.
Specific interaction between STIM1 and Calcium Release-Activated Calcium (CRAC) channel, ORAI1 invokes calcium signaling in cells by store-operated calcium entry (SOCE). STIM1-ORAI1 coupling machinery regulates Ca2+ influx in cells, which is critical for controlling several short and long-term functions [1,2]. STIM1 is a single pass ER membrane resident protein with a Ca2+ sensor inside the ER lumen and an extendable actuator in the cytoplasm. In the inactive state, the actuator domains fold back to form a compact structure. During SOCE, the luminal domains of STIM1 sense Ca2+ drop inside the ER lumen and transmit the information to the cytosolic actuator domains. The cytosolic fragment then takes an elongated conformation eventually leading to oligomerization and translocation to ER-PM junctions for gating ORAI1 to elicit Ca2+ influx. Fig 1A shows a schematic representation of amino acid positions and various domains of human STIM1 (herein referred to as ‘hSTIM1’). Structural details about intramolecular interactions , functional mutants , models for transmembrane helical packing [4,5], structure of EF-SAM in Ca2+ bound state , the plausible mechanism of Ca2+ sensing by EF-SAM  and the molecular structure of CAD/SOAR  domain have almost fully unravelled the conformational dynamics of STIM1 from the inactive to active state.
Complementation of split GFP reporters (and its variants) was already used to visualize protein-protein interaction in various eukaryotic systems including mammalian cells. Association of several split GFP pairs was known to fluoresce [16–19] but their fluorescence dictated by the self-assembly and maturation of split pairs apparently vary with the split architecture . This technique involves expressing complementary eGFP domains fused to the interacting proteins, which fluoresce when the interacting partners are stably bound. Full-length hSTIM1 molecules when carefully engineered using complementary eGFP reporters (ehSTIM1) fluoresce upon self-association of luminal domains driven by low Ca2+ condition inside ER lumen. This simple approach will confirm that luminal domains interact together if ehSTIM1 has to be activated. Secondly, fluorescent intensity provides a direct readout to enumerate ehSTIM1 oligomerization states after a drop in ER Ca2+ concentration.
Taken together, this work confirms three findings; (i) luminal domains of engineered hSTIM1 monomer units interact triggering ehSTIM1 activation after a drop in Ca2+ concentration inside ER lumen, (ii) Activated ehSTIM1 occurs in a diverse set of oligomeric states ranging from 1–6 dimer units with increased probability of occurrence of hexamers near the PM (iii) The number density of hexameric species is lower than that of other species. Multiple ORAI1 gating states defined by the modulation of the binding affinities with ehSTIM1 can influence the channel currents as observed in Orai1 concatemers . Although these Ca2+ currents are very small, they are diverse secondary messengers capable of regulating various several cellular functions–cell division, gene expression, apoptosis, etc. Hence, spatial mapping of the distribution of the ehSTIM1 oligomers and quantitating the molecular density of PM targeted species are indispensable. Furthermore, there has been an intrinsic interest in the development of small molecule drugs for modulating the CRAC channel activity but most candidates have very poor selectivity . Enumerating the number density of ehSTIM1 oligomer species in the vicinity of the PM will improve our understanding to engineer efficient modulators of STIM1-ORAI1 coupling machinery. Also, the results presented here demonstrate the potential of live functional imaging to visualize protein-protein interactions at a diffraction-limited resolution in cells. The relevant molecular engineering and the accompanying functional imaging approaches can also be applied for dissecting other signaling pathways in cells.