Abstract

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.

Highlights

  • Specific interaction between STIM1 and Calcium Release-Activated Calcium (CRAC) channel, ORAI1 invokes calcium signaling in cells by store-operated calcium entry (SOCE)

  • The CRAC activation domain (CAD) or STIM1-ORI1 activating region (SOAR) and the polybasic (K ) tail are critical for the gating Ca2+ channel, ORAI1 (b) Three engineered hSTIM1 constructs namely, eGFPS1-hSTIM1, eGFPS2- hSTIM1 and enhanced Green Fluorescent Protein (eGFP)-hSTIM1 were used for functional imaging experiments. eGFPS1 and eGFPS2 represents 1-157aa and 158-240aa of an eGFP molecule respectively. eGFP-hSTIM1 is a positive control for the complementation assay. (c) Schematic of the self-assembly of split eGFP pairs by luminal domain association of eGFPS1-hSTIM1 and eGFPS2-hSTIM1 after Ca2+ drop inside ER lumen

  • A drop in the ER Ca2+ concentration causes the luminal domains of engineered eGFPS1-hSTIM1 and eGFPS2-hSTIM1 to approach thereby self-assembling the complimentary eGFP split pairs

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Summary

Introduction

Specific interaction between STIM1 and Calcium Release-Activated Calcium (CRAC) channel, ORAI1 invokes calcium signaling in cells by store-operated calcium entry (SOCE). Structural details about intramolecular interactions [3], functional mutants [4], models for transmembrane helical packing [4,5], structure of EF-SAM in Ca2+ bound state [6], the plausible mechanism of Ca2+ sensing by EF-SAM [7] and the molecular structure of CAD/SOAR [8] domain have almost fully unravelled the conformational dynamics of STIM1 from the inactive to active state In spite of this wealth of structural information, direct in-situ experimental evidence to confirm the association of luminal domains of hSTIM1 are fragmentary. In-situ cell-biology evidence [10,11,12] based on FRET (Forster Resonance Energy Transfer) assays were used to characterize the association of STIM1 luminal domains While these results provide an indication for the approaching of STIM1 luminal domains in cells, it is sufficiently not persuasive to confirm the EF-hand mediated hSTIM1 signaling in cells. I report activated ehSTIM1 dimers translocated to ER-PM junction can exist in several oligomeric states ranging from 1–6 dimers and these are characterized by intensity quantization at diffraction-limited optical resolution

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