Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy

Abstract

Sigma-1 receptor (Sig1R) is an endoplasmic reticulum (ER)-related membrane protein, that forms heteromers with other cellular proteins. As the mechanism of action of this chaperone protein remains unclear, the aim of the present study was to detect and analyze the intracellular dynamics of Sig1R in live cells using super-resolution imaging microscopy. For that, the Sig1R-yellow fluorescent protein conjugate (Sig1R-YFP) together with fluorescent markers of cell organelles were transfected into human ovarian adenocarcinoma (SK-OV-3) cells with BacMam technology. Sig1R-YFP was found to be located mainly in the nuclear envelope and in both tubular and vesicular structures of the ER but was not detected in the plasma membrane, even after activation of Sig1R with agonists. The super-resolution radial fluctuations approach (SRRF) performed with a highly inclined and laminated optical sheet (HILO) fluorescence microscope indicated substantial overlap of Sig1R-YFP spots with KDEL-mRFP, slight overlap with pmKate2-mito and no overlap with the markers of endosomes, peroxisomes, lysosomes, or caveolae. Activation of Sig1R with (+)-pentazocine caused a time-dependent decrease in the overlap between Sig1R-YFP and KDEL-mRFP, indicating that the activation of Sig1R decreases its colocalization with the marker of vesicular ER and does not cause comprehensive translocations of Sig1R in cells.

Specific detection and deletion of the sigma‐1 receptor widely expressed in neurons and glial cells in vivo

“…We also noticed that unlike previous reports (Mavlyutov et al, 2010), the cerebral cortex, hippocampus, thalamus and olfactory bulb area showed strong immunolabelling of S1Rs by this protocol (Figure S4A, a 1 -a 3 and B, b 1 -b 3 ). Although in images with higher magnifications a background staining of tiny puncta could be seen in WT and KO mice (Figure S5A), Ab #61994 immunolabelling clearly demonstrated the ER-like perinuclear ring structures of the S1R staining as reported in previous studies using EYFP-tagged S1Rs in cultured cells (Hayashi & Su, 2007;Kopanchuk et al, 2022) which was further confirmed by co-labelling an ER marker SERCA2 (Figure S5B). In addition, this protocol could immunolabel S1Rs in other organs such as liver (Figure S6A-D) and heart (Figure S6E-H).…”

Pharmacological Analysis of GABAA Receptor and Sigma1R Chaperone Interaction: Research Report I―Investigation of the Anxiolytic, Anticonvulsant and Hypnotic Effects of Allosteric GABAA Receptors’ Ligands

“…First, Sigma1R-dependent influence on the effects of GABA A R PAMs may be mediated by chaperone properties toward cytoplasmic membrane proteins involved in the regulation of anxiety and seizures [44]. Under misfolded proteins' accumulation conditions (ER stress) or ligand activation, Sigma1R is capable of intracellular translocation within lipid domains, including the plasma membrane region [12,38,81,82]. On the other hand, antagonistic action on the chaperone is able to reduce Sigma1R levels in the cell surface membrane and the expression of α and β subunits of GABA A Rs in vivo [83].…”

Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools

“…Imaging was performed using a microscope setup as described previously. 66 Briefly, wide-field epifluorescence and TIRF imaging were performed using an inverted microscope based on a Till iMIC body (Till Photonics/FEI, Munich, Germany), equipped with a UPLSAPO 20× oil (NA 0.85) and TIRF APON 60× oil (NA 1.49) objectives (Olympus Co., Tokyo, Japan). The samples were alternately excited using a 405 nm (120 mW) PhoxX laser diode (Omicron-Laserage, Rodgau, Germany) for Hoechst 34580 or at 561 nm (106 mW) for compound 16.…”

“…Cells were imaged after 30 min of incubation (37 °C, atmosphere with 5% CO 2 ). Imaging was performed using a microscope setup as described previously . Briefly, wide-field epifluorescence and TIRF imaging were performed using an inverted microscope based on a Till iMIC body (Till Photonics/FEI, Munich, Germany), equipped with a UPLSAPO 20× oil (NA 0.85) and TIRF APON 60× oil (NA 1.49) objectives (Olympus Co., Tokyo, Japan).…”