Plasma Membrane Localized GCaMP-MS4A12 by Orai1 Co-Expression Shows Thapsigargin- and Ca2+-Dependent Fluorescence Increases

Abstract

Uniquely expressed in the colon, MS4A12 exhibits store-operated Ca²⁺ entry (SOCE) activity. However, compared to MS4A1 (CD20), a Ca²⁺ channel and ideal target for successful leukaemia immunotherapy, MS4A12 has rarely been studied. In this study, we investigated the involvement of MS4A12 in Ca²⁺ influx and expression changes in MS4A12 in human colonic malignancy. Fluorescence of GCaMP-fused MS4A12 (GCaMP-M12) was evaluated to analyse MS4A12 activity in Ca²⁺ influx. Plasma membrane expression of GCaMP-M12 was achieved by homo- or hetero-complex formation with no-tagged MS4A12 (nt-M12) or Orai1, respectively. GCaMP-M12 fluorescence in plasma membrane increased only after thapsigargin-induced depletion of endoplasmic reticulum Ca²⁺ stores, and this fluorescence was inhibited by typical SOCE inhibitors and siRNA for Orai1. Furthermore, GCaMP-MS4A12 and Orai1 co-transfection elicited greater plasma membrane fluorescence than GCaMP-M12 co-transfected with nt-M12. Interestingly, the fluorescence of GCaMP-M12 was decreased by STIM1 over-expression, while increased by siRNA for STIM1 in the presence of thapsigargin and extracellular Ca²⁺. Moreover, immunoprecipitation assay revealed that Orai1 co-expression decreased protein interactions between MS4A12 and STIM1. In human colon tissue, MS4A12 was expressed in the apical region of the colonic epithelium, although its expression was dramatically decreased in colon cancer tissues. In conclusion, we propose that MS4A12 contributes to SOCE through complex formation with Orai1, but does not cooperate with STIM1. Additionally, we discovered that MS4A12 is expressed in the apical membrane of the colonic epithelium and that its expression is decreased with cancer progression.

Keywords: GCaMP; MS4A12; Orai1; STIM1; colon cancer; store-operated Ca2+ entry.

Fig. 1. GCaMP-M12 complexes with nt-M12 localized in plasma membrane, and its fluorescence is dependent on store-operated Ca2+ influx.

(A and B) Localization of GCaMP-M12 in HEK cells upon confocal microscopy. The transmembrane domain of MS4A12 is indicated by blue rods, and GCaMP and Ca²⁺ are indicated by green and red dots, respectively. GCaMP-M12 fluorescence after treatment with 1 μM thapsigargin (TG) and extracellular 5 mM Ca²⁺ (left) and cell boundaries (differential interference contrast [DIC] image, right) were observed. Cytosolic localization of GCaMP-M12 expressed alone (A) or co-expressed with nt-M12 (B). Scale bars = 10 μm. (C-G) Fluorescence of GCaMP-M12 (green) to detect Ca²⁺ influx nearby MS4A12. Fluorescence changes in GCaMP6s (ΔF at each time point over basal fluorescence [F₀; ΔF/F₀]) were traced. Average ΔF/F₀ of all measurements was obtained from three independent experiments (each analysing 4-20 cells). The first and last images in each row show the before and last scene of recording, respectively. The dotted outlined cell in the first image indicates cells expressing GCaMP-M12, and the last scene image was merged with the transmission image (bar, 20 μm) to show the state of cell fluorescence. (C) Spontaneous Ca²⁺ influx when extracellular 5 mM Ca²⁺ was applied without any stimulation. (D) To induce store depletion, 5 μM thapsigargin was added, and 5 mM Ca²⁺ was extracellularly added to allow Ca²⁺ influx. No changes were observed in most cells upon thapsigargin treatment, after which 5 mM Ca²⁺ evoked increased GCaMP-M12 fluorescence. (E) 5 μM 2-APB was added to test whether the increased 5 mM Ca²⁺ with thapsigargin evoked Ca²⁺ influx was blocked. (F) 500 nM BTP2 was pre-incubated to block Ca²⁺ influx through Orai1 before performing B. (G) A summary of Ca²⁺ influx activity in (C-F).

Fig. 2. Orai1 knockdown decreases, while co-expression with Orai1 increases, thapsigargin (TG)- and Ca2+-dependent increases in GCaMP-M12 fluorescence.

(A) Plasma membrane localization of GCaMP-M12 by co-expression with Orai1, similar to that by co-expression with nt-M12. (B and C) Thapsigargin- and Ca²⁺-dependent fluorescence changes in GCaMP-M12 under Orai1-co-expression. (B) Thapsigargin- and Ca²⁺-dependent fluorescence changes in GCaMP-M12 co-expressed with Flag-Orai1 and summary in (C). **P ≤ 0.01. (D-G) Thapsigargin- and Ca²⁺-dependent fluorescence changes in GCaMP-M12 upon Orai1 knockdown (co-expressed with nt-M12). Images of each indicated time point (D) and fluorescence changes (F) for thapsigargin- and Ca²⁺-dependent GCaMP-M12 co-transfected with nt-MS4A12 in scramble and siOrai1-transfected cells. Summary is in (G). *P ≤ 0.05. siRNA efficiency of Orai1 protein was confirmed by Western blot with Orai1-specific antibody (E). (H) Immunoprecipitation assay to observe protein-protein interactions for MS4A12 and Orai1. Flag and GFP antibodies were used to detect Orai1 and MS4A12, respectively. Anti-Flag antibody for Orai1 was used in immunoprecipitation for GCaMP-M12. (I) Co-localization of GCaMP-M12 and mCherry-Orai1 observed by live cell imaging in normal and thapsigargin-stimulated conditions. Scale bars = 10 μm.

Fig. 3. STIM1 overexpression inhibits thapsigargin (TG)- and Ca2+-dependent increases in GCaMP-M12 fluorescence, and interaction between MS4A12 and STIM1 are reduced by Orai1.

(A-C) Thapsigargin- and Ca²⁺-dependent fluorescence changes GCaMP-M12 (co-expressed with nt-M12) in a STIM1 overexpressed condition. (A) Images at each indicated time point. STIM1 co-expressed cells are indicated by yellow dotted lines, and non-STIM1-expressing cells are indicated by white dotted lines. (B) Traces of fluorescence changes. Summary is in (C). ***P ≤ 0.001. (D-G) Thapsigargin- and Ca²⁺-dependent fluorescence changes in GCaMP-M12 (with nt-M12) under a STIM1 knockdown condition. siRNA efficiency for STIM1 was confirmed by Western blot with STIM1-specific antibody. (F) The fluorescence change traces. Summary is in (G). **P ≤ 0.01. (H) MS4A12 whole protein interaction with STIM1^WT in normal and thapsigargin-treated conditions. (I) Truncated MS4A12, in which the intracellular c-terminal region was deleted, shows failed protein-protein interaction with STIM1. (J) Truncated STIM1 proteins were used to detect the MS4A12 interacting region of STIM1. (K) MS4A12 interaction with STIM1^D76A was decreased by Orai1 over-expression, while STIM1^D76A interacted with Orai1. (L) MS4A12 (red) and STIM1 (green) localization in normal and thapsigargin conditions of HEK293 cells analyzed by immunostaining. White arrows indicate their co-localized region. Scale bars = 10 μm.

Fig. 4. Decreased mRNA and protein expression of MS4A12 in colon cancer.

(A) mRNA expression of hMS4A12 and hOrai1 in normal and tumour tissue from patients with colon cancer and a human colon cancer cell line. 18s rRNA expression to indicate cDNA levels and integrity in each sample. N indicates normal and T indicates tumour regions of colon specimens. (B) Decreased hMS4A12 and increased hOrai1 mRNA expression in cancer tissue analysed by quantitative PCR (Q-PCR). ***P ≤ 0.001 compared to normal. (C) Protein expression of MS4A12 in normal and cancer tissues. Actin was used as a loading control. Flag-MS4A12 plasmid expressing HEK lysate was used as a positive control for Western blotting.