Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Functional analysis of the N-FLAG-tagged Orai1 channel. A, thapsigargin (TG)-induced [Ca2+]i rises in Orai1-expressing HEK293 cells. Left, average time course of Ca2+ responses induced by 2 μm TG in Orai1 (gray triangles)-, N-FLAG-tagged Orai1 (black circles)-, and control vector (white circles)-transfected HEK293 cells (n = 27–28). The perfusion solution was first changed to 0.5 mm EGTA containing Ca2+-free HBS (EGTA), and 2 μm TG was applied to the cells in the absence of extracellular Ca2+. Eleven minutes after the application of TG, 2 mm Ca2+ was added to the extracellular solution. Right, maximum [Ca2+]i rises (Δ[Ca2+]i) induced by TG in EGTA and those after re-addition of 2 mm external Ca2+. B, TG-induced [Ca2+]i rises in Orai1 and STIM1-coexpressing HEK293 cells. Left, average time course of Ca2+ responses induced by 2 μm TG in Orai1 plus STIM1 (gray triangles)- and N-FLAG-tagged Orai1 plus STIM1 (black circles)-cotransfected HEK293 cells and control vector (white circles)-transfected HEK293 cells (n = 25–41). Right, Δ[Ca2+]i induced by TG in EGTA and those after re-addition of 2 mm external Ca2+. C, activation of CRAC current induced by 40 μm IP3 in Orai1- and STIM1-coexpressing cells. Left, representative current-voltage relationships in Orai1 plus STIM1 (a)- and N-FLAG-tagged Orai1 plus STIM1 (b)-cotransfected HEK293 cells and control vector (c)-transfected HEK293 cells. Data represent leak-subtracted current densities (pA/picofarad) evoked by 50-ms voltage ramps from -100 to +100 mV. Right, average CRAC current densities at -80 mV in Orai1 plus STIM1 (n = 11), N-FLAG-tagged Orai1 plus STIM1 (n = 9) and control (n = 9) cells. **, p < 0.01; and ***, p < 0.001.

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques: Functional Assay, Expressing, Control, Plasmid Preparation, Transfection, Activation Assay

Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Predicted topology of Orai1 and a docking model with STIM1. A, schematic diagram of N-FLAG Orai1. Orai1 is a highly glycosylated protein with glycan moiety of ∼8 kDa per subunit. The Orai1 polypeptide has a cytosolic N terminus, four TM segments (yellow columns: S1–S4), and a cytosolic C terminus with a putative coiled-coil sequence (green box) involved in binding with STIM1. The FLAG tag (red box) was introduced in the N terminus. B, illustration of putative docking between Orai1 and STIM1. The three-dimensional reconstruction in this study suggests that the cytoplasmic domain of Orai1 is long enough for direct association with STIM1. STIM1 contains a coiled-coil motif (green box) in the cytoplasm, a single TM segment (yellow column), a sterile α motif domain (purple box), and an EF-hand motif (blue crescent) in the ER lumen.

Article Snippet: For functional analysis, Orai1 encoding sequence was also subcloned into pCI-neo (Promega, Madison, WI), and mouse STIM1 encoding sequence was subcloned from pApuro-FLAG-tagged mouse STIM1 ( 31 ) into pCI-neo.

Techniques: Glycoproteomics, Sequencing, Binding Assay, FLAG-tag, Sterility

Journal: Nature Communications

Article Title: STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing

doi: 10.1038/s41467-020-20826-5

Figure Lengend Snippet: Membrane blebbing of DLD1 cells expressing either Sec61β-mCherry and GFP-PLCδ-PH ( a , Supplementary Movie ) or Orai1-mCherry and GFP-tagged STIM1 ( b ). Arrowheads show the ER-PM contact sites. Indicated times are relative to the first image. Results shown are representative of three independent experiments. Scale bar, 2 μm. c – f DLD1 cells expressing Lifeact-RFP were treated with the SOCE inhibitors AnCoA4 (50 µM) and SKF96365 (10 µM). c Representative images from three independent experiments. Indicated times are relative to drug treatment. Scale bar, 10 μm. The number ( d , N = 20 cells), area ( e , N = 20 blebs) and retraction velocity ( f , N = 20 blebs) of membrane blebs in vehicle-treated (control) and drug-treated (4-bromo-A23187) DLD1 cells over 10 min from three independent experiments. Individual data points are plotted with the means ± SD. **** P < 0.0001 (One-way ANOVA with Tukey’s post-hoc multiple comparison test). Source data are provided as a Source Data file.

Article Snippet: Following expression vectors were purchased from Addgene; pGP-CMV-GCaMP6s-CAAX (Addgene No.52228), Sec61β-mCherry (Addgene No. 49155), pGP-CMV-GCaMP6s (Addgene No.40753), EGFP-E-Syt1 (Addgene No.66830), GFP-PIP5K gamma (Addgene No. 22299). cDNAs encoding full-length STIM1, Orai1, Mena, VASP, STIMATE, and MRLC1 were amplified by RT-PCR, fused to the sequence encoding EGFP, Scarlet or HA, and ligated into the pCAGGS-neo vector.

Techniques: Expressing

Journal: Nature Communications

Article Title: STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing

doi: 10.1038/s41467-020-20826-5

Figure Lengend Snippet: a – f Membrane blebbing in DLD1 cells expressing Lifeact-RFP with either WT or dominant negative (E106Q) Orai1. a Representative still images from five independent experiments. b Tricolor maps showing angular coordinates along the horizontal axis and time on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. Results shown are representative of three independent experiments. c Fluorescence intensities of GCaMP6s in “bleb” and “cell body” cytoplasm were quantified in DLD1 cells expressing either WT or E106Q Orai1. The ratio of “bleb” to “cell body” intensities are plotted over time. Data presented are means ± SD based on the values from five independent experiments. The number ( d , N = 20 cells), area ( e , N = 20 blebs), and retraction velocity ( f , N = 10 blebs) of membrane blebs in WT or E106Q Orai1-expressing cells. g – l Membrane blebbing in DLD1 cells expressing Lifeact-RFP with either WT or dominant active (DA, D76A) STIM1. g Representative still images from three independent experiments. Arrowhead indicate persistent ER-PM contact site. h Tricolor maps showing angular coordinates along the horizontal axis and time on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. Results shown are representative of three independent experiments. i Histograms of bleb expansion and retraction velocities in WT an DA STIM1-expressing cells. Three independent measurements are plotted for each condition. The number ( j , N = 20 cells), area ( k , N = 20 blebs), and retraction velocity ( l , N = 10 blebs) of membrane blebs in WT or DA STIM1-expressing cells. Individual data points are plotted with the means ± SD. m Schematic of PM-ER contact site formation in the early stages of bleb formation. Details of the model are described in the text. a and g Scale bar, 10 μm. d – f , j – l **** P < 0.0001 (Two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Article Snippet: Following expression vectors were purchased from Addgene; pGP-CMV-GCaMP6s-CAAX (Addgene No.52228), Sec61β-mCherry (Addgene No. 49155), pGP-CMV-GCaMP6s (Addgene No.40753), EGFP-E-Syt1 (Addgene No.66830), GFP-PIP5K gamma (Addgene No. 22299). cDNAs encoding full-length STIM1, Orai1, Mena, VASP, STIMATE, and MRLC1 were amplified by RT-PCR, fused to the sequence encoding EGFP, Scarlet or HA, and ligated into the pCAGGS-neo vector.

Techniques: Expressing, Dominant Negative Mutation, Fluorescence

Journal: Nature Communications

Article Title: STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing

doi: 10.1038/s41467-020-20826-5

Figure Lengend Snippet: a Representative still images of membrane blebbing in DLD1 cells expressing Lifeact-RFP and GFP-E-Syt1 from five independent experiments. See also Supplementary Movie . b , c DLD1 cells expressing Lifeact-RFP with either GFP-E-Syt1 ( b ) or GCaMP6s ( c ) were treated with the actin polymerization inhibitor Latrunculin B (LatB, 10 µM). Arrowheads show disrupted actin cortex. Results shown are representative of three independent experiments. See also Supplementary Movie . d – f Biochemical analyses of ezrin’s role in STIM1-Orai1 complex formation. d Orai1-HA was co-expressed with either GFP or GFP-ezrin. Inputs and HA immunoprecipitates were immunoblotted with antibodies against HA and GFP. e STIM1-FLAG and Orai1-HA were co-expressed with either GFP or GFP-ezrin. Inputs and FLAG immunoprecipitates were immunoblotted with antibodies against FLAG, HA, and GFP. f GFP expression levels (shaded in green) and co-precipitated Orai1-HA (individual measurements) were quantified and normalized to GFP-expressing control. Data presented are means ± SD based on the values from four independent experiments. g , h Proximity ligation assay (PLA) for in situ detection of Orai1-STIM1 interaction. Representative images from five independent experiments are shown in g . h Quantification of PLA signals in expanding (low Lifeact intensity) and retracting (high Lifeact intensity) blebs based on data shown in g and Supplementary Fig. . STIMATE-STIM1 and Claudin-3-STIM1 are positive and negative controls, respectively. N = 25 independent blebs. Individual data points are plotted with the means ± SD. i Membrane blebbing in Ezrin KO cells expressing GFP- PLCδ-PH and Sec61β-mCherry (upper panels) and in WT DLD1 cells expressing constitutive active Rnd3 (S240A) and Sec61β-mCherry (lower panels). Arrowheads indicate persistent ER-PM contact sites. Results shown are representative of three independent experiments. j Representative images from three independent experiments of DLD1 cells expressing constitutive active Ezrin (T567E) and Sec61β-mCherry. a , g (middle and bottom panels), i , j (three right panels) Scale bar, 2 μm. b , c , g (top panels), and j (left panel) Scale bar, 10 μm. f , h **** P < 0.0001 (Two-sided, unpaired Student’s t test). Times shown are relative to drug treatment ( b, c ) or the first image ( i ). Source data are provided as a Source Data file.

Article Snippet: Following expression vectors were purchased from Addgene; pGP-CMV-GCaMP6s-CAAX (Addgene No.52228), Sec61β-mCherry (Addgene No. 49155), pGP-CMV-GCaMP6s (Addgene No.40753), EGFP-E-Syt1 (Addgene No.66830), GFP-PIP5K gamma (Addgene No. 22299). cDNAs encoding full-length STIM1, Orai1, Mena, VASP, STIMATE, and MRLC1 were amplified by RT-PCR, fused to the sequence encoding EGFP, Scarlet or HA, and ligated into the pCAGGS-neo vector.

Techniques: Expressing, Proximity Ligation Assay, In Situ