length stim1  (Addgene inc)

Journal: bioRxiv

Article Title: Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

doi: 10.64898/2026.01.16.700022

Figure Lengend Snippet: All FRET distributions were obtained in 0.5 mM EGTA. FiÄed Gaussian curves (gray) and their sum (blue) are superimposed on the data (red). ( A ) CC1α1-CC1α1’ FRET histogram of WT STIM1 (242:242’; n=437). Fit parameters (peak FRET and fracMonal area): 0.29 (25%), 0.65 (67%), 0.88 (8%). The smallest distance (4.2 nm) is greater than expected for a coiled-coil. ( B ) TM-TM’ FRET histogram of WT STIM1 (222:222’; n=83). Fit parameters (peak FRET and fracMonal area): 0.38 (32%), 0.73 (62%), 0.86 (6%). The minimum TM-TM’ distance (4.3 nm) is greater than expected for a coiled-coil. ( C ) CC1α1-CC1α1’ FRET histogram of STIM1-LL (242:242’; n=254). Fit parameters (peak FRET and fracMonal area): 0.36 (18%), 0.80 (60%), 0.97 (22%). The smallest distance (3.3 nm) is consistent with a coiled-coil. ( D ) TM-TM’ FRET histogram of STIM1-LL (222:222’; n=203). Fit parameters (peak FRET and fracMonal area): 0.32 (17%), 0.77 (70%), 0.98 (13%). The minimum TM-TM’ distance (3.1 nm) is consistent with a coiled-coil.

Article Snippet: For insect cell expression, STIM1 constructs were cloned in the pFastBac1 vector by PCR from full-length human STIM1 (Origene) as follows.

Techniques:

Journal: bioRxiv

Article Title: Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

doi: 10.64898/2026.01.16.700022

Figure Lengend Snippet: All FRET measurements were obtained for WT STIM1 in 0.5 mM EGTA. In each panel, simulated dye positions are superimposed on the 3HB structures (pdb: 6YEL). ( A ) smFRET histogram for 242:291 (n=189). Fit parameters (peak FRET and fracMonal area): 0.21 (17%), 0.51 (67%), 0.79 (16%). ( B ) smFRET histogram for 253:291 (n=275). Fit parameters (peak FRET and fracMonal area): 0.36 (36%), 0.71 (51%), 0.89 (13%). ( C ) smFRET histogram for 242:309 (n=158). Fit parameters (peak FRET and fracMonal area): 0.23 (19%), 0.72 (52%), 0.89 (29%). ( D ) smFRET histogram for 291:325 (n=210). Fit parameters (peak FRET and fracMonal area): 0.19 (18%), 0.42 (11%), 0.75 (60%), 0.88 (10%). ( E ) Comparison of the minimal inter-dye distances measured from smFRET and the NMR-derived 3HB structure of monomeric CC1. Intrasubunit distances within CC1 are consistent with formation of the 3-helix bundle.

Article Snippet: For insect cell expression, STIM1 constructs were cloned in the pFastBac1 vector by PCR from full-length human STIM1 (Origene) as follows.

Techniques: Comparison, Derivative Assay

Journal: bioRxiv

Article Title: Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

doi: 10.64898/2026.01.16.700022

Figure Lengend Snippet: ( A ) EGTA greatly increases the separation between CC1α3-CC1α3’ (337:337’). ( B ) Western blot shows diamide treatment of STIM1-S339C expressed in HEK 293 cells induces disulfide-linked dimers that are sensitive to DTT. Molecular weights in kDa are indicated. ( C-E ) S339C crosslinking prevents release of CAD from the CC1α1 clamp. ( C ) smFRET histogram for 242:242’ in 2 mM Ca 2+ + diamide (n=69). Fit parameters (peak FRET and fracMonal area): 0.36 (67%), 0.80 (33%). ( D ) smFRET histogram for 242:242’ in EGTA + diamide (n=229). Fit parameters (peak FRET and fracMonal area): 0.32 (58%), 0.67 (31%), 0.84 (12%). ( E ) smFRET histogram for 242:242’ in EGTA following DTT treatment (n=210). Fit parameters (peak FRET and fracMonal area): 0.28 (21%), 0.65 (69%), 0.89 (11%). ( F ) Effect of diamide treatment on puncta formation by STIM1-S339C following store depletion with TG. Mean (± sem) of 27 (WT) or 15 (S339C) cells.

Article Snippet: For insect cell expression, STIM1 constructs were cloned in the pFastBac1 vector by PCR from full-length human STIM1 (Origene) as follows.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

doi: 10.64898/2026.01.16.700022

Figure Lengend Snippet: ( A ) 178:178’ FRET in the presence of saturating Ca 2+ for WT STIM1 (blue; n=271)) and STIM1-LL (red; n=253). Promotion of the CC1 coiled-coil moves the SAM domains closer together despite the presence of Ca 2+ . ( B ) 178:178’ FRET in the presence of EGTA for WT STIM1 (blue; n=282) and STIM1-LL (red; n=189). ( C ) 178:178’ FRET in EGTA for STIM1-339C in the presence of diamide (blue; n=371) or DTT (red: n=206). Locking the CC1α2/3 brake impedes the movement of SAM domains in EGTA.

Article Snippet: For insect cell expression, STIM1 constructs were cloned in the pFastBac1 vector by PCR from full-length human STIM1 (Origene) as follows.

Techniques:

Journal: bioRxiv

Article Title: Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

doi: 10.64898/2026.01.16.700022

Figure Lengend Snippet: FRET measurements were obtained in 2 mM Ca 2+ (blue) or 0.5 mM EGTA (red). ( A ) Distance between the N-termini (349:349’) of the two CAD protomers increases in EGTA; n=221 (Ca 2+ ), n=247 (EGTA). ( B ) Distance between the CAD apices (378:378’) increases in EGTA; n=226 (Ca 2+ ), n=219 (EGTA). ( C ) Distance between the C-termini (431:431’) increases in EGTA; n=204 (Ca 2+ ), n=213 (EGTA). R304W restores the high FRET peak in Ca 2+ ; n=290. ( D ) The N– and C-termini of each CAD protomer (349:431) separate in EGTA; n=223 (Ca 2+ ), n=221 (EGTA). ( E ) The N-terminus of each protomer stays associated with the C terminus of its neighbor (349:431’) in EGTA; n=232 (Ca 2+ ), n=186 (EGTA). ( F ) Crystal structure of human STIM1 CAD showing the dye locations in A-E (3TEQ.pdb). Arrows illustrate movement of the paired N and C termini of the two protomers in EGTA consistent with distance meaurements in A-E.

Article Snippet: For insect cell expression, STIM1 constructs were cloned in the pFastBac1 vector by PCR from full-length human STIM1 (Origene) as follows.

Techniques:

Journal: bioRxiv

Article Title: Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

doi: 10.64898/2026.01.16.700022

Figure Lengend Snippet: ( A ) Western blot shows diamide treatment of STIM1-S361C expressed in HEK 293 cells induces disulfide-linked dimers that are sensitive to DTT. Molecular weights in kDa are indicated. ( B ) AlphaFold2 model of the STIM1 resting state showing the Y361C crosslinking site and dye locations for FRET measurements. ( C ) smFRET histograms for 242:242’ in STIM1-Y361C in 2 mM Ca 2+ + diamide (blue), EGTA + diamide (green), and EGTA + DTT (red). ( D ) smFRET histograms for 431:431’ in STIM1-Y361C in 2 mM Ca 2+ +diamide (blue), EGTA + diamide (green), and EGTA + DTT (red). ( E ) Effect of diamide treatment on puncta formation by STIM1-Y361C following store depletion with TG. Mean (± sem) of 27 (WT) or 17 (Y361C) cells.

Article Snippet: For insect cell expression, STIM1 constructs were cloned in the pFastBac1 vector by PCR from full-length human STIM1 (Origene) as follows.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

doi: 10.64898/2026.01.16.700022

Figure Lengend Snippet:

Article Snippet: For insect cell expression, STIM1 constructs were cloned in the pFastBac1 vector by PCR from full-length human STIM1 (Origene) as follows.

Techniques: Comparison, Construct

Journal: eLife

Article Title: Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

doi: 10.7554/eLife.66194

Figure Lengend Snippet: ( A ) 36 smFRET-derived distances were used to reconstruct the orientations of CC1 domains relative to CAD, yielding two classes of solutions with the CC1α2/α3 domains in a ’stacked' ( left ) or 'wedged' ( right ) configuration. In both classes, CC1α1 domains are in close parallel apposition to the CC3 domains on the adjacent subunit in a domain-swapped configuration, with the CC1α2 and CC1α3 domains forming a compact parallel structure directed away from CAD. The average of 50 model solutions is shown for each class (see for individual solutions). The complete list of smFRET values and distance constraints used to generate the models is shown in . ( B ) smFRET-derived models suggest hydrophobic stabilization of the CC1α2/α3 complex by antiparallel apposition of CC1α2 and CC1α3´. ( C ) The crystal structure of CC1 peptides depicts an antiparallel interaction of CC1α2 and CC1α3´ domains ( left , dashed box ), in which hydrophobic residues form a tightly packed dimer interface ( top right , adapted from 4O9B.pdb). ( Bottom right ) Tight antiparallel packing of CC1α2/α3´ in ctSTIM1 was confirmed by inter-subunit crosslinks with EDC ( red lines ) (see also ). ( D ) Parallel apposition of hydrophobic residues on CC1α1 and CC3´. Many of these were previously identified by mutagenesis to stabilize the inactive state of STIM1, including L248, L251, L258, L261, L416, V419, and L423. ( E ) A model of the hydrophobic CC1α1:CC3´ interface obtained by computational peptide docking (see Materials and methods).

Article Snippet: For samples that were encapsulated in liposomes for surface immobilization, DNA encoding ctSTIM1 (aa 233–685) was amplified by PCR from full-length human STIM1 (Origene), appending an N-terminal NcoI cleavage site, and a C-terminal TEV protease recognition sequence (SENLYFQG) followed by a HindIII cleavage site.

Techniques: Derivative Assay, Mutagenesis

Journal: eLife

Article Title: Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

doi: 10.7554/eLife.66194

Figure Lengend Snippet: ( A ) Cysteine crosslinking throughout the CC1 domain of ctSTIM1 mutants in vitro produced by copper phenanthroline and detected by non-reducing SDS-PAGE (see Materials and methods). Crosslinking efficiency peaked at aa 307 in the CC1α2/α3 linker region. ( B ) Full-length STIM1 (flSTIM1) mutants H266C, A268C, T307C, and N309C, were transiently over-expressed in HEK293 cells for diamide-induced cysteine crosslinking in vivo. Western-blot analysis shows strong crosslinking of flSTIM1-A268C and flSTIM1-T307C only after activation by store depletion (0 mM Ca+ CPA). Little or no crosslinking occurred at nearby residues (H266C or N309C). All crosslinking was reversed by DTT. ( C ) Crosslinking efficiencies in the inactive ( black ) and active ( white ) states for residues aa 262–269 at the C-terminal end of CC1α1. Crosslinking at several sites within this region increased somewhat in the active state, but crosslinking for A268C was particularly strong, suggesting a close, stable apposition at that position. Figure 7—figure supplement 1—source data 1. Raw unedited and uncropped labeled gel and western blots for .

Article Snippet: For samples that were encapsulated in liposomes for surface immobilization, DNA encoding ctSTIM1 (aa 233–685) was amplified by PCR from full-length human STIM1 (Origene), appending an N-terminal NcoI cleavage site, and a C-terminal TEV protease recognition sequence (SENLYFQG) followed by a HindIII cleavage site.

Techniques: In Vitro, Produced, SDS Page, In Vivo, Western Blot, Activation Assay, Labeling

Journal: eLife

Article Title: Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

doi: 10.7554/eLife.66194

Figure Lengend Snippet: ( A ) Western-blot analysis of diamide-induced cysteine crosslinking of flSTIM1-WT, flSTIM1-A268C, flSTIM1-T307C and flSTIM1-S339C in HEK293 cells, under resting (2 mM Ca 2+ ) or store-depleted (0 mM Ca 2+ + CPA) conditions. ( B ) Summary of flSTIM1 cysteine crosslinking before ( black ) and after ( white ) store depletion measured in individual paired experiments. While crosslinking at aa 268 and aa 307 strongly increased in the activated state, crosslinking at aa 339 occurred independently of STIM1 activation (see also ). ( C ) Effects of flSTIM1 cysteine crosslinking on deactivation of SOCE following store refilling. WT flSTIM1 and cysteine mutants were co-expressed with Orai1 for cytosolic calcium imaging. In cells expressing WT flSTIM1 and store-depleted by transient exposure to ionomycin (io, 1 µM), addition of 2 mM Ca 2+ elevated [Ca 2+ ] i due to SOCE, followed by a decline as SOCE deactivated from store refilling ( top left, black ). In contrast, diamide-induced crosslinking of A268C or T307C flSTIM1 mutants stabilized the active state, as evidenced by persistent calcium influx after ionomycin wash-out and store refilling ( bottom left and right, red ). Crosslinking of S339C did not affect deactivation of SOCE upon store refilling ( top right ). Each trace shows the mean and s.e.m. of the following numbers of cells (control/diamide) from at least two independent experiments: WT (91/106), A268C (31/47), T307C (38/46), S339C (44/41). ( D ) Schematic illustration of CC1 cysteine crosslinking in the resting ( left ) and activated ( right ) states of flSTIM1 (only CC1 and CAD are shown for clarity). In the resting state, CC1α1 and CC1α3 domains are kept apart, preventing crosslinking at locations upstream of aa 339. Upon store depletion, release of the CC1α1 domains from CAD promotes alignment of CC1 domains along their entire length, enabling crosslinking at aa 268 and 307. Figure 7—source data 1. Raw unedited and uncropped labeled western blots for (WT).

Article Snippet: For samples that were encapsulated in liposomes for surface immobilization, DNA encoding ctSTIM1 (aa 233–685) was amplified by PCR from full-length human STIM1 (Origene), appending an N-terminal NcoI cleavage site, and a C-terminal TEV protease recognition sequence (SENLYFQG) followed by a HindIII cleavage site.

Techniques: Western Blot, Activation Assay, Imaging, Expressing, Control, Labeling

Journal: eLife

Article Title: Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

doi: 10.7554/eLife.66194

Figure Lengend Snippet:

Article Snippet: For samples that were encapsulated in liposomes for surface immobilization, DNA encoding ctSTIM1 (aa 233–685) was amplified by PCR from full-length human STIM1 (Origene), appending an N-terminal NcoI cleavage site, and a C-terminal TEV protease recognition sequence (SENLYFQG) followed by a HindIII cleavage site.

Techniques: Recombinant, Plasmid Preparation, Protease Inhibitor, Software, Microscopy, Imaging

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