length stim1  (Addgene inc)

Journal:

Article Title: Novel Role for STIM1 as a Trigger for Calcium Influx Factor Production * S⃞

doi: 10.1074/jbc.M709575200

Figure Lengend Snippet: Expression of exogenous STIM1 restores CIF production and SOCE in deficient NG115 cells. a, representative Western blot shows >10-fold increase in the amount of STIM1 protein in NG115 cells transfected with exogenous STIM1 (+STIM1WT) compared with cells transfected with empty vector (Control). b, bar graph shows the activity of CIF extracts from TG-treated NG115 cells transfected with either empty vector (Control) or with STIM1 (+STIM1WT), which were tested in experiments similar to those shown in Fig. 2. Summary from seven to nine oocyte injections of CIF extracts from three different cultures of control and +STIM1WT cells. The asterisks denote significant difference (p = 0.003). c, representative traces show the average changes (±S.E.) in intracellular Ca2+ (F340/F380) recorded simultaneously in a number of individual NG115 cells in which exogenous STIM1 was expressed (+STIM1WT). The cells were pretreated with TG (TG, 1 μm, n = 38) or with 0.5% Me2SO (Basal, n = 50) for 5 min in Ca2+-free solution before 2 mm Ca2+ was added (as indicated by the arrow). d, summary data from experiments in Figs. 3(a and b) and 4c. Maximum SOCE in control NG108 cells (n = 230 from four cultures), control NG115 cells (n = 215 from four cultures), and in NG115 cells expressing exogenous STIM1 (+STIM1WT) (n = 92 from three different cultures). The asterisk denotes significant differences between control NG115 and +STIM1WT NG115 cells (p < 0.001).

Article Snippet: Full-length STIM1 cDNA plasmid was purchased from ATCC in the pOTB7 vector.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Activity Assay

Journal:

Article Title: Novel Role for STIM1 as a Trigger for Calcium Influx Factor Production * S⃞

doi: 10.1074/jbc.M709575200

Figure Lengend Snippet: Down-regulation of STIM1 impairs CIF production in smooth muscle cells. a, top panel shows pseudocolored ratiometric images demonstrating Ca2+ entry in a Xenopus oocyte (∼1-mm diameter) pre-loaded with fura-2 following injection of 28 nl of active CIF extract (prepared from TG-treated SMC transfected with scrambled siRNA). The asterisk in the first frame denotes the point of injection. Representative traces below show the changes in intracellular Ca2+ (F340/F380) in individual oocytes following injection of CIF extracts from untreated SMC (Basal), or TG-treated SMC transfected with either scrambled siRNA (Control), siRNA against STIM1 (-STIM1), or wild-type STIM1 construct (+STIM1WT). b, representative Western blots (on the top) show that SMC transfection with STIM1 siRNA (-STIM1, left blot) resulted in about 50% (48 ± 5%) reduction in STIM1 protein expression, whereas transient overexpression of wild-type STIM1 (+STIM1WT, right blot) increased protein levels to 264 ± 34% compared with controls. The bar graphs below compare the activity of CIF extracts (maximum ΔRatio ± S.E., as measured in the assay shown in c) obtained from SMC with different levels of STIM1 expression. The left panel shows the activity of CIF extracts from untreated SMC (Basal) and TG-treated SMC transfected with either scrambled siRNA (Control) or siRNA against STIM1 (-STIM1). The right panel shows the activity of CIF from TG-treated SMC transfected with either no DNA (Control), or STIM1 construct (+STIM1WT). Each bar summarizes results from seven to seventeen oocytes injected with extracts obtained from three or four different preparations of SMC for each condition. The asterisks denote significant differences between -STIM1 and its control (p = 0.003) and between +STIMWT and its control (p < 0.001), respectively.

Article Snippet: Full-length STIM1 cDNA plasmid was purchased from ATCC in the pOTB7 vector.

Techniques: Injection, Transfection, Control, Construct, Western Blot, Expressing, Over Expression, Activity Assay

Journal:

Article Title: Novel Role for STIM1 as a Trigger for Calcium Influx Factor Production * S⃞

doi: 10.1074/jbc.M709575200

Figure Lengend Snippet: Differences in SOCE (a and b), iPLA2β activity (c and d), CIF production (e), and STIM1 expression (f) in the neuronal cell lines NG108 and NG115. a and b, representative traces show the average changes (±S.E.) in intracellular Ca2+ (F340/F380) recorded simultaneously in a number of individual NG108 (a) and NG115 (b) cells. The cells were pretreated with TG (TG, 1 μm, n = 40 for NG108, n = 41 for NG115) or with 0.5% Me2SO (Basal, n = 31 for NG108, n = 37 for NG115) for 5 min in Ca2+-free solution before 2 mm Ca2+ was added (indicated by the arrows). c and d, summary data showing the activity of iPLA2 (absorbance/mg of protein) in the homogenates of NG108 (c) and NG115 (d) cells with no treatment (Basal), with TG treatment before homogenization (TG, 1 μm for 5 min), or with EGTA treatment (EGTA, 10 mm for 5 min) after homogenization (as a control for the ability of iPLA2 to get activated independently of store depletion). A summary of four to eight measurements from three different cultures of NG108 and NG115 cells is shown. e, summary bar graphs show the activity of CIF extracts from TG-treated NG108 and NG115 cells, which were tested in experiments similar to those shown in Fig. 1c. CIF was extracted from five different cultures of NG108 and seven cultures of NG115. The asterisks denote significant differences between the CIF activity of NG108 and NG115 cells (p < 0.001). f, bar graph shows the activity of CIF extracted from isolated ER vesicles from NG108 and NG115 cells. Depletion of Ca2+ from isolated ER vesicles was done by their treatment with TG, as described under “Materials and Methods.” CIF activity was tested in experiments similar to those shown in Fig. 2a. Each bar summarizes the results from four to seven oocyte injections with CIF obtained from three similar ER preparations. g, representative Western blot shows that NG115 cells contain only 11 ± 4% of STIM1 protein compared with NG108 cells.

Article Snippet: Full-length STIM1 cDNA plasmid was purchased from ATCC in the pOTB7 vector.

Techniques: Activity Assay, Expressing, Homogenization, Control, Isolation, Western Blot

Journal:

Article Title: Novel Role for STIM1 as a Trigger for Calcium Influx Factor Production * S⃞

doi: 10.1074/jbc.M709575200

Figure Lengend Snippet: Down-regulation of STIM1 impairs TG-induced SOCE in smooth muscle cells. a, representative traces show the average changes in intracellular Ca2+ (F340/F380) recorded simultaneously in a number of individual SMC. The cells were pretreated with TG (1 μm) for 5 min in Ca2+-free solution before 2 mm Ca2+ was added (as indicated by the arrow). TG-induced Ca2+ influx (averages ± S.E.) is shown in cells transfected with either scrambled siRNA (Control: n = 31) or siRNA against STIM1 (-STIM1, n = 33). TG-induced Ca2+ release from the stores was similar in both cell types (ΔRatio = 0.028 ± 0.004 in Control; ΔRatio = 0.032 ± 0.007 in -STIM1). b, summary data showing the maximum TG-induced Ca2+ influx in SMC in experiments described in a. Each bar summarizes results 103–215 individual cells from three to five preparations. Basal corresponds to Ca2+ entry in cells treated with 0.5% Me2SO without TG. Please notice that, in contrast with traces in a, the data here show the ΔRatio (±S.E.) values, which were calculated as the difference between the peak F340/F380 ratio after extracellular Ca2+ was added and its level right before Ca2+ addition. The asterisks denote significant differences between control and -STIM1 (p = 0.007).

Article Snippet: Full-length STIM1 cDNA plasmid was purchased from ATCC in the pOTB7 vector.

Techniques: Transfection, Control

Journal:

Article Title: Novel Role for STIM1 as a Trigger for Calcium Influx Factor Production * S⃞

doi: 10.1074/jbc.M709575200

Figure Lengend Snippet: Exogenously expressed YFP-STIM1 is able to form puncta in deficient NG115 cells. Live fluorescent images of representative NG115 cells expressing YFP-STIM1, before (upper panels) and after (lower panels) TG treatment (1 μm, 5 min). The images on the left show the bottom plane, and those on the right the middle plane (4.5 μm from the bottom) of the same cells. The histograms on the right show fluorescence intensities in cross-section of the cells (as indicated by the rectangles) before and after TG application. The entire deconvolved z stack is presented in supplemental Fig. S2. Translocation of YFP-STIM1 in NG115 cells in time is also shown in supplemental Fig. S3 and the supplemental movie.

Article Snippet: Full-length STIM1 cDNA plasmid was purchased from ATCC in the pOTB7 vector.

Techniques: Expressing, Fluorescence, Translocation Assay

Journal:

Article Title: Novel Role for STIM1 as a Trigger for Calcium Influx Factor Production * S⃞

doi: 10.1074/jbc.M709575200

Figure Lengend Snippet: The time course of CIF production and puncta formation. The average time courses of puncta formation in NG115 cells expressing YFP-STIM1 (puncta) and CIF production (CIF) in Jurkat T-lymphocytes following application of TG. The images of NG115 cells were analyzed using ImageJ program (the particle analysis function) after the convolve filter application. The number of puncta was normalized and plotted against time. Each point shows the means ± S.E. from several experiments. The most sensitive detection of early CIF production was achieved using Jurkat cells (∼200 million cells/each time point, repeated in three different cell preparations).

Article Snippet: Full-length STIM1 cDNA plasmid was purchased from ATCC in the pOTB7 vector.

Techniques: Expressing, Particle Size Analysis

Journal:

Article Title: Novel Role for STIM1 as a Trigger for Calcium Influx Factor Production * S⃞

doi: 10.1074/jbc.M709575200

Figure Lengend Snippet: Molecular requirements for STIM1 function as a trigger for CIF production: the role of SAM domain and glycosylation sites. a, the location of three mutations (deletions) within SAM domain of STIM1 and their effects on the predicted tertiary structure and the orientation of the Asn131 and Asn171 glycosylation sites. Structural modeling of the intraluminal EF-hand-SAM domain part of STIM1 was performed on wild-type STIM1 and on mutant STIM1 constructs in which amino acids 150–166 (Δ150–166), 163–169 (Δ163–169), or 168–180 (Δ168–180) had been deleted by site-directed mutagenesis (see “Materials and Methods” for details). The Asn131 and Asn171 glycosylation sites are shown as red hexagons. Color-coded asterisks indicate the location of the specific deletions in the sequence and structure of the mutant SAM domains. b, bar graph shows the activity of CIF extracts from TG-treated (1 μm, 5 min) NG115 cells transfected with either empty vector (Control), wild-type STIM1 (WT), deletion mutant STIM1 constructs (Δ150–166, Δ163–169, or Δ168–180), or with a mutant STIM1 in which both glycosylation sites were mutated from asparagine to glutamine (N131Q and N171Q, shown as NQ). The bars summarize the result from four to eleven oocyte injections with CIF obtained from two or three different cultures of transfected NG115 cells. The asterisks denote significant differences between WT and Δ150–166 (p = 0.011), WT and Δ168–180 (p < 0.001), and WT and NQ (p = 0.002), respectively. c, summary data showing the maximum TG-induced Ca2+ influx in NG115 cell transfected with either no DNA (Control), with wild-type STIM1 (WT), or with the double-glycosylation mutant STIM1 (N131Q and N171Q, shown as NQ, and described in b). Each bar summarizes results from 47–142 NG115 cells from three different transfections. The asterisks denote significant differences between WT and NQ (p = 0.014).

Article Snippet: Full-length STIM1 cDNA plasmid was purchased from ATCC in the pOTB7 vector.

Techniques: Glycoproteomics, Mutagenesis, Construct, Sequencing, Activity Assay, Transfection, Plasmid Preparation, Control

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: bioRxiv

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

doi: 10.1101/2020.12.17.423361

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 CC1α2/3 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 Supplementary Figure 4B for individual solutions). ( B ) smFRET-derived models suggest hydrophobic stabilization of the CC1α2/3 complex by antiparallel apposition of CC1α2 and CC1α3’. ( C ) The crystal structure of CC1 peptides defines an antiparallel interaction of CC1α2/α3’ domains ( left , dashed box ), in which heptad-like hydrophobic sequences 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 ( solid lines ) and BS 3 ( dashed line ) (see also Supplementary Figure 6). ( D ) Parallel apposition of hydrophobic residues on CC1α1 and CC3’. Several of these were previously identified by mutagenesis to stabilize the inactive state of STIM1, including L248, L251, L416, and L423. ( E ) A model of the CC1α1:CC3’ interface obtained by computational peptide docking (see Methods). In the lowest-energy solution, the helices engage in direct mutual hydrophobic interactions.

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: bioRxiv

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

doi: 10.1101/2020.12.17.423361

Figure Lengend Snippet: Full-length STIM1 (flSTIM1) mutants were transiently over- expressed in HEK293 cells for diamide-induced cysteine crosslinking in vivo . Western-blot analysis of cysteine crosslinking of flSTIM1-T307C ( left ) and flSTIM1-S339C ( right ), 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 independent of STIM1 activation (see also A and Supplementary Figure 8B). ( C ) WT flSTIM1 and cysteine mutants were co-expressed with Orai1 for cytosolic calcium imaging (see Methods). For WT flSTIM1 cells, after store depletion by transient exposure to ionomycin (io), readdition of Ca 2+ elevated [Ca 2+ ]i due to SOCE, followed by a decline as SOCE deactivated from store refilling ( top left, black ). Diamide ( red ) did not affect the WT response. In contrast, diamide-induced crosslinking of T307C stabilized the active state, as evidenced by persistent calcium influx after ionomycin wash-out and store refilling ( bottom left, red ). Similar results were obtained for crosslinking A268C ( bottom right , see also Supplementary Figure 8). Crosslinking of S339C did not inhibit 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), S339C (44/41), T307C (38/46), A268C (31/47). ( D ) Schematic illustration of CC1 cysteine crosslinking in the resting ( left ) and activated ( right ) states of flSTIM1. In the resting conformation, 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.

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 Vivo, Western Blot, Activation Assay, 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