Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Mitochondria depletion abolishes agonist-induced Ca2+ plateau in airway smooth muscle cells: potential role of H2O2.

doi: 10.1152/ajplung.00134.2009

Figure Lengend Snippet: Fig. 9. Stromal interaction molecule-1 (STIM1) shRNAi abolishes histamine-induced [Ca2]i plateau. Representa- tive fura 2 fluorescence obtained from 2 M histamine- stimulated RASMCs that cultured in the absence (A and B) or presence of pyruvate and uridine (C and D) as well as EB-pretreated cells (E and F). RASMCs were transfected with either nonspecific shRNAi/GFP construct or STIM1- specific shRNAi/GFP construct. After fura 2 loading, the transfected RASMC was first identified by GFP expres- sion under a fluorescent microscope with a wavelength of 485 nm for excitation and 520 nm for emission. Then the cell was excited alternatively by 340/380 nm, and the emitted fluorescence of 510 nm was continuously moni- tored before and after the challenge of 2 M histamine. STIM1 knockdown abolished histamine-induced [Ca2]i plateau in control RASMCs cultured in the absence (B) or presence (D) of Py and Ur vs. nonspecific shRNAi con- struct (A and C), respectively, whereas EB pretreatment abolished the [Ca2]i plateau (E and F) (n 18–20 cells for each).

Article Snippet: Effective shRNAi specifically against stromal interaction molecule-1 (STIM1) and its nonspecific control were purchased from Origene (Rockville, MD).

Techniques: Cell Culture, Transfection, Construct, Microscopy, Knockdown, Control

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Mitochondria depletion abolishes agonist-induced Ca2+ plateau in airway smooth muscle cells: potential role of H2O2.

doi: 10.1152/ajplung.00134.2009

Figure Lengend Snippet: Fig. 10. H2O2 reverses EB pretreatment-abolished [Ca2]i plateau in an STIM1-dependent way. Represen- tative fura 2 fluorescence obtained from 2 M hista- mine-stimulated RASMCs pretreated by EB to deplete mitochondria. EB-pretreated RASMCs were transfected with either nonspecific shRNAi/GFP construct or STIM1-specific shRNAi/GFP construct. After fura 2 loading, the transfected RASMC was first identified by GFP expression under a fluorescent microscope with wavelength of 485 nm for excitation and 520 nm for emission. Then, the cell was excited alternatively by 340/380 nm, and the emitted fluorescence of 510 nm was continuously monitored before and after the chal- lenge of 2 M histamine. EB pretreatment abolished histamine-induced [Ca2]i plateau; the subsequent co- exposure of 0.1 M H2O2 reversed EB pretreatment- abolished [Ca2]i plateau in control (A) and nonspecific shRNAi-transfected RASMCs (B), but not in STIM1- knockdown cells (C) (n 18–20 cells for each).

Article Snippet: Effective shRNAi specifically against stromal interaction molecule-1 (STIM1) and its nonspecific control were purchased from Origene (Rockville, MD).

Techniques: Transfection, Construct, Expressing, Microscopy, Control, Knockdown

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, Control

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Impairment of mitochondrial unfolded protein response contribute to resistance declination of H 2 O 2 ‐induced injury in senescent MRC‐5 cell model

doi: 10.1002/kjm2.12146

Figure Lengend Snippet: Effects of STIM1‐Orai1‐dependent SOCE induced [Ca 2+ ]i signals at different passage numbers of MRC‐5 cells after treatment with or without H 2 O 2 . Left panel of A, Analysis of SOCE‐induced [Ca 2+ ]i concentrations after the readdition of 2 mM Ca 2+ to Ca 2+ store depletion cells. Depletion of [Ca 2+ ]i stores by 20 nM thapsigargin. The resulting calcium signals represent the mean [Ca 2+ ]i changes of 36 cells with five replicates for each group ( N = 5). Right panel of A, Calcium signals were estimated by quantifying the areas under the calcium signal curve after an extracellular calcium pulse. B, Changes in STIM1 and Orai1 protein expression after treatment with or without 100 μM H 2 O 2 for 4 hours at indicated passages. GAPDH was used as control for equal loading. Values are mean ± SEM ( N = 6). * P < .05, ** P < .01, and *** P < 0.001

Article Snippet: Target proteins were probed with the following antibodies: anti‐Hsp60 and anti‐Hsp10 (1:1000; Enzo Life Science, New York), anti‐ClpP, anti‐SMP30, and antiubiquitin proteins (1:1000; Santa Cruz Biotech, Heidelberg, Germany), anti‐STIM1 (1:1000; OriGene Technologies, China), and anti‐Orai1 (1:1000; Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Control

Journal: The Journal of Cell Biology

Article Title: The elementary unit of store-operated Ca 2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions

doi: 10.1083/jcb.200604015

Figure Lengend Snippet: Regions of CRAC-mediated Ca 2+ influx overlap with STIM1 puncta. (A) ΔF/F 0 image from a Jurkat cell collected 10–60 ms after a hyperpolarizing voltage step to −122 mV. (B) Linear interpolation of the ΔF/F 0 image in A to equalize the number of pixels to the Cherry-STIM1 image in D. (C and F) 10 μM La 3+ inhibited both ΔF/F 0 and I CRAC in this cell, confirming that ΔF/F 0 arises from CRAC channels. (D) TIRF image of Cherry-STIM1 in the same cell. A gray contour line outlines the fluo-5F footprint depicted in A. (E) Pseudocolored contour lines of Ca 2+ influx density from B overlaid on the Cherry-STIM1 image show the overlap of Ca 2+ influx sites with Cherry-STIM1 puncta.

Article Snippet: Cherry-STIM1 plasmid was constructed by inserting mCherry (a gift from R. Tsien, University of California, San Diego, La Jolla, CA) after the signal sequence of human STIM1 (Origene). mCherry was amplified by PCR from mCherry-pRSET-B ( ) with primers 5′-ttattaggtaccatggtgagcaagggc-3′ and 5′-ataataggtacccttgtacagctcgtccat-3′ to append KpnI sites onto each end.

Techniques:

Journal: The Journal of Cell Biology

Article Title: The elementary unit of store-operated Ca 2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions

doi: 10.1083/jcb.200604015

Figure Lengend Snippet: Cytochalasin D reorganizes STIM1 puncta in Jurkat cells. (A) TIRF images of Cherry-STIM1 in an intact cell. Initially, Cherry-STIM1 was diffusely distributed within the ER (left) but redistributed into puncta after 5 min of treatment with 0 mM Ca o 2+ plus 1 mM EGTA and 1 μM TG (middle). After a subsequent 15-min exposure to 5 μM cytochalasin D, the puncta coalesced into large, sparsely distributed structures (right). The complete image sequence is shown in Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200604015/DC1 ). (B) An IRM image of the cell footprint after 16 min of cytochalasin D treatment shows persistent adherence of the cell to the coverslip. (C–E) Electron micrographs from cells treated with TG plus cytochalasin D as in A, showing the distribution of HRP-STIM1 in ER tubules (arrows) located next to the plasma membrane (pm). n, nucleus; m, mitochondrion. Bars, 200 nm. (F) Cytochalasin D does not affect the integrated fluorescence of Cherry-STIM1 puncta near the plasma membrane. The fluorescence increases in response to store depletion (TG) because of the formation of puncta but is constant after 15 min of subsequent cytochalasin D treatment ( n = 7 cells). (G) Cytochalasin D does not affect maintenance of I CRAC . I CRAC density was measured at −122 mV in Cherry-STIM1–transfected cells after >15 min of pretreatment with TG ± 5 μM cytochalasin D ( n = 4 cells each). Because I CRAC activates well before cytochalasin D causes any noticeable changes in ER structure, this experiment tests its effect on the maintenance rather than the activation of I CRAC .

Article Snippet: Cherry-STIM1 plasmid was constructed by inserting mCherry (a gift from R. Tsien, University of California, San Diego, La Jolla, CA) after the signal sequence of human STIM1 (Origene). mCherry was amplified by PCR from mCherry-pRSET-B ( ) with primers 5′-ttattaggtaccatggtgagcaagggc-3′ and 5′-ataataggtacccttgtacagctcgtccat-3′ to append KpnI sites onto each end.

Techniques: Sequencing, Clinical Proteomics, Membrane, Fluorescence, Transfection, Activation Assay

Journal: The Journal of Cell Biology

Article Title: The elementary unit of store-operated Ca 2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions

doi: 10.1083/jcb.200604015

Figure Lengend Snippet: Open CRAC channels colocalize with STIM1 puncta in cytochalasin D–treated cells. TIRF images of ΔF/F 0 (A) and Cherry-STIM1 fluorescence (B) in a Jurkat cell treated for 15 min with 0-Ca 2+ Ringer's plus 1 mM EGTA, 1 μM TG, and 5 μM cytochalasin D. ΔF/F 0 was linearly interpolated as in . A gray contour line outlines the fluo-5F footprint in B. (C) Pseudocolored contour lines of Ca 2+ influx density overlaid on the Cherry-STIM1 image show that Ca 2+ influx sites are associated with bright STIM1 puncta. (D) Overlay of the pseudocolored ΔF/F 0 map on a surface plot of Cherry-STIM1 fluorescence. (E) Comparison of the spatial extent of Cherry-STIM1 and ΔF/F 0 at a single Ca 2+ influx site. The intensity profiles through the center of a single Cherry-STIM1 punctum and its associated Ca 2+ influx site (inset; taken from the boxed area in A) are shown. The radial spread of ΔF/F 0 is centered on the Cherry-STIM1 profile. Convolution of the Cherry-STIM1 fluorescence curve with the diffusional spread function for fluo-5F yields a curve (black line) that is similar in extent to ΔF/F 0 .

Article Snippet: Cherry-STIM1 plasmid was constructed by inserting mCherry (a gift from R. Tsien, University of California, San Diego, La Jolla, CA) after the signal sequence of human STIM1 (Origene). mCherry was amplified by PCR from mCherry-pRSET-B ( ) with primers 5′-ttattaggtaccatggtgagcaagggc-3′ and 5′-ataataggtacccttgtacagctcgtccat-3′ to append KpnI sites onto each end.

Techniques: Fluorescence, Comparison

Journal: The Journal of Cell Biology

Article Title: The elementary unit of store-operated Ca 2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions

doi: 10.1083/jcb.200604015

Figure Lengend Snippet: Orai1 colocalizes with STIM1 at peripheral ER–plasma membrane junctions after store depletion. TIRF imaging of a Jurkat cell transiently coexpressing Cherry-STIM1 and GFP-myc-Orai1 before and after store depletion. (A) In cells bathed in 2 mM Ca 2+ , Cherry-STIM1 is distributed throughout the ER (left), whereas GFP-myc-Orai1 is distributed throughout the cell footprint (middle). The merged image (right) shows little overlap of Orai1 (green) and STIM1 (red). (B) After a 5-min treatment with 1 μM TG in 0-Ca 2+ Ringer's plus 1 mM EGTA, both Cherry-STIM1 (left) and GFP-myc-Orai1 (middle) colocalize at discrete puncta, as shown in the merged image (right). Similar results were seen in 14 out of 16 cells. (C) After a subsequent 15-min exposure to 5 μM cytochalasin D, Cherry-STIM1 (left) and GFP-myc-Orai1 (middle) remained localized in coalesced puncta (right).

Article Snippet: Cherry-STIM1 plasmid was constructed by inserting mCherry (a gift from R. Tsien, University of California, San Diego, La Jolla, CA) after the signal sequence of human STIM1 (Origene). mCherry was amplified by PCR from mCherry-pRSET-B ( ) with primers 5′-ttattaggtaccatggtgagcaagggc-3′ and 5′-ataataggtacccttgtacagctcgtccat-3′ to append KpnI sites onto each end.

Techniques: Clinical Proteomics, Membrane, Imaging

Journal: The Journal of Cell Biology

Article Title: The elementary unit of store-operated Ca 2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions

doi: 10.1083/jcb.200604015

Figure Lengend Snippet: Local activation of CRAC channels by STIM1 at ER–plasma membrane junctions. Store depletion causes STIM1 to accumulate in preexisting and newly formed regions of junctional ER, whereas Orai1 accumulates in apposed regions of the plasma membrane. CRAC channels open only in the close vicinity of the STIM1 puncta. The convergence of STIM1 and Orai1 at ER–plasma membrane junctions creates the elementary unit of SOCE.

Article Snippet: Cherry-STIM1 plasmid was constructed by inserting mCherry (a gift from R. Tsien, University of California, San Diego, La Jolla, CA) after the signal sequence of human STIM1 (Origene). mCherry was amplified by PCR from mCherry-pRSET-B ( ) with primers 5′-ttattaggtaccatggtgagcaagggc-3′ and 5′-ataataggtacccttgtacagctcgtccat-3′ to append KpnI sites onto each end.

Techniques: Activation Assay, Clinical Proteomics, Membrane