Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Photostimulation was applied at 470 nm (4.0 mW/cm 2 ). Data were shown as mean ± sem. Scale bar, 5 µm. a Domain architecture of the human STIM1. SP, signal peptide; EF-SAM, EF-hand and sterile alpha-motif; TM, transmembrane domain; CC1, coiled-coil domain 1; SOAR, STIM-Orai activating region; P/S, proline/serine-rich region; TRIP, the S/TxIP microtubule-binding motif; PB, polybasic tail. b Schematic of STIM1–ORAI1 coupling at the ER–PM junction that mediates store-operated Ca 2+ entry. c – e Use of the iLID-sspB optical dimerizer to trigger STIM1ct activation and Ca 2+ influx through endogenous ORAI channels. c Schematic of the design. iLID or sspB was fused to the N-terminus of STIM1ct at residue 233. d Confocal images showing photoswitchable Ca 2+ influx in HeLa cells co-transfected with a red Ca 2+ sensor (R-GECO 1.2) and the iLID/sspB fused STIM1ct chimeras. Cells were exposed to two repeated dark-light cycles. e Quantitative analysis of Ca 2+ signals in response to repeated photostimulation ( n = 40 cells from three independent experiments). The half-lives ( t 1/2 ) of on and off kinetics were fitted with one phase exponential decay (“±” means 95% confidence interval). f – h Use of the CRY2-CIBN optical dimerizer to photo-activate STIM1ct and Ca 2+ influx. f Schematic of the design. CRY2 was used to photo-crosslink CIBN-STIM1ct and trigger STIM1ct activation to induce Ca 2+ entry. g Confocal images showing light-induced co-localization of mCherry (mCh)-tagged CIBN-STIM1ct with YFP-ORAI1 in HeLa cells. h Reversible Ca 2+ responses monitored by R-GECO 1.2 ( n = 30 cells). Blue bar, photostimulation at 470 nm with a power density of 4 mW/cm 2 . i – k ER-tethered CRY2-STIM1ct mimics STIM1 puncta formation at ER–PM junctions to evoke localized Ca 2+ influx. i Schematic of the design. j Confocal images illustrating light-induced clustering of ER-resident CRY2-STIM1ct at the footprint of HeLa cells. Enlarged views of the boxed regions were shown on the right. k Cytosolic Ca 2+ signals reported by R-GECO1.2 in HeLa cells subjected to two repeated dark-light cycles ( n = 30).

Article Snippet: Similarly, other photosensory modules such as the PHR domain (CRY2 1–498 ) of Arabidopsis thaliana CRY2 (Addgene; #70159) and its binding partner CIBN (CIB1 1–180 ; Addgene; #47458) were also amplified and inserted into mCh/YFP-STIM1 233–685 .

Techniques: Sterility, Binding Assay, Activation Assay, Residue, Transfection

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a The 3D structure of the juxta-ER membrane region of CC1 (selected residues 241–271; PDB entry: 4O9B), with the predicted helical heptad repeats indicated below the cartoon. b , c Summary of the anticipated outcomes in HeLa cells transfected with the full-length or truncated STIM1ct variants. CRY2-fusion constructs were depicted in panel c . d – f Light-triggered Ca 2+ responses for the indicated CRY2-STIM1ct variants. d GCaMP6s was used to report the basal level of Ca 2+ and light-induced changes of Ca 2+ influx in HeLa cells. e Box-whisker plots showing the GCaMP6s signals before (gray) and after photostimulation (blue) for the indicated CRY2-STIM1ct variants. Data were presented as median and the interquartile range with 5–95 percentile distribution. f The basal Ca 2+ levels (left Y axis, black dots) and fold-change of Ca 2+ signals (right Y axis, red dots) plotted against the starting residues of tested CRY2-STIM1ct variants ( X -axis). n = 40 cells from three independent experiments.

Article Snippet: Similarly, other photosensory modules such as the PHR domain (CRY2 1–498 ) of Arabidopsis thaliana CRY2 (Addgene; #70159) and its binding partner CIBN (CIB1 1–180 ; Addgene; #47458) were also amplified and inserted into mCh/YFP-STIM1 233–685 .

Techniques: Membrane, Transfection, Construct, Whisker Assay

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of an optogenetic clustering assay to examine real-time protein–protein interactions in living cells. b Summary of mCh-tagged baits and YFP-tagged preys used to map critical domains in STIM1ct that dictate STIM1 oligomerization. c Representative confocal images showing the intracellular distribution of the bait (mCh-CRY2-STIM1 233–448 ) and two different preys (P1—top panel, YFP-STIM1 233–658 ; P3—bottom panel, YFP-STIM1 233–342 ) before and after blue light stimulation in HeLa cells. d Time courses showing the kinetics of light-induced clustering (F cluster /F neighbor ) of the bait and its co-clustering with P1 (blue), but not with P3 (green), as seen in panel c . n = 18 cells. ( e ) Quantification of the degrees of light-inducible co-clustering for the five indicated preys in HeLa cells co-transfected with the bait. n = 18 cells from three independent experiments. f Summary of the optogenetic co-clustering assay results for the bait–prey combinations shown in panel b . “+” means co-clustering notably observed after photo-illumination; “−” means no appreciable cluster formation before and after blue light stimulation. g – i Light inducible co-clustering to dissect the STIM1 luminal EF-SAM domain. g Schematic showing the design of bait–prey constructs. h – i Representative confocal images of HeLa cells co-expressing ( h ) mCh-CRY2-EF (B4, STIM1 32–128, EF-hand ; red) with YFP-EF (P6, green), or ( i ) mCh-CRY2-SAM (B5, STIM1 128–200, SAM ; red) with YFP-SAM (P7; red) under dark (left) and blue light (right). The selected regions (dashed boxes) were enlarged to aid visualization. Scale bar 5 µm.

Article Snippet: Similarly, other photosensory modules such as the PHR domain (CRY2 1–498 ) of Arabidopsis thaliana CRY2 (Addgene; #70159) and its binding partner CIBN (CIB1 1–180 ; Addgene; #47458) were also amplified and inserted into mCh/YFP-STIM1 233–685 .

Techniques: Protein-Protein interactions, Transfection, Construct, Expressing

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of the high-throughput screening pipeline. The cytosol-to-PM translocation and Ca 2+ influx (GCaMP6s as readout) were used as two readouts. b Sequence alignment of human SOAR1 and SOAR2 domains and the 3D structure of SOAR1 (PDB entry: 3TEQ). Key residues at the interdimer interface or involved in ORAI1-binding were indicated by dots and triangles, respectively. Selected key residues were highlighted in the 3D structure. c Quantification of Ca 2+ responses (GCaMP6s) and PM translocation (mCherry signals) of selected CRY2-STIM1ct mutants before (dark dots) and after (blue dots) photostimulation. HeLa-GCaMP6s stable cells were co-transfected with each of the indicated mCh-CRY2-STIM1ct mutants and ORAI1-CFP. d Time courses showing the kinetics of light-induced Ca 2+ influx for WT and the indicated mCh-CRT2-STIM1ct variants. n = 60 cells. e – g Representative confocal images ( e ) and quantification of intracellular Ca 2+ signals, n = 60 cells. Box-whisker plots indicated the median, and the interquartile range with 5–95 percentile distribution. f , as well light-induced PM translocation, n = 8 cells ( g ), in HeLa cells expressing WT or the indicated mCh-CRY2-STIM1ct mutants. h Representative confocal images of HEK293 S1-KO cells expressing the GFP-tagged full-length STIM1-T393F mutant before and after TG-induced store depletion. i SOCE monitored by R-GECO1.2 in HEK293 S1-KO cells expressing GFP-STIM1 WT or the mutant T393F. n = 90 cells. j Summary of the degrees of Ca 2+ influx and PM translocation of cancer-associated mutations found in the SOAR domains of STIM1. HeLa cells were transfected with the indicated mCh-CRT2-STIM1ct mutants. Gain-of-function (H395Y and R424W; red) and loss-of-function (L402R, R426L/C, R429C; green) mutations were both identified. n = 60 cells.

Article Snippet: Similarly, other photosensory modules such as the PHR domain (CRY2 1–498 ) of Arabidopsis thaliana CRY2 (Addgene; #70159) and its binding partner CIBN (CIB1 1–180 ; Addgene; #47458) were also amplified and inserted into mCh/YFP-STIM1 233–685 .

Techniques: High Throughput Screening Assay, Translocation Assay, Sequencing, Binding Assay, Transfection, Whisker Assay, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Diagram of the STIM1 C-terminal fragment (residues 443–685) that contains both the S/TxIP EB1-binding motif and the positively charged polybasic tail (PB) that interacts with PM-resident PIPs. Mutations used in this study to perturb STIM1ct-target interactions were highlighted in red. b , c Schematic ( b ) and representative confocal images ( c ) showing light-inducible bimodal distribution (tracking of MT plus ends or cytosol-to-PM translocation) of mCh-CRY2-STIM1 443–685 in COS-7 cells. d Time courses of light-triggered MT plus-end tracking (green) and PM translocation (blue) of mCh-CRY2-STIM1 443–685 . Upon blue light illumination, cytosolic mCherry signals rapidly reduced ( t 1/2 = 18.2 ± 5.0 s), accompanied with the increase of MT tip tracking ( t 1/2 = 21.5 ± 7.3 s) or PM decoration ( t 1/2 = 27.6 ± 6.2 s). n = 12 cells. e , f Representative confocal images ( e ) and quantifications of MT plus-end tracking ( f ) of mCh-CRY2-STIM1ct variants (top, WT; bottom, P645N in the context of STIM1 443–670 ) in COS-7 cells co-transfected with EB1-GFP (green). The bar graph showed the averaged values of MT tip-to-cytosol intensity ratio under dark and lit conditions. n = 16 cells from three independent experiments. g , h Representative confocal images in HeLa cells ( g ) and quantification of the cytosol-to-PM translocation ( h ) of the indicated CRY2-PB variants before and after blue light illumination. n = 16 cells from three independent experiments. i – k Schematic showing light inducible ER-MT interactions in COS-7 cells cotransfected ER-anchored mCh-CRY2-STIM1 443–670 (red) with ( j ) EB1-GFP (green) or ( k ) GFP-tubulin (green). j Representative confocal images showing ER morphology change following blue light illumination. The mCherry and GFP fluorescent intensities across the dashed line were plotted next to the images to indicate the degree of signal overlaps. k Confocal images showing the clustering of ER-resident mCh-CRY2-STIM1 443–670 along with GFP-tubulin (green) upon blue light illumination. The surface profiles of a selected area (oval) were presented to aid the visualization of subcellular distribution. l , m Light-inducible assembly of ER-plasma membrane contact sites (MCSs) mediated by ER-resident mCh-CRY2-PB (STIM1 671–685 ). l Schematic of the design. m Representative confocal images of the footprint of HeLa cells transfected with mCh-CRY2-PB variants before and after blue light illumination.

Article Snippet: Similarly, other photosensory modules such as the PHR domain (CRY2 1–498 ) of Arabidopsis thaliana CRY2 (Addgene; #70159) and its binding partner CIBN (CIB1 1–180 ; Addgene; #47458) were also amplified and inserted into mCh/YFP-STIM1 233–685 .

Techniques: Binding Assay, Translocation Assay, Transfection, Clinical Proteomics, Membrane