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: Nucleic Acids Research

Article Title: Optimized light-inducible transcription in mammalian cells using Flavin Kelch-repeat F-box1/GIGANTEA and CRY2/CIB1

doi: 10.1093/nar/gkx804

Figure Lengend Snippet: Comparison of optimized FKF1/GI-based system to optimized CRY2/CIB1-based system. ( A ) Schematic representation of optimized NLOV/GI-based light-induced transcription. ( B ) Schematic representation of optimized CRY2/CIB1-based light-induced transcription. ( C ) Comparison between the leakiness of NLOV/GI and CRY2/CIB1 when kept in the dark. (** P < 0.01, n = 4–14, mean ± s.d.). The NLOV/-GI-based system showed significantly lower signal under dark conditions compared to the CRY2/CIB1-based system. ( D ) Fluorescent images using the NLOV- and CRY2-based systems in HEK 293T cells to express red fluorescent proteins (RFP, mKate2) in live cells 24 h post-transfection. Hoechst 33 285 was used for nuclear staining. Scale bar, 20 μm. Red arrowheads, mKate2-positive cells. ( E ) Illumination protocol used in (F), I–N (blue LED, 447.5 nm, 0.5 mW, 6.25 μW/mm 2 ). ( F ) DNA ratio optimization of the CRY2/CIB1-based system showing an increase in normalized Luc signal when using 5:1 Cry2-VP16:CIB1 (* P < 0.05, n = 3, in three independent experiments, mean ± s.d.). ( G ) Illumination protocol used in I and J (6 h off) using a blue LED, 447.5 nm, 0.5mW, 6.25 μW/mm 2 . ( H ) Illumination protocol used in I and J (30 min EXP) using a blue light source (470 ± 20 nm, 1.2 mW). ( I and J ) Luc signal of NLOV/GI and CRY2/CIB1 respectively after variable illumination times followed by darkness. (n.s., non significant *** P < 0.001 n = 12 in two independent experiments for 6 h off and 30 min EXP, n = 27 and n = 58 for CRY2 and FKF1 Protocol B, respectively as compiled legacy data was used, mean ± s.d.). CRY2/CIB1 has a significant decrease in induction when kept in dark after illumination. ( K ) Western blotting using the NLOV/GI-based and CRY2/CIB1 systems expressing destabilized green fluorescent protein (dsGFP) in HEK 293T cells with Protocol B. A housekeeping molecule, β-tubulin, was examined as an internal control in the cells. D, dark. L, light. ( L ) Quantification of dsGFP proteins to compare the NLOV/GI-based and CRY2/CIB1 systems. The expression of dsGFP was normalized to β-tubulin expression (* P < 0.05, n = 8, mean ± s.d.). The CRY2/CIB1 system demonstrated a significantly increased induction of dsGFP when compared with the FKF1/GI-based system. ( M and N ) FACS results for NLOV/GI and CRY2/CIB1 systems transfected into HEK 293T cells and human patient primary skin fibroblasts, respectively (n.s., non significant * P < 0.05, ** P < 0.01, n = 6, mean ± s.d.). CRY2/CIB1 has a higher percentage of GFP+ cells in dark and light condition (Protocol B) in the human patient fibroblasts. Representative raw FACS analyses are shown in .

Article Snippet: Published LITE2.0 CRY2 and CIB1 constructs ( ) (Addgene #47455 and 47458) were used as templates for PCR subcloning into pcDNA3.

Techniques: Comparison, Transfection, Staining, Western Blot, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Optimized light-inducible transcription in mammalian cells using Flavin Kelch-repeat F-box1/GIGANTEA and CRY2/CIB1

doi: 10.1093/nar/gkx804

Figure Lengend Snippet: Optimization of the CRY2/CIB1-based system to control transcription with light. ( A ) The illumination protocol used is shown. ( B ) Optimization of fusion combinations of CRY2 (C2), CIB1 (C1), LITE2.0 constructs, Gal4DBD and VP16 ( n = 7–8 in three independent experiments, mean ± s.d.).

Article Snippet: Published LITE2.0 CRY2 and CIB1 constructs ( ) (Addgene #47455 and 47458) were used as templates for PCR subcloning into pcDNA3.

Techniques: Control, Construct