lck mturquoise2 ilid  (Addgene inc)

Journal: eLife

Article Title: Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

doi: 10.7554/elife.84364

Figure Lengend Snippet: Figure 1. Optimization of an optogenetic recruitment tool. (A) Normalized membrane to cytosol intensity ratio for the rapamycin system (green) HeLa cells expressing Lck-FRB-T2098L-mTurquoise-IRES-sYFP2xNES-FKBP12, for the improved light-induced dimer (iLID) system (purple) expressing Venus- iLID-CaaX and SspB-mScarlet-I and for the enhanced Magnets (eMags) system (blue) expressing eMagA-EGFP-CaaX and eMagB-tagRFP stimulated with either 100 nM rapamycin (indicated by gray bar) or 488 nm laser light (indicated by cyan bar). The thin lines represent measurements of single cells, the thick lines represent the mean, and the ribbon represents the 95% confidence interval. The number of cells per condition is: iLID n=17, eMags n=12, rapamycin n=11. The data is from two biological replicates based on independent transfections. (B) Change in membrane to cytosol intensity ratio as percentage for the localization of the PIP3 sensor. All Hela cells were expressing the PIP3 location sensor mCherry-Akt-PH and for the iLID system (purple) Venus-iLID-CaaX, iSH-iRFP-SspB, for the eMags system (blue) eMagA-eGFP-CaaX and eMagB-iSH-iRFP670 and for the rapamycin system (green) Lck-FRB-mTurquoise2 and mNeonGreen-FKBP12-iSH. Stimulated with either 100 nM rapamycin or 488 nm laser light each frame. Comparing the ratio of membrane over cytosol intensity of the PIP3 sensor for 0 s pre-activation and 50 s of activation. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar.

Article Snippet: C1- Lck- mTurquoise2- iLID was created by ligating the backbone Lck- mTurquoise2 (addgene #98822) and the PCR- amplified insert iLID, from Venus- iLIDCaaX (addgene #604110) with primers shown in Table 1, both digested with BsrGI and XhoI.

Techniques: Membrane, Expressing, Transfection, Activation Assay

Journal: eLife

Article Title: Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

doi: 10.7554/elife.84364

Figure Lengend Snippet: Figure 2. Photo-activation of optogenetically recruitable RhoGEFs (Opto-RhoGEFs) controls permeability and vascular barrier strength. (A) Fluorescence intensity measured in a transwell assay for a monolayer of blood outgrowth endothelial cells (BOECs) stably expressing Lck- mTurquoise2-iLID, solely as a control, and SspB-HaloTag-TIAM1(DHPH) treated with FITC 0.3 kDa, FITC dextran 10 kDa, and FITC dextran 70 kDa. Photo-activated with blue LED light for 10 min as indicated by cyan background in the graph or kept in the dark indicated by white background. Dots represent individual transwell dishes. The black bar indicates the mean. The number of transwell dishes per condition is: Control Lck-iLID=9, OptoTIAM dark = 6, OptoTIAM light = 9. The data is from three experiments. (B) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (gray), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with electrical cell- substrate impedance sensing (ECIS) at 4000 Hz, representing paracellular permeability, every 10 s. Cyan bars indicated photo-activation with blue LED light (1 min, 5 min, 10 min, 3×15 min, 120 min). Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. The number of wells per condition was: OptoTIAM = 6, OptoITSN = 6, OptoP63=6, control = 4. (C) Representative zoom-ins from confocal

Article Snippet: C1- Lck- mTurquoise2- iLID was created by ligating the backbone Lck- mTurquoise2 (addgene #98822) and the PCR- amplified insert iLID, from Venus- iLIDCaaX (addgene #604110) with primers shown in Table 1, both digested with BsrGI and XhoI.

Techniques: Activation Assay, Permeability, Fluorescence, Transwell Assay, Stable Transfection, Expressing, Control

Journal: eLife

Article Title: Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

doi: 10.7554/elife.84364

Figure Lengend Snippet: Figure 3. Role of junctions in optogenetically recruitable RhoGEF (Opto-RhoGEF)-induced changes in vascular barrier strength. (A) Linearity index for a blood outgrowth endothelial cell (BOEC) monolayer stably expressing Lck-mTurquoise2-iLID and either SspB-HaloTag-TIAM1(DHPH) (purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) for the frame before photo-activation (pre) and the last frame of photo-activation (Activation). Images of the junctions are in Figure 2—figure supplement 2 and example of linearity index analysis in Figure 3—figure supplement 1A. Each

Article Snippet: C1- Lck- mTurquoise2- iLID was created by ligating the backbone Lck- mTurquoise2 (addgene #98822) and the PCR- amplified insert iLID, from Venus- iLIDCaaX (addgene #604110) with primers shown in Table 1, both digested with BsrGI and XhoI.

Techniques: Stable Transfection, Expressing, Activation Assay

Journal: eLife

Article Title: Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

doi: 10.7554/elife.84364

Figure Lengend Snippet: Figure 4. Characterization of global photo-activation of blood outgrowth endothelial cells (BOECs) expressing optogenetically recruitable RhoGEFs (Opto-RhoGEFs). (A) Cell area change during 10 min photo-activation with a 442 nm laser line for BOECs stably expressing Lck-mTurquoise2-iLID, solely as control, and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH). The images show an overlay of the cell area before and after the photo-activation and colors indicate either contraction or extension. (B) Normalized cell area over time for the cells depicted in A. Control = gray, OptoTIAM=purple, OptoITSN=blue, OptoP63=green. The cyan bar indicates photo-activation with 442 nm laser light. Thin lines represent individual cells, thick lines represent the mean values, and ribbons represent their 95% confidence interval. The number of analyzed cells is: Control

Article Snippet: C1- Lck- mTurquoise2- iLID was created by ligating the backbone Lck- mTurquoise2 (addgene #98822) and the PCR- amplified insert iLID, from Venus- iLIDCaaX (addgene #604110) with primers shown in Table 1, both digested with BsrGI and XhoI.

Techniques: Activation Assay, Expressing, Stable Transfection, Control

Journal: eLife

Article Title: Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

doi: 10.7554/elife.84364

Figure Lengend Snippet: Figure 5. Characterization of local photo-activation of blood outgrowth endothelial cells (BOECs) expressing optogenetically recruitable RhoGEFs (Opto-RhoGEFs). (A) Cell area change during two 10 min local photo-activations with 442 nm laser line for BOECs stably expressing Lck-mTurquoise2- iLID and either SspB-HaloTag-TIAM1(DHPH)/ITSN1(DHPH) or p63RhoGEF(DH). The images show an overlay of the cell area before and after the activation and colors indicate either contraction or extension. The gray dashed box indicates the area of activation. (B) Activation area covered by cell, pre- and post-10 min activation with 442 nm laser line, for BOECs stably expressing Lck-mTurquoise2-iLID and either SspB-HaloTag-TIAM1(DHPH) (purple)/ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green). Small dots represent individual activation areas, which are connected by lines. Larger dots represent the mean of each replicate indicated by different colors. The number of activation areas for OptoTIAM=53, for OptoITSN=85, OptoP63=61. (C) Representative confocal microscopy images of BOECs stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye. Additionally, stained for F-actin with SiR-actin. Scale bars: 10 µm. Times are min:s from the start of the photo-activation. Cyan bar indicates 442 nm photo-activation and gray box indicates area of activation.

Article Snippet: C1- Lck- mTurquoise2- iLID was created by ligating the backbone Lck- mTurquoise2 (addgene #98822) and the PCR- amplified insert iLID, from Venus- iLIDCaaX (addgene #604110) with primers shown in Table 1, both digested with BsrGI and XhoI.

Techniques: Activation Assay, Expressing, Stable Transfection, Confocal Microscopy, Staining