Journal: Nature Communications

Article Title: Spatiotemporal coupling of caveolae mechanosensing and RhoA-GEFs regulates cell polarity and directional migration

doi: 10.1038/s41467-025-67090-z

Figure Lengend Snippet: a–e Representative time-lapse images of Hs578t cells expressing optogenetic constructs (Lck-mTurquoise2-iLID, SspB-HaloTag-ARHGEF25-DH (ARHGEF25), and Cav1-RFP), in the following conditions: ( a ) WT, ( b ) cavin1-KO, ( c ) cavin1-KO+cavin1, ( d ) Cav1-KO, ( e ) Cav1-KO+wtCav1. Insets show zoomed ROI corresponding to optogenetic activation. Timepoints span from -2 min (pre-activation) to +16 min (opto-activation). Normalized intensities of ARHGEF25 (orange), Cav1 (gray), and cell area (blue) in the opto-activated ROI are plotted over time. Data are presented as mean values ± SEM., n = 21 (WT), 16 (cavin1-KO), 22 (cavin1-KO+cavin1), 19 (Cav1-KO), and 23 (Cav1-KO+wtCav1) cells from 3 independent experiments. f Representative time-lapse images of WT- and cavin1-KO Hs578t cells expressing Halo-ARHGEF25 and Cav1-RFP. White arrow indicate direction of migration. g Quantification of ARHGEF25 and Cav1 fluorescence intensity at the front and rear. h Quantification at the cell periphery. Data are presented as mean values ± SEM., n = 32 (WT) and 43 (cavin1-KO) cells from 3 independent experiments. Statistical tests: Two-tailed, Wilcoxon matched-pairs signed-rank test ( g ); Two-tailed, Mann-Whitney t-test ( h ); ****P < 0.0001; ns = not significant. i Representative time-lapse images of random contracting events in WT- and cavin1-KO Hs578t cells expressing Halo-ARHGEF25 and Cav1-RFP. Plots show normalized intensity and cell area: ARHGEF25 (black), Cav1 (purple), and cell area (blue). Data are presented as mean values ± SEM., n = 90 (WT) and 42 (cavin1-KO) contraction events pooled from 3 independent experiments. Scale bars, 10 µm. F, front; R, rear. Fluo., fluorescence. Source numerical data are provided.

Article Snippet: HaloTag-3xrGBD (Addgene #176108) (RhoA biosensor), ARHGEF25 plasmid for optogenetics, pLV-SspB-HaloTag-p63DH (Addgene #176129), Lck-mTurquoise2-iLID (Addgene #176125), Tiam-1 GEF and pLV-SspB-HaloTag-DHPH-TIAM1 plasmids for optogenetics (Addgene#176114) were gifts from Dr. Dorus Gadella (University of Amsterdam, Netherlands).

Techniques: Expressing, Construct, Activation Assay, Migration, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

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: 10.1101/2022.10.17.512253

Figure Lengend Snippet: ( A ) Normalized Membrane to Cytosol Intensity Ratio for the rapamycin system (green) HeLa cells expressing Lck-FRB-T2098L-mTurquoise-IRES-sYFP2xNES-FKBP12, for the iLID system (purple) expressing Venus-iLID-CaaX and SspB-mScarlet-I and for the eMags system (blue) expressing eMagA-EGFP-CaaX and eMagB-tagRFP stimulated with either 100 nM rapamycin (indicated by grey bar) or 488 nm laser light (indicated by cyan bar). The thin lines represent measurements of single cells, the thick line represent the mean and the ribbon represent 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 and 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. The number of cells per condition is: iLID = 29, eMags = 20, rapamycin = 15. ( C ) Representative confocal images for the in B described conditions. The PIP3 sensor intensity is depicted with the mpl-inferno look up table, where brighter colors represent higher fluorescent intensities. Scale bars: 10 µm. ( D ) Change in cytosolic intensity in percentage for HeLa cells expressing SspB-mScarlet-I and either Lck-mTurquoise2-iLID or Venus-iLID-CaaX measured 20 s after stimulation with 1 pulse of 440 nm laser light at 20% for 1 s. Images acquired at a spinning disk microscope. Each dot represents and 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. The number of cells per condition is: CaaX=43, Lck=44. The data is from three biological replicates based on independent transfections. ( E ) Schematic of the light-induced heterodimerization of iLID. Upon photo-activation the Ja helix in the iLID unfolds, SsrA becomes available for binding by SspB which is recruited form the cytosol to the location of SsrA, which is localized at the plasma membrane. The GEF fused to the SspB activates the Rho GPTase at the plasma membrane, which is binding GTP and thereby activates its signaling cascade. That results in the remodeling of the actin cytoskeleton and change in cell morphology. The combination of fluorescent markers and GEFs fused to SspB in this study is indicated in the bottom. ( F ) Normalized cytosolic intensity for the HaloTag-3xrGBD Rho sensor (purple) or control HaloTag (gray) stained with JF635 nm upon the photo activation (indicated by cyan bar) of SspB-mCherry-p63RhoGEF(DH), expressed in HeLa cells together with Lck-mTurquoise2-iLID. Thin lines represent individual cells, thick lines represent the mean and ribbons represent their 95% confidence interval. The number of cells per condition: Rho sensor HaloTag-3xrGBD=18, Control HaloTag=30. The data is from two biological replicates based on independent transfections. ( G ) Normalized cytosolic intensity for the dimericTomato-wGBD Cdc42 sensor (purple) or control dimericTomato (gray) upon the photo activation (indicated by cyan bar) of SspB-HaloTag-ITSN1(DHPH) stained with JF635 nm, expressed in HeLa cells together with Lck-mTurquoise2-iLID. Thin lines represent individual cells, thick lines represent the mean and ribbons represent their 95% confidence interval. The number of cells per condition: Cdc42 sensor dimericTomato-wGBD=6, Control dimericTomato=7. The data is from two biological replicates based on independent transfections.

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

Techniques: Membrane, Expressing, Transfection, Activation Assay, Microscopy, Binding Assay, Clinical Proteomics, Control, Staining

Journal: bioRxiv

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: 10.1101/2022.10.17.512253

Figure Lengend Snippet: ( 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 are 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 . Control=grey, Opto-TIAM=purple, Opto-ITSN=blue, Opto-P63=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=9, Opto-TIAM=9, Opto-ITSN=9, Opto-P63=9. The data is from two independent experiments and at least three independent photo-activations. ( C ) Representative confocal microscopy images of BOECs stably expressing Lck-mTurquoise2-iLID (not shown), solely as control, 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 recording. Cyan bar indicates 442 nm photo-activation. Gray box indicates zoom. Gray dashed line indicates cell edge.

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

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

Journal: bioRxiv

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: 10.1101/2022.10.17.512253

Figure Lengend Snippet: ( A ) Fluorescence intensity measured in a transwell assay for a monolayer of 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, OpotTIAM_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 (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (1 min, 5 min, 10 min, 3x 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 microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white). Scale bars: 25 µm. Times are min:s from the start of the recording. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer. Whole field of view is shown in S4.2 .

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

Techniques: Fluorescence, Transwell Assay, Stable Transfection, Expressing, Control, Activation Assay, Confocal Microscopy, Staining, Labeling

Journal: bioRxiv

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: 10.1101/2022.10.17.512253

Figure Lengend Snippet: ( A )Linearity Index for a 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 Fig. S2.2 and example of linearity index analysis in Fig. S3.1A . Each dot represents and 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. The number of cells is: Opto-TIAM pre=55, Opto-TIAM Activation=52, Opto-ITSN pre=71, Opto-ITSN Activation=76, Opto-P63 pre=74, Opto-P63 Activation=51. The data is from two independent experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (60 min, 15 min). Gray bar with dashed lines indicates the addition of VE-cadherin blocking antibody in medium (darker color line) or medium as a control (lighter color line). At the end of the gray bar the medium is replaced for all conditions. Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. Four wells were measured for each condition. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white in merge, grey inverted in single channel). Left panel shows untreated cells, right panel shows cells treated with the VE-cadherin blocking antibody. Scale bars: 25 µm. Times are min:s from the start of the recording. Gray bar indicates the condition before photo activation. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer.

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

Techniques: Stable Transfection, Expressing, Activation Assay, Control, Blocking Assay, Confocal Microscopy, Staining, Labeling

Journal: bioRxiv

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: 10.1101/2022.10.17.512253

Figure Lengend Snippet: ( 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 Opto-TIAM=53, for Opto-ITSN=85, Opto-P63=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-iLID-CaaX (addgene #604110) with primers shown in , both digested with BsrGI and XhoI.

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