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mneongreen 3xrgbd (Addgene inc)

Name: mneongreen 3xrgbd
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The optimized dimericTomato-2xrGBD Rho sensor has the best location efficiency. (A) Spinning disk still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD Rho sensor (upper panel) or CMVdel-dimericTomato-2xrGBD Rho sensor (lower panel) and H1R (not shown), which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. The images match with the perturbation that is indicated for the plot in C. Times are min:s from the start of the recording. Scale bars: 25 µm. (B) Change in cytosolic intensity for CMV-mNeonGreen, CMV-eGFP-1xrGBD, CMVdel-3xmNeonGreen-1xrGBD, CMVdel-mNeonGreen-1xrGBD, -2xrGBD and <t>-3xrGBD,</t> and CMVdel-dimericTomato-1xrGBD and -2xrGBD (schematics on left) in HeLa cells expressing H1R, upon stimulation with 100 μM histamine. 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. The gray dashed line indicates no change in cytosolic intensity. The data is from two biological replicates based on two independent transfections. The number of cells per condition is: 3xmNG-1xrGBD=27, dT-1xrGBD=32, dT-2xrGBD=33, eGFP-1xrGBD=40, mNG=39, mNG-1xrGBD=28, mNG-2xrGBD=34, mNG-3xrGBD=26. (C) Time traces of the change in cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in gray and for the dimericTomato-2xrGBD sensor in black. mNG, mNeonGreen; dT, dimericTomato; rGBD, rhotekin G protein binding domain.

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1) Product Images from "Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor"

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

Journal: Journal of Cell Science

doi: 10.1242/jcs.258823

Figure Legend Snippet: The optimized dimericTomato-2xrGBD Rho sensor has the best location efficiency. (A) Spinning disk still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD Rho sensor (upper panel) or CMVdel-dimericTomato-2xrGBD Rho sensor (lower panel) and H1R (not shown), which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. The images match with the perturbation that is indicated for the plot in C. Times are min:s from the start of the recording. Scale bars: 25 µm. (B) Change in cytosolic intensity for CMV-mNeonGreen, CMV-eGFP-1xrGBD, CMVdel-3xmNeonGreen-1xrGBD, CMVdel-mNeonGreen-1xrGBD, -2xrGBD and -3xrGBD, and CMVdel-dimericTomato-1xrGBD and -2xrGBD (schematics on left) in HeLa cells expressing H1R, upon stimulation with 100 μM histamine. 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. The gray dashed line indicates no change in cytosolic intensity. The data is from two biological replicates based on two independent transfections. The number of cells per condition is: 3xmNG-1xrGBD=27, dT-1xrGBD=32, dT-2xrGBD=33, eGFP-1xrGBD=40, mNG=39, mNG-1xrGBD=28, mNG-2xrGBD=34, mNG-3xrGBD=26. (C) Time traces of the change in cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in gray and for the dimericTomato-2xrGBD sensor in black. mNG, mNeonGreen; dT, dimericTomato; rGBD, rhotekin G protein binding domain.

Techniques Used: Expressing, Transfection, Protein Binding

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Journal: Journal of Cell Science

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

doi: 10.1242/jcs.258823

Figure Lengend Snippet: The optimized dimericTomato-2xrGBD Rho sensor has the best location efficiency. (A) Spinning disk still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD Rho sensor (upper panel) or CMVdel-dimericTomato-2xrGBD Rho sensor (lower panel) and H1R (not shown), which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. The images match with the perturbation that is indicated for the plot in C. Times are min:s from the start of the recording. Scale bars: 25 µm. (B) Change in cytosolic intensity for CMV-mNeonGreen, CMV-eGFP-1xrGBD, CMVdel-3xmNeonGreen-1xrGBD, CMVdel-mNeonGreen-1xrGBD, -2xrGBD and -3xrGBD, and CMVdel-dimericTomato-1xrGBD and -2xrGBD (schematics on left) in HeLa cells expressing H1R, upon stimulation with 100 μM histamine. 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. The gray dashed line indicates no change in cytosolic intensity. The data is from two biological replicates based on two independent transfections. The number of cells per condition is: 3xmNG-1xrGBD=27, dT-1xrGBD=32, dT-2xrGBD=33, eGFP-1xrGBD=40, mNG=39, mNG-1xrGBD=28, mNG-2xrGBD=34, mNG-3xrGBD=26. (C) Time traces of the change in cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in gray and for the dimericTomato-2xrGBD sensor in black. mNG, mNeonGreen; dT, dimericTomato; rGBD, rhotekin G protein binding domain.

Article Snippet: The following plasmids, generated in this study, are available on Addgene ( http://www.addgene.org/ ): 129625 , dTomato-2xrGBD (C1 vector); 176098 , dimericTomato-2xrGBD (pLV vector); 129624 , mNeongreen-2xrGBD; 176091 , mNeonGreen-3xrGBD; 129633 , mNeonGreen-aGBD (anillin); 129634 , mNeonGreen-pGBD (PKN1 codon optimized); 176094 , H2A-mTurquoise2-CDC42-G12V-ΔCaaX; 176095 , H2A-mTurquoise2-RAC1-G12V-ΔCaaX; and 176097 , H2A-mTurquoise2-RHOA-G14V-ΔCaaX.

Techniques: Expressing, Transfection, Protein Binding

Journal: Journal of Cell Science

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

doi: 10.1242/jcs.258823

Techniques: Expressing, Transfection, Protein Binding

The GBDs of anillin and PKN1 are not suitable for a relocation Rho sensor. (A) Change in cytosolic intensity for CMVdel-mNeonGreen-1xpGBD, -2xpGBD and -3xpGBD, and CMVdel-dimericTomato-2xpGBD co expressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. 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. The number of samples per condition is: dT-2xpGBD=16, mNG-1xpGBD=24, mNG-2xpGBD=28, mNG-3xpGBD=14. (B) Spinning disk images showing colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-2xpGBD in HeLa cells. Scale bars: 20 µm. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. 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. The number of cells from a single replicate per condition is: H2A-dT-2xpGBD=19, H2A-RhoA-dT2xpGBD=16. (C) Change in cytosolic intensity for CMVdel-mNeonGreen-1xaGBD, -2xaGBD and -3xaGBD, CMVdel-dimericTomato-1xaGBD and eGFP-anillin(AHD+PH) coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. 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. The number of cells per condition is: dT-1xaGBD=14, eGFP-AHD+PH=27, mNG-1xaGBD=17, mNG-2xaGBD=10, mNG-3xaGBD=13. (D) Spinning disk images showing colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-1xaGBD and mScarlet-I-anillin(AHD+PH) in HeLa cells. Scale bars: 20 µm. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX-expressing HeLa cells. The dashed line indicates a ratio of one. 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 from a single replicate per condition is: H2A-aGBD=12, H2A-RhoA-aGBD=10, H2A-RhoA-AHD+PH=13. (E) Amino acid sequence alignment for aGBD, rGBD and pGBD from MUSCLE and depicted with the clustalX color code, where green is polar, blue is hydrophobic, purple is negative charge, red is positive charge, yellow is prolines, orange is glycines, cyan is aromatic and white is unconserved. (F) Crystal structures of PKN1 G-protein binding domain (purple) and anillin G-protein binding domain (yellow), bound to RhoA GTP (gray). On the left, a structural alignment of PKN1 and anillin by their G-protein binding domains. On the right, a structural alignment of the bound RhoA molecules, showing the two binding positions at the RhoA molecule (PDB: anillin, 4XOI ; PKN1, 1CXZ ).

Journal: Journal of Cell Science

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

doi: 10.1242/jcs.258823

Figure Lengend Snippet: The GBDs of anillin and PKN1 are not suitable for a relocation Rho sensor. (A) Change in cytosolic intensity for CMVdel-mNeonGreen-1xpGBD, -2xpGBD and -3xpGBD, and CMVdel-dimericTomato-2xpGBD co expressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. 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. The number of samples per condition is: dT-2xpGBD=16, mNG-1xpGBD=24, mNG-2xpGBD=28, mNG-3xpGBD=14. (B) Spinning disk images showing colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-2xpGBD in HeLa cells. Scale bars: 20 µm. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. 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. The number of cells from a single replicate per condition is: H2A-dT-2xpGBD=19, H2A-RhoA-dT2xpGBD=16. (C) Change in cytosolic intensity for CMVdel-mNeonGreen-1xaGBD, -2xaGBD and -3xaGBD, CMVdel-dimericTomato-1xaGBD and eGFP-anillin(AHD+PH) coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. 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. The number of cells per condition is: dT-1xaGBD=14, eGFP-AHD+PH=27, mNG-1xaGBD=17, mNG-2xaGBD=10, mNG-3xaGBD=13. (D) Spinning disk images showing colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-1xaGBD and mScarlet-I-anillin(AHD+PH) in HeLa cells. Scale bars: 20 µm. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX-expressing HeLa cells. The dashed line indicates a ratio of one. 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 from a single replicate per condition is: H2A-aGBD=12, H2A-RhoA-aGBD=10, H2A-RhoA-AHD+PH=13. (E) Amino acid sequence alignment for aGBD, rGBD and pGBD from MUSCLE and depicted with the clustalX color code, where green is polar, blue is hydrophobic, purple is negative charge, red is positive charge, yellow is prolines, orange is glycines, cyan is aromatic and white is unconserved. (F) Crystal structures of PKN1 G-protein binding domain (purple) and anillin G-protein binding domain (yellow), bound to RhoA GTP (gray). On the left, a structural alignment of PKN1 and anillin by their G-protein binding domains. On the right, a structural alignment of the bound RhoA molecules, showing the two binding positions at the RhoA molecule (PDB: anillin, 4XOI ; PKN1, 1CXZ ).

Techniques: Binding Assay, Expressing, Sequencing, Protein Binding

A ) Still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD RhoA sensor (upper panel) or CMVdel-dimericTomato-2xrGBD RhoA sensor (lower panel) and H1R (not shown) which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. B ) Change in cytosolic intensity for mNeonGreen-1xrGBD/ - 2xrGBD/ -3xrGBD, 3xmNeonGreen-1xrGBD and dimericTomato-1xrGBD/ -2xrGBD in Hela cells expressing H1R, upon stimulation with 100 μM histamine. Each dot represents an individual cell. The median of the data is shown as vertical, black line and the grey dashed line indicates no change in cytosolic intensity. The number of samples per condition is: 3xmNG-1xrGBD=16, mNG-1xrGBD=28, dT-1xrGBD=15, mNG-2xrGBD=34, mNG-3xrGBD=14, dT-2xrGBD=14. C ) Time traces of the normalized cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in grey and for the dimericTomato-2xrGBD sensor in black. Abbreviations: mNG: mNeonGreen, dT: dimericTomato, rGBD: rhotekin G protein binding domain

Journal: bioRxiv

Article Title: Visualizing endogenous RhoA activity with an improved localization-based, genetically encoded biosensor

doi: 10.1101/2021.02.08.430250

Figure Lengend Snippet: A ) Still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD RhoA sensor (upper panel) or CMVdel-dimericTomato-2xrGBD RhoA sensor (lower panel) and H1R (not shown) which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. B ) Change in cytosolic intensity for mNeonGreen-1xrGBD/ - 2xrGBD/ -3xrGBD, 3xmNeonGreen-1xrGBD and dimericTomato-1xrGBD/ -2xrGBD in Hela cells expressing H1R, upon stimulation with 100 μM histamine. Each dot represents an individual cell. The median of the data is shown as vertical, black line and the grey dashed line indicates no change in cytosolic intensity. The number of samples per condition is: 3xmNG-1xrGBD=16, mNG-1xrGBD=28, dT-1xrGBD=15, mNG-2xrGBD=34, mNG-3xrGBD=14, dT-2xrGBD=14. C ) Time traces of the normalized cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in grey and for the dimericTomato-2xrGBD sensor in black. Abbreviations: mNG: mNeonGreen, dT: dimericTomato, rGBD: rhotekin G protein binding domain

Article Snippet: The following plasmids are available on addgene ( http://www.addgene.org/ ): # 129633: mNeongreen-aGBD(anillin), # 129634: mNeongreen-pGBD(PKN1 codon optimized), # 129624: mNeongreen-2xrGBD, # 129625: dTomato-2xrGBD.

Techniques: Expressing, Protein Binding

A ) Change in cytosolic intensity for CMVdel-mNeonGreen-1xpGBD/ -2xpGBD/ -3xpGBD, CMVdel-dimericTomato-2xpGBD coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as vertical, black line. The number of samples per condition is: dT-2xpGBD=16, mNG-1xpGBD=24, mNG-2xpGBD=28, mNG-3xpGBD=14. B ) Colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-2xpGBD in HeLa cells. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. Each dot represents an individual cell. The median of the data is shown as a vertical, black line. The number of samples per condition is:H2A-dT-2xpGBD=19, RhoA-dT2xpGBD=16. C ) Change in cytosolic intensity for CMVdel-mNeonGreen-1xaGBD/ -2xaGBD/ -3xaGBD, CMVdel-dimericTomato-1xaGBD coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as vertical, black line. The number of samples per condition is: dT-1xaGBD=14, eGFP-AHD+PH=27, mNG-1xaGBD=17, mNG-2xaGBD=7, mNG-3xaGBD=9. D ) Colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-1xaGBD and mScarlet-I-AHD+PH in HeLa cells. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. The median of the data is shown as a vertical, black line. The number of samples per condition is: H2A-aGBD=12, RhoA-aGBD=10, RhoA-AHD=9. E ) Amino acid sequence alignment for aGBD, rGBD and pGBD by MUSCLE depicted with the clustalX color code. F ) On the left a structural alignment of PKN1 and Anillin by their RhoA binding domains. On the right a structural alignment of PKN1 and anillin by RhoA, showing the two binding positions at the RhoA molecule. Anillin and the bound RhoA are depicted in dark and light yellow, respectively. PKN1 and the bound RhoA are depicted in light and dark blue, respectively. (PDB: Anillin = 4xOI, PKN1 = 1cxz).

Journal: bioRxiv

Article Title: Visualizing endogenous RhoA activity with an improved localization-based, genetically encoded biosensor

doi: 10.1101/2021.02.08.430250

Figure Lengend Snippet: A ) Change in cytosolic intensity for CMVdel-mNeonGreen-1xpGBD/ -2xpGBD/ -3xpGBD, CMVdel-dimericTomato-2xpGBD coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as vertical, black line. The number of samples per condition is: dT-2xpGBD=16, mNG-1xpGBD=24, mNG-2xpGBD=28, mNG-3xpGBD=14. B ) Colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-2xpGBD in HeLa cells. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. Each dot represents an individual cell. The median of the data is shown as a vertical, black line. The number of samples per condition is:H2A-dT-2xpGBD=19, RhoA-dT2xpGBD=16. C ) Change in cytosolic intensity for CMVdel-mNeonGreen-1xaGBD/ -2xaGBD/ -3xaGBD, CMVdel-dimericTomato-1xaGBD coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as vertical, black line. The number of samples per condition is: dT-1xaGBD=14, eGFP-AHD+PH=27, mNG-1xaGBD=17, mNG-2xaGBD=7, mNG-3xaGBD=9. D ) Colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-1xaGBD and mScarlet-I-AHD+PH in HeLa cells. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. The median of the data is shown as a vertical, black line. The number of samples per condition is: H2A-aGBD=12, RhoA-aGBD=10, RhoA-AHD=9. E ) Amino acid sequence alignment for aGBD, rGBD and pGBD by MUSCLE depicted with the clustalX color code. F ) On the left a structural alignment of PKN1 and Anillin by their RhoA binding domains. On the right a structural alignment of PKN1 and anillin by RhoA, showing the two binding positions at the RhoA molecule. Anillin and the bound RhoA are depicted in dark and light yellow, respectively. PKN1 and the bound RhoA are depicted in light and dark blue, respectively. (PDB: Anillin = 4xOI, PKN1 = 1cxz).

Techniques: Binding Assay, Expressing, Sequencing

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