Source 1primary paper2019Small GTPases
Toolkit/single cell FRET measurements
single cell FRET measurements
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Single cell FRET measurements with Rho GTPase biosensors are a quantitative cell-based assay used in primary human endothelial cells to monitor guanine nucleotide exchange factor activity toward Cdc42 and Rac1. In the cited study, the method was applied to compare the cellular activities of overexpressed endothelial GEFs.
Usefulness & Problems
Why this is useful
This assay is useful for measuring Rho GTPase pathway activation in individual primary human endothelial cells rather than relying on bulk readouts. It enabled comparative profiling of endothelial GEF efficiency toward Cdc42 and Rac1 in a cellular context.
Problem solved
The method addresses the problem of determining which endothelial GEFs increase Cdc42 activity, and whether they also affect Rac1, in primary human endothelial cells. It provides a way to characterize GEFs that may directly or indirectly activate Cdc42 using a live-cell biosensor readout.
Problem links
Need conditional control of signaling activity
DerivedSingle cell FRET measurements with Rho GTPase biosensors constitute a quantitative cell-based assay used in primary human endothelial cells to monitor guanine nucleotide exchange factor (GEF) efficiency toward Cdc42 and Rac1. In the cited study, the method enabled comparison of the cellular activities of overexpressed full-length endothelial GEFs.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Mechanisms
biosensor-based detection of rho gtpase activationbiosensor-based detection of rho gtpase activationförster resonance energy transferförster resonance energy transfer (fret)Techniques
Functional AssayTarget processes
signalingImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: sensor
The reported implementation used Rho GTPase biosensors and single-cell FRET measurements in primary human endothelial cells. The study context involved comparison of overexpressed full-length endothelial GEFs, but the supplied evidence does not provide construct architecture, fluorophore pair, instrumentation, or analysis workflow details.
The supplied evidence supports use in primary human endothelial cells for Cdc42 and Rac1 monitoring, but does not establish performance in other cell types, organisms, or signaling systems. The evidence also comes from a single cited study, with limited methodological detail on dynamic range, sensitivity, or reproducibility.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
PLEKHG2, FGD1, PLEKHG1, and PREX1 induced the most efficient Cdc42 activation among the tested endothelial GEFs, and FGD1 showed the highest selectivity.
Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
The study characterized endothelial GEFs that may directly or indirectly activate Cdc42.
Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
A new single cell-based analysis was developed to enable quantitative comparison of cellular activities of overexpressed full-length GEFs.
A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs.
Approval Evidence
1 source1 linked approval claimfirst-pass slug single-cell-fret-measurements
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Source:
assay applicationsupports
Single cell FRET measurements with Rho GTPase biosensors were used in primary human endothelial cells to monitor GEF efficiency toward Cdc42 and Rac1.
By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1.
Source:
Comparisons
Source-backed strengths
The assay was performed at single-cell resolution in primary human endothelial cells using Rho GTPase biosensors, supporting quantitative comparison of GEF efficiency toward Cdc42 and Rac1. In the reported application, it resolved activity differences among endothelial GEFs, including strong Cdc42 activation by PLEKHG2, FGD1, PLEKHG1, and PREX1, with FGD1 showing the highest selectivity.
Compared with IRAP-pHluorin translocation assay
single cell FRET measurements and IRAP-pHluorin translocation assay address a similar problem space because they share signaling.
Shared frame: same top-level item type; shared target processes: signaling
Strengths here: looks easier to implement in practice.
Compared with multicomponent, ligand-functionalized microarrays
single cell FRET measurements and multicomponent, ligand-functionalized microarrays address a similar problem space because they share signaling.
Shared frame: same top-level item type; shared target processes: signaling
single cell FRET measurements and root-specific transcriptomic dataset comparison for ethylene responses address a similar problem space because they share signaling.
Shared frame: same top-level item type; shared target processes: signaling
Ranked Citations
- 1.