Source 1primary paper2012The Plant Cell
Toolkit/fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy
fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy
Also known as: FRET-FLIM
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) is an assay method that combines fluorescence resonance energy transfer with fluorescence lifetime imaging to detect molecular proximity in living cells. In the cited Arabidopsis study, it was used to support a physical interaction between CRY2 and SPA1 in nuclei.
Usefulness & Problems
Why this is useful
This assay is useful for testing whether two labeled biomolecules are in close proximity in living cells, using fluorescence lifetime changes as the readout. The supplied evidence specifically supports its use for assessing nuclear interaction between Arabidopsis CRY2 and SPA1.
Problem solved
FRET-FLIM helps address the problem of determining whether proteins are physically associated in a cellular context rather than only inferred from genetics. In the cited work, it contributed evidence relevant to whether CRY2 and SPA1 interact in nuclei.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Techniques
Functional AssayTarget processes
No target processes tagged yet.
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: sensor
The available evidence indicates use in living cells and specifically in nuclei in an Arabidopsis study. No further implementation details, such as construct design, fluorophore pairs, instrumentation, or analysis pipeline, are provided in the supplied material.
The provided evidence is sparse and does not report donor-acceptor fluorophores, lifetime shifts, controls, sensitivity, or false-positive/false-negative behavior. It also does not establish whether the observed proximity reflects direct binding versus colocalization within a complex.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
phyA influences cry2 stability because a phyA mutant has enhanced cry2 levels, particularly under low fluence rate blue light.
a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light
phyA influences cry2 stability because a phyA mutant has enhanced cry2 levels, particularly under low fluence rate blue light.
a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light
phyA influences cry2 stability because a phyA mutant has enhanced cry2 levels, particularly under low fluence rate blue light.
a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light
phyA influences cry2 stability because a phyA mutant has enhanced cry2 levels, particularly under low fluence rate blue light.
a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light
phyA influences cry2 stability because a phyA mutant has enhanced cry2 levels, particularly under low fluence rate blue light.
a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light
phyA influences cry2 stability because a phyA mutant has enhanced cry2 levels, particularly under low fluence rate blue light.
a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light
phyA influences cry2 stability because a phyA mutant has enhanced cry2 levels, particularly under low fluence rate blue light.
a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light
SPA proteins contribute to cry2 degradation because cry2 degradation under continuous blue light is alleviated in spa mutants in a fluence rate-dependent manner.
In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner.
SPA proteins contribute to cry2 degradation because cry2 degradation under continuous blue light is alleviated in spa mutants in a fluence rate-dependent manner.
In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner.
SPA proteins contribute to cry2 degradation because cry2 degradation under continuous blue light is alleviated in spa mutants in a fluence rate-dependent manner.
In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner.
SPA proteins contribute to cry2 degradation because cry2 degradation under continuous blue light is alleviated in spa mutants in a fluence rate-dependent manner.
In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner.
SPA proteins contribute to cry2 degradation because cry2 degradation under continuous blue light is alleviated in spa mutants in a fluence rate-dependent manner.
In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner.
SPA proteins contribute to cry2 degradation because cry2 degradation under continuous blue light is alleviated in spa mutants in a fluence rate-dependent manner.
In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner.
SPA proteins contribute to cry2 degradation because cry2 degradation under continuous blue light is alleviated in spa mutants in a fluence rate-dependent manner.
In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner.
cry2 stability is controlled by SPA and phyA.
Our results suggest that cry2 stability is controlled by SPA and phyA
cry2 stability is controlled by SPA and phyA.
Our results suggest that cry2 stability is controlled by SPA and phyA
cry2 stability is controlled by SPA and phyA.
Our results suggest that cry2 stability is controlled by SPA and phyA
cry2 stability is controlled by SPA and phyA.
Our results suggest that cry2 stability is controlled by SPA and phyA
cry2 stability is controlled by SPA and phyA.
Our results suggest that cry2 stability is controlled by SPA and phyA
cry2 stability is controlled by SPA and phyA.
Our results suggest that cry2 stability is controlled by SPA and phyA
cry2 stability is controlled by SPA and phyA.
Our results suggest that cry2 stability is controlled by SPA and phyA
cry2 physically interacts with SPA1 in nuclei of living cells.
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells.
cry2 physically interacts with SPA1 in nuclei of living cells.
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells.
cry2 physically interacts with SPA1 in nuclei of living cells.
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells.
cry2 physically interacts with SPA1 in nuclei of living cells.
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells.
cry2 physically interacts with SPA1 in nuclei of living cells.
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells.
cry2 physically interacts with SPA1 in nuclei of living cells.
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells.
cry2 physically interacts with SPA1 in nuclei of living cells.
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells.
In etiolated Arabidopsis seedlings exposed to blue light, cry2 protein level strongly decreases and cry2 is phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
In etiolated Arabidopsis seedlings exposed to blue light, cry2 protein level strongly decreases and cry2 is phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
In etiolated Arabidopsis seedlings exposed to blue light, cry2 protein level strongly decreases and cry2 is phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
In etiolated Arabidopsis seedlings exposed to blue light, cry2 protein level strongly decreases and cry2 is phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
In etiolated Arabidopsis seedlings exposed to blue light, cry2 protein level strongly decreases and cry2 is phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
In etiolated Arabidopsis seedlings exposed to blue light, cry2 protein level strongly decreases and cry2 is phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
In etiolated Arabidopsis seedlings exposed to blue light, cry2 protein level strongly decreases and cry2 is phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome.
Approval Evidence
1 source1 linked approval claimfirst-pass slug fluorescence-resonance-energy-transfer-fluorescence-lifetime-imaging-microscopy
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies
Source:
physical interactionsupports
cry2 physically interacts with SPA1 in nuclei of living cells.
Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells.
Source:
Comparisons
Source-backed strengths
The method is described as a living-cell assay for molecular proximity and was applied in Arabidopsis nuclei. The supplied evidence supports its use as interaction-supporting evidence for CRY2-SPA1, but does not provide quantitative performance metrics.
Compared with Langendorff perfused heart electrical recordings
fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy and Langendorff perfused heart electrical recordings address a similar problem space.
Shared frame: same top-level item type
Strengths here: looks easier to implement in practice.
Compared with native green gel system
fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy and native green gel system address a similar problem space.
Shared frame: same top-level item type
Strengths here: looks easier to implement in practice.
Compared with TR-FRET assay
fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy and TR-FRET assay address a similar problem space.
Shared frame: same top-level item type; shared mechanisms: fluorescence resonance energy transfer
Ranked Citations
- 1.