Category Ranking
98%
Total Visits
921
Avg Visit Duration
2 minutes
Citations
20
Article Abstract
Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.3791/61602 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
A fluorescence lifetime-based FLIM-timer for measuring the protein turnover of transcription factor Nrf2 in live cells.
Sci Rep
August 2025
Division of Cellular Medicine, University of Dundee School of Medicine, Dundee, DD1 9SY, UK.
Measuring protein turnover in cells has been greatly assisted by fluorescent timers (FT). However, FT quantification requires relatively high fluorescence intensity samples, prohibiting their use for proteins with low or non-uniform expression like transcription factor Nrf2, the master regulator of redox homeostasis. To visualise changes in stability/turnover of Nrf2, we constructed a genetically encoded tag combining sfGFP and mCherry and used intensity-independent Fluorescence Lifetime Imaging (FLIM) to measure Förster Resonance Energy Transfer (FRET) within the tag (named FLIM-timer).
View Article and Find Full Text PDFCalyculin A Induces Premature Chromosome Condensation and Chromatin Compaction in G-Phase HeLa Cells without Histone H1 Phosphorylation.
bioRxiv
July 2025
Department of Chemistry, University of Wisconsin-Oshkosh, 800 Algoma Blvd, Oshkosh, WI 54901 U.S.A.
We show here that treatment of HeLa cells with calyculin A, an inhibitor of Protein Phosphatases 1 and 2A, induces premature chromosome condensation (PCC) at any point in interphase of the cell cycle. Chromosomes in G-phase PCC closely resemble metaphase chromatids in the light microscope, and measurements using FLIM-FRET show that they have the same level of chromatin compaction as metaphase chromosomes. However, histone H1 is not phosphorylated in G- or early S-phase PCC.
View Article and Find Full Text PDFHyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate the excitability of dorsal root ganglion (DRG) neurons, particularly in the context of neuropathic pain. Cholesterol, a major component of lipid-ordered membrane domains (OMDs), has recently been identified as a critical modulator of HCN channel function. Using FLIM-FRET-based OMD probes and a fluorescent cholesterol sensor GRAM-W, we investigated the effects of cholesterol supplementation on nociceptor DRG neurons from a rat model of spared nerve injury (SNI).
View Article and Find Full Text PDFMultiplexed confocal FLIM for dynamic molecular imaging in live cells.
Discov Imaging
May 2025
Department of Engineering Physics, McMaster University, Hamilton, ON L8S 4L8 Canada.
Quantitative measurements of the dynamics of biomolecular interactions allow biologists to develop a better understanding of biological processes that are critical to new diagnostic tools, drug discovery, and personalized treatments of diseases. Such measurements require multidimensional (spatial, spectral, and temporal) imaging with a high frame rate. Conventional single point confocal microscopy can produce 3D images at video rate but faces difficulties in accurately measuring fluorescence lifetime images (FLIM) while maintaining low excitation power to avoid phototoxicity and photobleaching in live cells.
View Article and Find Full Text PDFFluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates.
J Cell Biol
January 2025
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
Article Synopsis
- Ribonucleoprotein (RNP) condensates create distinct liquid phases that make it hard to study their functions versus the surrounding environment.
- Researchers implemented fluorescence lifetime imaging microscopy (FLIM) alongside phasor plot filtering to better differentiate these condensates and assess protein interactions using FLIM-Förster resonance energy transfer (FRET).
- They found that interactions among decapping complex subunits in P-bodies change based on conditions, particularly observing altered interactions during oxidative stress, highlighting the dynamic nature of these protein networks in live cells.