High Resolution Fluorescence Lifetime Maps from Minimal Photon Counts
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ABSTRACT:
SUBMITTER: Fazel M
PROVIDER: S-EPMC9278809 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fluorescence lifetime imaging microscopy presents a powerful tool in biology and biophysics because it allows the investigation of the local environment of a fluorochrome in living cells in a quantitative manner. Furthermore, imaging Förster-type resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy enables protein-protein interactions and intermolecular distances to be mapped under physiological conditions. Quantitative and precise data analysis methods are required to access the richness of information that is contained in FRET data on biological samples. Lifetime detection in the frequency-domain yields two lifetime estimations. The lifetime moments analysis (LiMA) provides a quantitative measure of the lifetime distribution broadness by exploiting the analytical relationship between the phase- and demodulation-lifetime estimations and relating them to the weighted average and variance of the lifetime distribution. The LiMA theoretical framework is validated by comparison with global analysis and by applying it to a constrained two-component FRET system using simulations and experiments. Furthermore, a novel LIMA-based error analysis and a more intuitive formalism for global analysis are presented. Finally, a new method to resolve a FRET system is proposed and experimentally applied to the investigation of protein-protein interactions.
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Project description:BackgroundThe human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.ResultsResults show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three alpha helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the alpha-helices could perturb the leucine zipper like motifs. The DeltaQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.ConclusionWe report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three alpha helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.
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