Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

Devauges, Viviane; Matthews, Daniel R.; Aluko, Justin; Nedbal, Jakub; Levitt, James A.; Poland, Simon P.; Coban, Oana; Weitsman, Gregory; Monypenny, James; Ng, Tony; Ameer-Beg, Simon

doi:10.1371/journal.pone.0110695

Cited by 10 publications

(3 citation statements)

References 94 publications

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“…The g factor accounts for any systemic differences introduced by the optics or detectors into the parallel vs. perpendicular channel and this was calibrated in our system by measurement of fluorescein, a fast-rotating molecule whose emission polarization should be isotropic. Depolarization effects caused by use of a high numerical aperture objective were taken-into-account by incorporation of an additional correction factor into r that was derived from referencing eGFP anisotropy values acquired with a 60X water objective to eGFP anisotropy values acquired with a ×10 air objective 50 . After application of this correction factor to FAIM measurements acquired in live DiVA cells expressing free eGFP we find that in our system the fluorescence anisotropy value of eGFP in the absence of homo-FRET ( r eGFP ) is 0.32.…”

Section: Methodsmentioning

confidence: 99%

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

et al. 2020

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Tumor suppressor p53-binding protein 1 (53BP1) is a DNA repair protein essential for the detection, assessment, and resolution of DNA double strand breaks (DSBs). The presence of a DSB is signaled to 53BP1 via a local histone modification cascade that triggers the binding of 53BP1 dimers to chromatin flanking this type of lesion. While biochemical studies have established that 53BP1 exists as a dimer, it has never been shown in a living cell when or where 53BP1 dimerizes upon recruitment to a DSB site, or upon arrival at this nuclear location, how the DSB histone code to which 53BP1 dimers bind regulates retention and self-association into higher-order oligomers. Thus, here in live-cell nuclear architecture we quantify the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response by coupling fluorescence fluctuation spectroscopy (FFS) with the DSB inducible via AsiSI cell system (DIvA). From adopting this multiplexed approach, we find that preformed 53BP1 dimers relocate from the nucleoplasm to DSB sites, where consecutive recognition of ubiquitinated lysine 15 of histone 2A (H2AK15ub) and di-methylated lysine 20 of histone 4 (H4K20me2), leads to the assembly of 53BP1 oligomers and a mature 53BP1 foci structure.

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Section: Methodsmentioning

confidence: 99%

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

et al. 2020

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“…Conversely, time-resolved fluorescence anisotropy can detect FRET between identical fluorescent proteins. In fact, this technique has been used in living cells to study the effect of protein dimerization and aggregation on the cell functioning ( 19 , 21 , 22 , 23 ). Although oligomerization in the biological milieu may involve a mixture of monomers and dimers, as well as higher-order oligomers, at various distances and orientations, and does not typically involve a covalent linker, the short FRET component in the anisotropy decay is a qualitative tell-tale sign of the occurrence of oligomerization.…”

Section: Introductionmentioning

confidence: 99%

Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer

Teijeiro-Gonzalez

Crnjar

Beavil

et al. 2021

Biophysical Journal

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Fö rster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (hE exp FRET i ¼ 0.25 5 0.05) and those calculated from the molecular dynamics simulations (hE MD FRET i ¼ 0.18 5 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors hk 2 i ¼ 0.17 5 0.16 and hjk j i ¼ 0.35 5 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time-and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET.

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“…(v) Fluorescence anisotropy of the acceptor is typically very low if excited through the donor by FRET allowing quantification of the FRET efficiency [4,26].…”

Section: Theory Of Fret In Briefmentioning

confidence: 99%

Quo vadis FRET? Förster’s method in the era of superresolution

Szabó

Szendi-Szatmári

Szöllősi

et al. 2020

Methods Appl. Fluoresc.

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Although the theoretical foundations of Förster resonance energy transfer (FRET) were laid in the 1940s as part of the quantum physical revolution of the 20th century, it was only in the 1970s that it made its way to biology as a result of the availability of suitable measuring and labeling technologies. Thanks to its ease of application, FRET became widely used for studying molecular associations on the nanometer scale. The development of superresolution techniques at the turn of the millennium promised an unprecedented insight into the structure and function of molecular complexes. Without downplaying the significance of superresolution microscopies this review expresses our view that FRET is still a legitimate tool in the armamentarium of biologists for studying molecular associations since it offers distinct advantages and overcomes certain limitations of superresolution approaches.

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Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

Cited by 10 publications

References 94 publications

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer

Quo vadis FRET? Förster’s method in the era of superresolution

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