Multi‐dimensional time‐correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells

Duncan, Rory R.; Bergmann, Axel; Cousin, Michael A.; Apps, David K.; Shipston, Michael J.

doi:10.1111/j.0022-2720.2004.01343.x

Cited by 136 publications

(122 citation statements)

References 56 publications

(88 reference statements)

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“…We chose a fluorophore pair for FRET that consisted of a mCerulean donor and an enhanced yellow fluorescent protein (eYFP) acceptor. FRET was calculated using fluorescence lifetime imaging microscopy (FLIM) by time-correlated single photon counting (TCSPC) (23,24). Fluorescence lifetime refers to the amount of time a valence electron from a fluorophore remains in the excited state before returning to ground state and emitting a photon.…”

Section: Resultsmentioning

confidence: 99%

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

Caron

Desmond

Xia

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

129

150

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Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion within the huntingtin gene that encodes a polymorphic glutamine tract at the amino terminus of the huntingtin protein. HD is one of nine polyglutamine expansion diseases. The clinical threshold of polyglutamine expansion for HD is near 37 repeats, but the mechanism of this pathogenic length is poorly understood. Using Förster resonance energy transfer, we describe an intramolecular proximity between the N17 domain and the downstream polyproline region that flanks the polyglutamine tract of huntingtin. Our data support the hypothesis that the polyglutamine tract can act as a flexible domain, allowing the flanking domains to come into close spatial proximity. This flexibility is impaired with expanded polyglutamine tracts, and we can detect changes in huntingtin conformation at the pathogenic threshold for HD. Altering the structure of N17, either via phosphomimicry or with small molecules, also affects the proximity between the flanking domains. The structural capacity of N17 to fold back toward distal regions within huntingtin requires an interacting protein, protein kinase C and casein kinase 2 substrate in neurons 1 (PACSIN1). This protein has the ability to bind both N17 and the polyproline region, stabilizing the interaction between these two domains. We also developed an antibody-based FRET assay that can detect conformational changes within endogenous huntingtin in wild-type versus HD fibroblasts. Therefore, we hypothesize that wild-type length polyglutamine tracts within huntingtin can form a flexible domain that is essential for proper functional intramolecular proximity, conformations, and dynamics.polyglutamine diseases | conformational switching | FLIM-FRET | neurodegeneration | fluorescence lifetime imaging microscopy H untington disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene (1). The pathogenic threshold of CAG expansion is ∼37 repeats, with increased repeats leading to an earlier age-onset of HD (2-4). This CAG mutation results in an expanded polyglutamine tract in the amino terminus of the gene's protein product, huntingtin (1). To date, no phenotypes at the level of huntingtin molecular biology or animal models can be attributed to polyglutamine lengths near the human pathogenic disease threshold (5).Polyglutamine or glutamine-rich domains are also found in transcription factors such as the Sp-family and cAMP response element-binding protein (CREB) and are defined as proteinprotein interaction motifs used to scaffold and regulate transcription by RNA polymerase II (6, 7). In the transducin-like enhancer of split (TLE) corepressor proteins, the glutamine-rich domains are thought to allow dimerization (8). However, the role of polyglutamine in proteins such as huntingtin may be distinct from that of a protein-protein interaction or dimerization domain.The polyglutamine tract of huntingtin is flanked on...

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Section: Resultsmentioning

confidence: 99%

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

Caron

Desmond

Xia

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

129

150

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“…The Fe65 protein is a member of a family of multidomain adaptor proteins that form multiprotein complexes. It has been shown that the PTB2 domain of Fe65 interacts with APP as well as with the APP intracellular fragment (AICD) released upon c-secretase cleavage (24). Some reports show that Fe65-AICD complex translocate to the nucleus and participate in gene transcription events (25).…”

Section: Application Of Fret Spectral Imaging In Protein-protein Intementioning

confidence: 99%

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Kim

Kang

et al. 2011

Cytometry Pt A

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Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/ mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells. ' International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; two-photon spectral imaging; unmixing; protein-protein interaction; YFP; mCherry THE current advances in light microscopy and engineering of fluorescent proteins (FPs) with improved properties and altered colors have provided the cellular biologist with the ability to investigate fine molecular events in living cells (1,2). Properly combined, fluorescent molecules allow the study of protein conformational changes or inter protein interaction by means of fluorescence resonance energy transfer (FRET) (3,4). When an excited donor fluorophore is proximate (=10 nm) and suitably oriented to an acceptor molecule, FRET occurs through a nonradiative dipoledipole interaction. Currently, the most popular FP pairs for FRET analysis are cyan with yellow fluorescent protein (CFP/YFP) and green with red fluorescent protein (GFP/RFP) (5,6).Among the methodologies based on nonradiative resonance energy transfer, two approaches prevail, the fluorescence lifetime imaging microscopy (FLIM), and the fluorescence intensity-based FRET imaging. The FLIM is known to be cumbersome to require highly specialized equipment and as well to involve long acquisition time (7). To the contrary, fluorescence intensity-based FRET estimation provides

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“…In other photon-counting FLIM studies of FRET between CFP and YFP in cells, the extent of FRET has been described by biexponential kinetics, with two lifetimes that are different to the donor-only decays [23][24][25]. More complex kinetics were not observed.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

Millington

Grindlay

Altenbach

et al. 2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT High-Precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion proteinMichael Millington, [a, b] G. Joan Grindlay, [c] Kirsten Altenbach, [a, d] Robert K. Neely, [a, b] Walter Kolch, [ c, e] Mojca Bencina , [ f ] Nick D. Read, [a, d] Anita C. Jones, [a, b] David T. F.Dryden, [a, b]

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Multi‐dimensional time‐correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells

Cited by 136 publications

References 56 publications

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

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