Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins

Gautier, Isabelle; Tramier, Marc; Durieux, Christiane; Coppey, Jacques; Pansu, Robert; Nicolas, Jean‐Claude; Kemnitz, Klaus; Coppey-Moisan, Maïté

doi:10.1016/s0006-3495(01)76265-0

Cited by 198 publications

(189 citation statements)

References 33 publications

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“…The rotational correlation time ( ) is the time constant of this slow anisotropy decay component and serves as a measure of molecular rotation (25). This value was similar to rotational correlation times measured for GFP (23,28). The two-photon limiting anisotropy of V␣(1-315) (the anisotropy at t ϭ 0) was found to be 0.48.…”

Section: Catalytic Domains Are Arranged As Pairs In the Intact Autoinsupporting

confidence: 67%

“…A FRET approach seemed reasonable because it has already been shown that tagging the N and C termini of CaMKII␣ with FPs did not alter catalytic activity or the ability of the kinase to form holoenzymes (18), thus FPs attached to either the catalytic or association domains of CaMKII␣ could be used to decipher the structure of the holoenzyme. We specifically chose time-resolved fluorescence anisotropy as the method to measure emFRET because in addition to measuring the proximity between fluorophores, this method can also estimate the number of FPs participating in energy migration and measure the molecular rotation of an FP when attached to CaMKII␣ (22,23).…”

Section: Catalytic Domains Are Arranged As Pairs In the Intact Autoinmentioning

confidence: 99%

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Structural rearrangement of CaMKIIα catalytic domains encodes activation

Thaler

Koushik²,

Puhl³

et al. 2009

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

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Section: Catalytic Domains Are Arranged As Pairs In the Intact Autoinsupporting

confidence: 67%

Section: Catalytic Domains Are Arranged As Pairs In the Intact Autoinmentioning

confidence: 99%

Structural rearrangement of CaMKIIα catalytic domains encodes activation

Thaler

Koushik²,

Puhl³

et al. 2009

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

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“…FRET can also be detected by fluorescence anisotropy (Runnels and Scarlata 1995;Gautier et al 2001;Clayton et al 2002;Lidke et al 2003) which uses linearly polarized light to detect the orientation of the molecules. When there is no energy transfer, the orientation of excited molecule is highly correlated with the orientation of the emitting molecule.…”

Section: Fö Rster Resonance Energy Transfer (Fret)mentioning

confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

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“…Another method to get a single-color biosensor to perform multiplex could be based on homo-FRET measured by anisotropy [55,56]. HomoFRET occurs when a fluorophore transfers its energy to a closely identical fluorophore.…”

Section: New Methodological Insights For Multiplexing Kinase Biosensorsmentioning

confidence: 99%

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

Sizaire¹

2017

Protein Phosphorylation

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Fluorescence microscopy is widely used in biology to localize, to track, or to quantify proteins in single cells. However, following particular events in living cells with good spatio-temporal resolution is much more complex. In this context, Forster resonance energy transfer (FRET) biosensors are tools that have been developed to monitor various events such as dimerization, cleavage, elasticity, or the activation state of a protein. In particular, genetically encoded FRET biosensors are strong tools to study mechanisms of activation and activity of a large panel of kinases in living cells. Their principles are based on a conformational change of a genetically encoded probe that modulates the distance between a pair of fluorescent proteins leading to FRET variations. Recent advances in fluorescence microscopy such as fluorescence lifetime imaging microscopy (FLIM) have made the quantification of FRET efficiency easier. This review aims to address the different kinase biosensors that have been developed, how they allow specific tracking of the activity or activation of a kinase, and to give an overview of the future challenging methods to simultaneously track several biosensors in the same system.

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Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins

Cited by 198 publications

References 33 publications

Structural rearrangement of CaMKIIα catalytic domains encodes activation

Structural rearrangement of CaMKIIα catalytic domains encodes activation

Fluorescence Techniques to Study Lipid Dynamics

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

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