Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins

Laviv, Tal; Kim, Benjamin B.; Chu, Jun; Lam, Amy; Lin, Michael Z.; Yasuda, Ryohei

doi:10.1038/nmeth.4046

Cited by 100 publications

(97 citation statements)

References 24 publications

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“…Another multiparameter study detected fluorescent lifetime changes of TN-L15, a genetic FRET calcium sensor, alongside homo-FRET detection by anisotropy of an AKT domain as an indicator of 3′-phosphoinositide accumulation (Warren et al 2015). Recently, both the spectral and lifetime characteristics of a newly developed FRET donor, the monomeric cyan-excitable red fluorescent protein (mCyRFP1), were exploited to simultaneously monitor two signaling events (Laviv et al 2016). …”

Section: Introductionmentioning

confidence: 99%

FRET from single to multiplexed signaling events

Bunt

Wouters

2017

Biophys Rev

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Förster resonance energy transfer (FRET) is a powerful tool for the visualization of molecular signaling events such as protein activities and interactions in cells. In its different implementations, FRET microscopy has been mainly used for monitoring single events. Recently, there has been a trend of extending FRET imaging towards the simultaneous detection of multiple events and interactions. The concomitant increase in experimental complexity requires a deeper understanding of the biophysical background of FRET. The presence of multiple acceptors for one donor affects the well-known formalism for FRET between two molecules, increasing distance sensitivity through mechanisms that have become known as the ‘antenna’ and ‘surplus’ effect. We will discuss the nature of these effects and present the imaging methods that have been used to unravel the combined transfer rates in the multi-protein interactions of multiplexed FRET experiments. Multiplexing strategies are becoming invaluable analytical tools for the elucidation of biological complexes and for the visualization of decision points in cellular signaling networks in physiological and pathological conditions.

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Section: Introductionmentioning

confidence: 99%

FRET from single to multiplexed signaling events

Bunt

Wouters

2017

Biophys Rev

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“…Thus, our structure-functional favors a working model in which a diffusible gradient of Ran-GTP emanating from the chromosomes, together with the centromere localization of TIP60, is critical to execute Ran function at the kinetochore–microtubule and spindle geometry in mitosis. It would be of great interest, in follow-up studies, to quantify the spatiotemporal dynamics of TIP60-elicited acetylation relative to Ran GTPase gradient in dividing cells using fluorescence resonance energy transfer-based sensors and delineate their precise molecular function in mitosis (Chu et al, 2012; Laviv et al, 2016). …”

Section: Discussionmentioning

confidence: 99%

Mitosis-specific acetylation tunes Ran effector binding for chromosome segregation

Bao

Liu

et al. 2017

Journal of Molecular Cell Biology

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Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. The Ran GTPase plays a key role in mitotic spindle assembly. However, how the generation of a chemical gradient of Ran-GTP at the spindle is coupled to mitotic post-translational modifications has never been characterized. Here, we solved the complex structure of Ran with the nucleotide release factor Mog1 and delineated a novel mitosis-specific acetylation-regulated Ran–Mog1 interaction during chromosome segregation. Our structure-guided functional analyses revealed that Mog1 competes with RCC1 for Ran binding in a GTP/GDP-dependent manner. Biochemical characterization demonstrated that Mog1-bound Ran prevents RCC1 binding and subsequent GTP loading. Surprisingly, Ran is a bona fide substrate of TIP60, and the acetylation of Lys134 by TIP60 liberates Mog1 from Ran binding during mitosis. Importantly, this acetylation-elicited switch of Ran binding to RCC1 promotes high level of Ran-GTP, which is essential for chromosome alignment. These results establish a previously uncharacterized regulatory mechanism in which TIP60 provides a homeostatic control of Ran-GTP level by tuning Ran effector binding for chromosome segregation in mitosis.

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“…To overcome both limitations, a novel red-shifted fluorophore mCyRFP1 has been developed with a high Stokes shift [53]. This fluorophore has an excitation spectrum in the range of the GFP emission spectrum (around 500 nm), but its emission spectrum is shifted compared to GFP.…”

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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Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins

Cited by 100 publications

References 24 publications

FRET from single to multiplexed signaling events

FRET from single to multiplexed signaling events

Mitosis-specific acetylation tunes Ran effector binding for chromosome segregation

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

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