scholarly journals Interaction of Poxvirus Intracellular Mature Virion Proteins with the TPR Domain of Kinesin Light Chain in Live Infected Cells Revealed by Two-Photon-Induced Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Microscopy

2010 ◽  
Vol 84 (24) ◽  
pp. 12886-12894 ◽  
Author(s):  
Ananya Jeshtadi ◽  
Pierre Burgos ◽  
Christopher D. Stubbs ◽  
Anthony W. Parker ◽  
Linda A. King ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Surface Protein ◽  
Fluorescence Lifetime Imaging ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Infected Cells ◽  
Tpr Domain

ABSTRACT Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis

2015 ◽  
Vol 7 (10) ◽  
pp. 4071-4089 ◽  
Author(s):  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Dominic Alibhai ◽  
Sunil Kumar ◽  
Yuriy Alexandrov ◽  
...  
Keyword(s):  
Content Analysis ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
High Content Analysis ◽  
Förster Resonance

An HCA-FLIM instrument is presented alongside exemplar oligomerisation, intermolecular and intramolecular FRET assays that require robust measurement of small lifetime changes.

scholarly journals Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Anca Margineanu ◽  
Jia Jia Chan ◽  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Delphine Flatters ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Förster Resonance

Abstract We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. K D values broadly agree with published biochemical measurements.

scholarly journals A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation

2013 ◽  
Vol 203 (3) ◽  
pp. 445-455 ◽  
Author(s):  
Cedric Espenel ◽  
Bipul R. Acharya ◽  
Geri Kreitzer
Keyword(s):  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Epithelial Differentiation ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Data ◽  
Lifetime Imaging ◽  
Phasor Plot

We showed previously that the kinesin-2 motor KIF17 regulates microtubule (MT) dynamics and organization to promote epithelial differentiation. How KIF17 activity is regulated during this process remains unclear. Several kinesins, including KIF17, adopt compact and extended conformations that reflect autoinhibited and active states, respectively. We designed biosensors of KIF17 to monitor its activity directly in single cells using fluorescence lifetime imaging to detect Förster resonance energy transfer. Lifetime data are mapped on a phasor plot, allowing us to resolve populations of active and inactive motors in individual cells. Using this biosensor, we demonstrate that PKC contributes to the activation of KIF17 and that this is required for KIF17 to stabilize MTs in epithelia. Furthermore, we show that EB1 recruits KIF17 to dynamic MTs, enabling its accumulation at MT ends and thus promoting MT stabilization at discrete cellular domains.

scholarly journals Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Kalina T. Haas ◽  
Maximilian W. Fries ◽  
Ashok R. Venkitaraman ◽  
Alessandro Esposito
Keyword(s):  
Fluorescence Lifetime ◽  
Cell Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging ◽  
Biochemical Reactions ◽  
Lifetime Imaging ◽  
Spectrally Resolved ◽  
Single Living Cells

Revealing mechanisms underpinning cell function requires understanding the relationship between different biochemical reactions in living cells. However, our capabilities to monitor more than two biochemical reactions in living cells are limited. Therefore, the development of methods for real-time biochemical multiplexing is of fundamental importance. Here, we show that data acquired with multicolor (mcFLIM) or spectrally resolved (sFLIM) fluorescence lifetime imaging can be conveniently described with multidimensional phasor transforms. We demonstrate a computational framework capable of demixing three Forster resonance energy transfer (FRET) probes and quantifying multiplexed biochemical activities in single living cells. We provide a comparison between mcFLIM and sFLIM suggesting that sFLIM might be advantageous for the future development of heavily multiplexed assays. However, mcFLIM—more readily available with commercial systems—can be applied for the concomitant monitoring of three enzymes in living cells without significant losses.

scholarly journals Fluorescence lifetime imaging for studying DNA compaction and gene activities

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Svitlana M. Levchenko ◽  
Artem Pliss ◽  
Xiao Peng ◽  
Paras N. Prasad ◽  
Junle Qu
Keyword(s):  
Fluorescence Lifetime ◽  
Cultured Cells ◽  
Genomic Structure ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Probes ◽  
Fluorescence Lifetime Imaging ◽  
Dna Compaction ◽  
Lifetime Imaging ◽  
Significant Difference

AbstractOptical imaging is a most useful and widespread technique for the investigation of the structure and function of the cellular genomes. However, an analysis of immensely convoluted and irregularly compacted DNA polymer is highly challenging even by modern super-resolution microscopy approaches. Here we propose fluorescence lifetime imaging (FLIM) for the advancement of studies of genomic structure including DNA compaction, replication as well as monitoring of gene expression. The proposed FLIM assay employs two independent mechanisms for DNA compaction sensing. One mechanism relies on the inverse quadratic relation between the fluorescence lifetimes of fluorescence probes incorporated into DNA and their local refractive index, variable due to DNA compaction density. Another mechanism is based on the Förster resonance energy transfer (FRET) process between the donor and the acceptor fluorophores, both incorporated into DNA. Both these proposed mechanisms were validated in cultured cells. The obtained data unravel a significant difference in compaction of the gene-rich and gene-poor pools of genomic DNA. We show that the gene-rich DNA is loosely compacted compared to the dense DNA domains devoid of active genes.

scholarly journals Targetable Conformationally Restricted Cyanines Enable Photon-Count Limited Applications

2021 ◽  
Author(s):  
Patrick Eiring ◽  
Ryan McLaughlin ◽  
Siddharth Matikonda ◽  
HAN ZHONGYING ◽  
Lennart Grabenhorst ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ring System ◽  
Fluorescence Lifetime Imaging ◽  
Conformationally Restricted ◽  
Lifetime Imaging ◽  
Tertiary Amide ◽  
Imaging Performance

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to parent species in a range of contexts including their use on a DNA-nano-antenna, in single-molecule Förster resonance energy transfer (FRET) applications, far-red fluorescence lifetime imaging microscopy (FLIM), and single-molecule localization microscopy. These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.

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