Fluorescence Lifetime Imaging of Biosensor Peptide Phosphorylation in Single Live Cells

Damayanti, Nur P.; Parker, Laurie L.; Irudayaraj, Joseph

doi:10.1002/anie.201209303

Cited by 44 publications

(44 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our findings are in concordance with the previous in vitro study on FAK nuclear localization and activation 23 . Quantitative analysis (Fig.…”

supporting

confidence: 94%

“…Herein we report on a novel approach to monitor FAK signaling in live cells by measuring the real-time kinase phosphorylation activity of FAK. We utilized Time-Correlated Single Photon Counting (TCSPC) Fluorescence Lifetime Imaging (FLIM) 18–23 to monitor FAK phosphorylation with a FAK peptide biosensor (FAKSOR) as depicted in Figure 2. Our design exploits the auto phosphorylation property of FAK at Tyr397 sites, used as the recognition motif (Fig.…”

mentioning

confidence: 99%

“…The working principle of FAKSOR is as follows: upon phosphorylation of the peptide sensor at the Tyr397 site by the cellular FAK, the phospho group in the FAKSOR (phopsho-Tyr) conforms to bind with the cellular phospho-binding domain of the kinase to alter the fluorescence lifetime of 5-FAM in a kinase phosphorylation dependent manner(Fig. 2a–d) due to a change in the solvatochromic microenvironment upon phosphorylation 23 .…”

mentioning

confidence: 99%

See 2 more Smart Citations

Monitoring focal adhesion kinase phosphorylation dynamics in live cells

Damayanti

Buno

Narayanan

et al. 2017

Analyst

Self Cite

View full text Add to dashboard Cite

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging.

show abstract

“…Our findings are in concordance with the previous in vitro study on FAK nuclear localization and activation 23 . Quantitative analysis (Fig.…”

supporting

confidence: 94%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Monitoring focal adhesion kinase phosphorylation dynamics in live cells

Damayanti

Buno

Narayanan

et al. 2017

Analyst

Self Cite

View full text Add to dashboard Cite

show abstract

“…37, 40, 41 The major advantage of our technique is the strong signal from single plasmonic probes bound to target sites due to the large LSPR cross-section, which makes the mapping and quantification of epigenetic marks with high SNR at the single-cell level a reality. As provided in Fig.…”

Section: Discussionmentioning

confidence: 99%

Single-Cell Quantification of Cytosine Modifications by Hyperspectral Dark-Field Imaging

2015

View full text Add to dashboard Cite

Epigenetic modifications on DNA, especially on cytosine, play a critical role in regulating gene expression and genome stability. It is known that the levels of different cytosine derivatives are highly dynamic and are regulated by a variety of factors that act on the chromatin. Here we report an optical methodology based on hyperspectral dark-field imaging (HSDFI) using plasmonic nanoprobes to quantify the recently identified cytosine modifications on DNA in single cells. Gold (Au) and silver (Ag) nanoparticles (NPs) functionalized with specific antibodies were used as contrast-generating agents due to their strong Local Surface Plasmon Resonance (LSPR) properties. With this powerful platform we have revealed the spatial distribution and quantity of 5-carboxylcytosine (5caC) at the different stages in cell cycle, and demonstrated that 5caC was a stably inherited epigenetic mark. We have also shown that the regional density of 5caC on a single chromosome can be mapped due to the spectral sensitivity of the nanoprobes in relation to the inter-particle distance. Notably, HSDFI enables an efficient removal of the scattering noises from non-specifically aggregated nanoprobes, to improve accuracy in the quantification of different cytosine modifications in single cells. Further, by separating the LSPR fingerprints of AuNPs and AgNPs, multiplex detection of two cytosine modifications was also performed. Our results demonstrate HSDFI as a versatile platform for spatial and spectroscopic characterization of plasmonic nanoprobe-labeled nuclear targets at the single-cell level for quantitative epigenetic screening.

show abstract

“…[3] Peptide reporters phosphorylated by kinases within cells are highly sensitive, quantitative, and possess great multiplexing potential when coupled to capillary electrophoresis (CE). To date, delivery of these reporters into the cell interior has relied on low throughput methods such as microinjection or on cell-penetrating peptides (CPPs) which largely deliver cargo to endosomal compartments.…”

mentioning

confidence: 99%

An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells

et al. 2016

View full text Add to dashboard Cite

Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system which utilizes a light-programmable, cell-permeable reporter deliverable en masse to many cells. The reporter's ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2-4,5-dimethoxy 2-nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single-cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity.

show abstract

Fluorescence Lifetime Imaging of Biosensor Peptide Phosphorylation in Single Live Cells

Cited by 44 publications

References 25 publications

Monitoring focal adhesion kinase phosphorylation dynamics in live cells

Monitoring focal adhesion kinase phosphorylation dynamics in live cells

Single-Cell Quantification of Cytosine Modifications by Hyperspectral Dark-Field Imaging

An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells

Contact Info

Product

Resources

About