Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells

Nur P. Damayanti, Kevin Buno, Yi Cui, Sherry L. Voytik-Harbin, Roberto Pili, Jennifer Freeman, Joseph M.K. Irudayaraj

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Phosphorylation is an important post-translational modification implicated in cellular signaling and regulation. However, current methods to study protein phosphorylation by various kinases lack spatiotemporal resolution or the ability to simultaneously observe in real time the activity of multiple kinases in live cells. We present a peptide biosensor strategy with time correlated single photon counting-fluorescence lifetime imaging (TCSPC-FLIM) to interrogate the spatial and temporal dynamics of VEGFR-2 and AKT phosphorylation activity in real time in live 2D and 3D cell culture models at single cell resolution. By recording the increase in fluorescence lifetime due to a change in the solvatochromic environment of the sensor upon phosphorylation, we demonstrate that spatiotemporal maps of protein kinase activity can be obtained. Our results suggest that fluorescence lifetime imaging of peptide biosensors can be effectively and specifically used to monitor and quantify phosphorylation of multiple kinases in live cells.

Original languageEnglish (US)
Pages (from-to)1225-1230
Number of pages6
JournalACS Sensors
Volume2
Issue number8
DOIs
StatePublished - Aug 25 2017

Fingerprint

phosphorylation
Phosphorylation
Phosphotransferases
Cells
sensors
Sensors
Fluorescence
bioinstrumentation
Biosensors
life (durability)
fluorescence
Peptides
peptides
resolution cell
Cell signaling
proteins
Proteins
Imaging techniques
Vascular Endothelial Growth Factor Receptor-2
cells

Keywords

  • FLIM
  • kinase phosphorylation
  • live 2D and 3D cultures
  • multiplex singe cell monitoring
  • peptide biosensor
  • zebrafish

ASJC Scopus subject areas

  • Bioengineering
  • Fluid Flow and Transfer Processes
  • Process Chemistry and Technology
  • Instrumentation

Cite this

Damayanti, N. P., Buno, K., Cui, Y., Voytik-Harbin, S. L., Pili, R., Freeman, J., & Irudayaraj, J. M. K. (2017). Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells. ACS Sensors, 2(8), 1225-1230. https://doi.org/10.1021/acssensors.7b00359

Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells. / Damayanti, Nur P.; Buno, Kevin; Cui, Yi; Voytik-Harbin, Sherry L.; Pili, Roberto; Freeman, Jennifer; Irudayaraj, Joseph M.K.

In: ACS Sensors, Vol. 2, No. 8, 25.08.2017, p. 1225-1230.

Research output: Contribution to journalArticle

Damayanti, NP, Buno, K, Cui, Y, Voytik-Harbin, SL, Pili, R, Freeman, J & Irudayaraj, JMK 2017, 'Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells', ACS Sensors, vol. 2, no. 8, pp. 1225-1230. https://doi.org/10.1021/acssensors.7b00359
Damayanti NP, Buno K, Cui Y, Voytik-Harbin SL, Pili R, Freeman J et al. Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells. ACS Sensors. 2017 Aug 25;2(8):1225-1230. https://doi.org/10.1021/acssensors.7b00359
Damayanti, Nur P. ; Buno, Kevin ; Cui, Yi ; Voytik-Harbin, Sherry L. ; Pili, Roberto ; Freeman, Jennifer ; Irudayaraj, Joseph M.K. / Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells. In: ACS Sensors. 2017 ; Vol. 2, No. 8. pp. 1225-1230.
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