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 language | English (US) |
|---|---|
| Pages (from-to) | 1225-1230 |
| Number of pages | 6 |
| Journal | ACS Sensors |
| Volume | 2 |
| Issue number | 8 |
| DOIs | |
| State | Published - Aug 25 2017 |
Fingerprint
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
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 journal › Article
}
TY - JOUR
T1 - Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells
AU - Damayanti, Nur P.
AU - Buno, Kevin
AU - Cui, Yi
AU - Voytik-Harbin, Sherry L.
AU - Pili, Roberto
AU - Freeman, Jennifer
AU - Irudayaraj, Joseph M.K.
PY - 2017/8/25
Y1 - 2017/8/25
N2 - 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.
AB - 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.
KW - FLIM
KW - kinase phosphorylation
KW - live 2D and 3D cultures
KW - multiplex singe cell monitoring
KW - peptide biosensor
KW - zebrafish
UR - http://www.scopus.com/inward/record.url?scp=85028301836&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85028301836&partnerID=8YFLogxK
U2 - 10.1021/acssensors.7b00359
DO - 10.1021/acssensors.7b00359
M3 - Article
VL - 2
SP - 1225
EP - 1230
JO - ACS Sensors
JF - ACS Sensors
SN - 2379-3694
IS - 8
ER -