Monitoring focal adhesion kinase phosphorylation dynamics in live cells

Abstract

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.

Bioinspired glycosaminoglycan hydrogels via click chemistry for 3D dynamic cell encapsulation

“…To measure how cells respond to the HCP hydrogels, in vitro studies were performed by encapsulating hMSCs during gelation of HCP hydrogels. Real‐time hMSC responses in response to microenvironment changes during hydrogel gelation were characterized by monitoring dynamics of FAK signaling in live single cells utilizing FLIM with a newly designed FAK peptide sensor (FAKSOR) [Figure (a)] . FAK, a cytoplasmic nonreceptor tyrosine kinase, is sensitive to changes in matrix stiffness and its activation plays a key role in transmitting extracellular cues to intracellular targets .…”

“…FAK, a cytoplasmic nonreceptor tyrosine kinase, is sensitive to changes in matrix stiffness and its activation plays a key role in transmitting extracellular cues to intracellular targets . As illustrated in Figure (a), FAKSOR was first incubated with hMSCs to enable cellular uptake using an optimized protocol . Then, hMSCs loaded with FAKSOR were suspended in HCP3400 hydrogel precursor solutions for FLIM measurements.…”

“…The real‐time monitoring of FAK phosphorylation of live single cells was conducted to examine cell‐hydrogel interactions utilizing a fluorescence lifetime imaging microscopy (FLIM) with FAK phosphorylation biosensors . In brief, ~5000 hMSCs were incubated with a ~20 μM permeable FAK phosphorylation biosensor in growth media for 15 min and washed three times with PBS.…”

“…hMSCs were further encapsulated within the hydrogels and evaluated for cell viability, dynamics of focal adhesion kinase (FAK, an early mechanosensor that responds to changes in matrix stiffness) signaling, morphology, and cytoskeleton. Interestingly, using a newly designed FAK peptide sensor, hMSCs were found to exhibit a three‐stage gelation‐dependent cellular response in real‐time FAK phosphorylation during 3D encapsulation (Figure ). These results provide important new insights into real‐time cellular responses during hydrogel‐based cell encapsulation, and lay the foundation for fine‐tuning hydrogel properties and cell–hydrogel interactions to promote in situ tissue regeneration.…”

Modulation of Gene Silencing by Cdc7p via H4 K16 Acetylation and Phosphorylation of Chromatin Assembly Factor CAF-1 in Saccharomyces cerevisiae

“…The final signal was further filtered by a band-pass filter (520 6 20 nm; Chroma) before reaching the single-photon avalanche photodiode detector (SPCM-AQR; Perkin-Elmer [Perkin Elmer-Cetus], Norwalk, CT). Collected photons were registered as a time-correlated single-photon counting (TCSPC) format for time-domain fluorescence lifetime calculation (Damayanti et al 2017). Fluorescence lifetime (t) was determined by fitting the decay rate of a TCSPC histogram as the photon frequency decreased to 1/e of the original: F(t)=F 0 exp(21/t).…”