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A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells
- Source :
- eLife, eLife, Vol 9 (2020)
- Publication Year :
- 2019
-
Abstract
- Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.<br />eLife digest Cells contain networks of signaling proteins that can respond to a variety of cues from the surrounding environment. Often the cell’s response to these cues is not just controlled by the level of protein, but by changing the activity of signaling proteins. For example, a signaling protein in humans and other mammals known as Fyn regulates a number of different processes, including when a cell grows, dies, or develops a specialist role. Defects in the activity of Fyn are associated with several diseases in humans including cancer and Alzheimer’s disease. However, it remains unclear how Fyn contributes to these diseases, or how the protein is able to precisely coordinate responses to multiple different cues in healthy individuals. This is largely because there are no readily available tools that are able to specifically detect where and when this protein is active in cells. Researchers often use fluorescent proteins called biosensors as tools to detect where specific proteins are located in living cells over time. Now, Mukherjee, Singh et al. have developed a new biosensor named FynSensor to monitor the active form of Fyn in mammalian cells. Microscopy imaging of FynSensor in several different cell types showed that although Fyn was present everywhere, it was only active in certain areas. In these areas the protein switched between an active and inactive state, with clear ‘pulses’ of signaling activity lasting a couple of minutes in response to specific cues. These areas of high Fyn activity behaved like signaling hubs in which several different cues integrate together before Fyn triggers an appropriate cell response. These results shed light on how Fyn is able to precisely control many different processes in cells. In the future, FynSensor could be used to rapidly screen for drug-like molecules to treat cancer, Alzheimer’s disease and other conditions linked with defects in Fyn activity. Furthermore, the FynSensor could be adapted to allow researchers to study other signaling proteins in humans and other animals.
- Subjects :
- 0301 basic medicine
Magnetic Resonance Spectroscopy
Mouse
medicine.medical_treatment
S. cerevisiae
Biosensing Techniques
Proto-Oncogene Proteins c-fyn
Mice
0302 clinical medicine
Fyn kinase
Yeasts
Fluorescence Resonance Energy Transfer
Biology (General)
Phosphorylation
compartmentalized
biology
Chemistry
General Neuroscience
General Medicine
Cell biology
Virus
Crosstalk (biology)
030220 oncology & carcinogenesis
Medicine
Intracellular
Signal Transduction
Research Article
Human
Cell physiology
QH301-705.5
Science
Integrin
Cell fate determination
biosensor
General Biochemistry, Genetics and Molecular Biology
Cell Line
Cell Physiological Phenomena
03 medical and health sciences
FYN
Biochemistry and Chemical Biology
medicine
Animals
Humans
Kinase activity
Fluorescent Dyes
General Immunology and Microbiology
pulsatile
Growth factor
E. coli
Cell Biology
src kinases
030104 developmental biology
HEK293 Cells
biology.protein
FRET
Subjects
Details
- ISSN :
- 2050084X
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- eLife
- Accession number :
- edsair.doi.dedup.....96542ae9d53dc583109063b7f441df6a