A biosensor generated via high-throughput screening quantifies cell edge Src dynamics
| Autor: | Lee M. Graves, Richard M. Allen, Dmitriy Gremyachinskiy, Klaus M. Hahn, Steven M. Lewis, Brian Kuhlman, Timothy C. Elston, Brian J. Dewar, Brian K. Kay, Akash Gulyani, Jianrong Wu, Eric A. Vitriol |
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| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular High-throughput screening Cell Intracellular Space Biosensing Techniques Plasma protein binding Biology 010402 general chemistry 01 natural sciences Article Mice 03 medical and health sciences High-Throughput Screening Assays medicine Animals Computer Simulation Molecular Biology Fluorescent Dyes 030304 developmental biology 0303 health sciences Cell Biology Fluorescence Fibronectins 0104 chemical sciences Monobody src-Family Kinases medicine.anatomical_structure Biochemistry NIH 3T3 Cells Biophysics Biosensor Protein Binding Proto-oncogene tyrosine-protein kinase Src |
| Zdroj: | Nature chemical biology |
| ISSN: | 1552-4469 1552-4450 |
| DOI: | 10.1038/nchembio.585 |
| Popis: | Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge. |
| Databáze: | OpenAIRE |
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