Publication Type:Journal Article
Source:Methods in cell biology, Volume 82, p.497-523 (2007)
Keywords:Amino Acid Sequence, Biosensing Techniques, Calibration, Cell Survival, Enzyme Activation, Fluorescence Resonance Energy Transfer, Fluorescent Dyes, Hela Cells, Humans, Mechanotransduction, Cellular, Molecular Sequence Data, Optical Tweezers, src-Family Kinases
The study of how cells respond to mechanical stimuli has recently leaped into the forefront of cell science with recent advances in molecular probes facilitating real-time measurements of cell signaling. In this chapter, we will detail the development of a "real-time" molecular probe designed to report the current fractional activated state of Src kinase by changing its spectral output in accordance to local Src states. Src kinase is widely understood to be a key player in the transduction of mechanical stimuli transduced through cell adhesions and focal complexes. To study the local and long-range Src response to localized stresses, an experimental protocol was developed whereby ligand-coated microspheres were adhered to the cell surface and pulled laterally by laser tweezers. This chapter contains a practical discussion of system design considerations and force calibration. Image processing, background subtraction, and the construction of an unbiased ratio image are discussed. Methods of analyzing the distribution of activated Src molecules are detailed with examples of cells with varying degrees of mechanostimulation.