title: Measuring in vivo signaling kinetics in a mitogen-activated kinase pathway using dynamic input stimulation.
abstract: Determining the in vivo kinetics of a signaling pathway is a challenging task. We can measure a property we termed pathway bandwidth to put in vivo bounds on the kinetics of the mitogen-activated protein kinase (MAPk) signaling cascade in Saccharomyces cerevisiae that responds to hyperosmotic stress [the High Osmolarity Glycerol (HOG) pathway]. Our method requires stimulating cells with square waves of oscillatory hyperosmotic input (1 M sorbitol) over a range of frequencies and measuring the activity of the HOG pathway in response to this oscillatory input. The input frequency at which the pathway's steady-state activity drops precipitously because the stimulus is changing too rapidly for the pathway to respond faithfully is defined as the pathway bandwidth. In this chapter, we provide details of the techniques required to measure pathway bandwidth in the HOG pathway. These methods are generally useful and can be applied to signaling pathways in S. cerevisiae and other organisms whenever a rapid reporter of pathway activity is available.
authors.affiliation: Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA. mmcclean@princeton.edu
authors.forename: Megan N
authors.initials: MN
authors.lastname: McClean
authors.forename: Pascal
authors.initials: P
authors.lastname: Hersen
authors.forename: Sharad
authors.initials: S
authors.lastname: Ramanathan
mesh_terms: D001671:Biochemistry / Q000379:methods*; D003470:Culture Media / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects; D044085:Microfluidics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D012984:Software; D012996:Solutions; D013997:Time Factors
chemical_list: D003470:Culture Media; D012996:Solutions; D020928:Mitogen-Activated Protein Kinases