Protocol

Yeast Genetic Networks

Volume 734 of the series Methods in Molecular Biology pp 101-119

Date:

Measuring In Vivo Signaling Kinetics in a Mitogen-Activated Kinase Pathway Using Dynamic Input Stimulation

  • Megan N. McCleanAffiliated withLewis-Sigler Institute for Integrative Genomics, Princeton University Email author 
  • , Pascal Hersen
  • , Sharad Ramanathan

* Final gross prices may vary according to local VAT.

Get Access

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.

Key words

MAP kinase Microfluidics HOG pathway Bandwidth Frequency-response Kinetics