Stress management: Gene regulation during osmotic challenge in S. Cerevisiae

Research Areas:

Gene regulation, Stress response, S. cerevisiae, Live-cell imaging, Green fluorescent protein

Imaging Needs:

Sub-micron resolution and good sensitivity at 509 nm

Imaging System (immunofluorescence):
  • Zeiss Axioplan 2 fluorescence microscope
  • Sony Spot Pursuit camera with Spot Basic software
  • Olympus Cell R system
  • Hamamatsu ORCA-R2 CCD camera
  • Cellasic microfluidic device for time-lapse microscopy
  • ImageJ software for fluorescence quantification
Measuring protein interactions in cells, whole organisms

Imaging nuclear proteins in living cells requires high sensitivity and precise localization. What if you want to measure interactions within an intact living organism? Find out how Takeaki Ozawa and colleagues screen for GPCR ligands in mice in Illuminating Activity—in vitro and in vivo. Read now.

THE QUESTION

How is the stress response orchestrated?

When challenged with excess heat, oxidation, acid, or salt, the yeast Saccharomyces cerevisiae responds by rapidly shutting down protein production and ramping up genes associated with a generalized stress response. Most of those stress-response genes are regulated by the related transcription factors Msn2 and Msn4, both of which are necessary for survival under persistent stress.

THE BARRIERS

The activity of Msn2 and Msn4 is in turn regulated by several routes: molecular modification (notably phosphorylation), recruitment to the chromatin they act upon, and transport into and out of the nucleus. Indeed, waves of Msn2 have been observed to oscillate in and out of the nucleus during persistent mild stress. The molecular mediators of these actions are not well understood.

Among potential candidates, a highly-conserved, muti-functional enzyme called protein phosphatase 2A (PP2A) has been reported to aid Msn2 accumulation in the nucleus during stress. However, PP2A’s exact role and mode of action remain a mystery.

THE SOLUTION

Yeast Protein Phosphatase 2A-Cdc55 Regulates the Transcriptional Response to Hyperosmolarity Stress by Regulating Msn2 and Msn4 Chromatin Recruitment
Wolfgang Reiter, Eva Klopf, Veerle De Wever, Dorothea Anrather, Andriy Petryshyn, Andreas Roetzer, Gerhard Niederacher, Elisabeth Roitinger, Ilse Dohnal, Wolfram Görner, Karl Mechtler,e Cécile Brocard, Christoph Schüller, Gustav Ammerera
Mol Cell Biol. 2013 Mar 33(5): 1057–1072. PMCID: PMC3623084.

Reiter, et al,1 performed live-cell fluorescence imaging to track nuclear accumulation of Msn2 and Msn4 during osmotic stress in S. cerevisiae expressing Msn2 or Msn4 tagged with green fluorescent protein (GFP). With the help of a Hamamatsu ORCA-R2 CCD camera, the researchers quantified fluorescence intensity in the nucleus at baseline and after 5–20 minutes of exposure to a high-salt environment.

Mutants for a specific regulatory subunit of PP2A showed abbreviated accumulation of Msn2 and Msn4 in the nucleus, compared with wild-type yeast. The intact subunit was required for full activation of the stress response, and for extended Msn2 chromatin recruitment. The authors propose a role of this PP2A subunit in Msn2 and Msn4 nuclear accumulation and chromatin association during the stress response.

THE POSSIBILITIES

Reiter, et al, measured small changes in fluorescence intensity in yeast cell nuclei using Hamamatsu’s ORCA-R2 CCD camera. Find out how Takeaki Ozawa and colleagues use the even more-sensitive ImagEM camera to detect receptor binding using luminescence in place of fluorescence in Illuminating Activity—in vitro and in vivo.

References

  1. Reiter, et al. Yeast Protein Phosphatase 2A-Cdc55 Regulates the Transcriptional Response to Hyperosmolarity Stress by Regulating Msn2 and Msn4 Chromatin Recruitment. Mol Cell Biol. 2013 Mar 33(5): 1057–1072. PMCID: PMC3623084.
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