Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging

Brian D. Slaughter, Joel W. Schwartz, Rong Li

Research output: Contribution to journalArticlepeer-review

Abstract

Fluorescence correlation spectroscopy (FCS), fluorescence crosscorrelation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with Kd >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation.

Original languageEnglish (US)
Pages (from-to)20320-20325
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number51
DOIs
StatePublished - Dec 18 2007

Keywords

  • Fluorescence correlation spectroscopy
  • Fluorescence cross-correlation spectroscopy
  • Fluorescence resonance energy transfer
  • Photon-counting histograms
  • Scaffold

ASJC Scopus subject areas

  • General

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