ArticleProceedings of the National Academy of Sciences of the United States of America2007
Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2007. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 81 papers.
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Who cites it
81 citing papers in PubMed, 136 citations in OpenAlex.
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- The RNA export factor Mex67 functions as a mobile nucleoporin.The Journal of cell biology · 2019Article
- FRET Microscopy in Yeast.Biosensors · 2019Review
- Single-cell quantification of the concentrations and dissociation constants of endogenous proteins.The Journal of biological chemistry · 2019Article
- Resolving fluorescent species by their brightness and diffusion using correlated photon-counting histograms.PloS one · 2019Article
- mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.Cell · 2018Article
- The effect of cell geometry on polarization in budding yeast.PLoS computational biology · 2018Article
- Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy.Methods (San Diego, Calif.) · 2018Article
- Scanning number and brightness yields absolute protein concentrations in live cells: a crucial parameter controlling functional bio-molecular interaction networks.Biophysical reviews · 2018Review
- Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.Journal of virology · 2017Article
- Single-cell dynamics and variability of MAPK activity in a yeast differentiation pathway.Proceedings of the National Academy of Sciences of the United States of America · 2016Article
- Dopamine Receptor Signaling in MIN6 β-Cells Revealed by Fluorescence Fluctuation Spectroscopy.Biophysical journal · 2016Article
- Enzyme Sequestration as a Tuning Point in Controlling Response Dynamics of Signalling Networks.PLoS computational biology · 2016Article
21 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation 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 K(d) >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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.