Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging.
TLDR
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.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.read more
Citations
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Real-time observation of transcription initiation and elongation on an endogenous yeast gene.
Daniel R. Larson,Daniel Zenklusen,Daniel Zenklusen,Bin Wu,Jeffrey A. Chao,Robert H. Singer,Robert H. Singer +6 more
TL;DR: A method of fluctuation analysis of fluorescently labeled RNA is described to measure dynamics of nascent RNA—including initiation, elongation, and termination—at an active yeast locus and finds no transcriptional memory between initiation events, and elongation speed can vary by threefold throughout the cell cycle.
Journal ArticleDOI
Fluorescence Correlation Spectroscopy in Living Cells
TL;DR: A step-by-step guide to the application of FCS to cellular systems, including methods for minimizing artifacts, optimizing measurement conditions and obtaining parameter values in the face of diverse and complex conditions of the living cell.
Journal ArticleDOI
Probing cellular protein complexes using single-molecule pull-down
Ankur Jain,Ruijie Liu,Biswarathan Ramani,Edwin Arauz,Yuji Ishitsuka,Yuji Ishitsuka,Kaushik Ragunathan,Jeehae Park,Jie Chen,Yang Kevin Xiang,Taekjip Ha,Taekjip Ha +11 more
TL;DR: An assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes is described, which can reveal how many proteins and of which kinds are present in the in vivo complex.
Journal ArticleDOI
mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding
Morgan Delarue,Gregory P. Brittingham,Stefan Pfeffer,Ivan V. Surovtsev,Ivan V. Surovtsev,Sudarshan Pinglay,Kevin Kennedy,Miroslava Schaffer,J.I. Gutierrez,Dajun Sang,Gregory Poterewicz,Jean K. Chung,Jürgen M. Plitzko,Jay T. Groves,Jay T. Groves,Christine Jacobs-Wagner,Benjamin D. Engel,Liam J. Holt +17 more
TL;DR: A role for mTORC1 is established in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation through genetically encoded multimeric nanoparticles.
Journal ArticleDOI
Fluorescence correlation spectroscopy.
Jonas Ries,Petra Schwille +1 more
TL;DR: The basic principle of FCS is introduced, its application to biological questions as well as its limitations and challenges are discussed, an overview of novel technical developments to overcome those challenges are presented, and speculations about the future applications of fluorescence fluctuation spectroscopy are concluded.
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