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

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.

Fig. 1.

Autocorrelation and cross-correlation analysis of GFP and mCherry-tagged proteins in live yeast cells. (A) Schematic of FCS in one-channel experiments (Left) and FCCS using red and green probes (Right). The autocorrelation curve reports on mobile concentration (N, related to inverse of amplitude of curve) and relative mobility (slower mobility correlates with larger τ_D). The amplitude of the cross-correlation curve is proportional to the codiffusion of the two species. (B) Normalized autocorrelation curves of GFP in solution (from Clontech, expressed in insect cells) compared with cytosolic GFP, nuclear GFP (nucleus located with Fus3-mCherry, image). Curves were normalized to identical particle number for comparison of mobility. A representative cell is shown, with * marking a representative location of a cytosolic measurement, and V marking the vacuole. (C) Controls to demonstrate the feasibility of fluorescence cross-correlation in yeast cells with chromosomally expressed GFP and mCherry-tagged proteins. For a positive control, cross-correlation (CC) was measured by using a yeast strain expressing Bat2-GFP-mCherry (Top). For a negative control, cross-correlation was measured for a yeast strain expressing Bat2-GFP and mCherry under the Bat2 promoter from two separate loci (Middle). A yeast strain expressing two subunits of the Arp2/3 complex (Bottom) provided an additional positive control where two distinct proteins are tagged with GFP and mCherry. Example curves are averages from many cells. Results are quantified in Fig. 2 C and D.

Fig. 2.

FCS and FCCS analysis of components of the MAPK pathway in live yeast cells. (A) Examples of nonnormalized FCS autocorrelation curves of Ste7-GFP, Kss1-GFP, and Fus3-GFP at 2 h after pheromone exposure (Left) and normalized FCS autocorrelation curves for MAPK proteins Ste5-GFP, Ste7-GFP, Ste11-GFP, Kss1-GFP, and Fus3-GFP, compared with cytosolic GFP and GFP in solution (Right). The inverse of the initial amplitude of the curve is related to the average number of molecules in the focal volume. The live-cell curves represent the average of 30–60 measurements from 10–20 cells. G₁ (unbudded) cells in a cycling population were analyzed. (B) Example autocorrelation and cross-correlation curves from FCCS experiments are shown for the Kss1/Ste7 pair (Upper Left), Ste5/Fus3 pair (Upper Right), GST-Ste5-GFP/Fus3-mCherry pair (Lower Left), next to curves for the control Ste5/Fus3 pair (Lower Right), taken with the FCCS experimental conditions described in Materials and Methods. Curves are the average across three cells. Inset compares the cross-correlation curves for the GST-Ste5, Fus3 pair compared with the Ste5, Fus3 pair in the lower graphs. (C) Cross-correlation between various GFP and mCherry-tagged protein pairs, presented as the percent cross-correlation as a percentage of the lower particle number of the two binding partners (see SI Text), after first calculating N_bound particles from the amplitudes of the three curves (28). For cross-correlation, uncertainty is represented as the standard error of the mean for a minimum of 30–80 measurements from 10–30 cells. (*) Measurements distinct from the negative control (Bat2-GFP/mCherry pair) with 95% confidence. (α) −2 h after pheromone stimulation. (D) Percent cross-correlation as described above for the interaction of wild-type or GST-tagged Ste5 with Fus3, with Ste5 either under the endogenous promoter, or overexpressed with the Gal1 promoter by 2- to 3-fold. Unless marked with Nc (referring to nuclear), the data represent cytosolic interaction.

Fig. 3.

PCH analysis of Ste5 dimerization in live yeast cells. (A) Example PCH data and fits for single fluorescence traces of GFP and GFP-GFP in live yeast cells. (B) Example brightness values of single cytosolic measurements obtained under identical experimental conditions for GFP, GFP-GFP, Ste5 (expressed with pGal1), Fus3, Ste7, and GST-Ste5 (expressed with pGal1). (C) Global fit of percent monomer and percent dimer of all of the data to a distribution of two species, with fixed brightness values equal to the averages for the GFP (monomer) and GFP-GFP (dimer) controls.

Fig. 4.

FRET analysis of Ste5 and Fus3 interaction in pheromone-stimulated cells. (A) Fluorescence images of Ste5-GFP (expressed under pGal1); Fus3-mStawberry, in pheromone treated cells. (B Upper) Intensity image of Ste5-GFP in a yeast cell with shmoo. A line-scan intensity plot is shown below for this cell, with pixel values averaged over the white lines shown in the image (Lower). The ratio image of the GFP intensity, after Fus3-mStrawberry bleaching, to GFP intensity before bleach. A ratio >1.0 is observed at the shmoo tip. The line scan of the ratio image is also shown for this particular cell, with pixel values averaged over the lines shown in the image. Green lines represent the average for the internal and tip areas. The nucleus is marked in both the images and line scans as white arrowheads, whereas the red stars mark the shmoo tip. (C) Pixels were grouped as either internal, including the nucleus (inside the black dotted circle in the example), total perimeter of the cell, or perimeter at the shmoo tip (along the dotted line in the example). The box plot represents the average (dot), standard error (rectangle centered on average), and median of the ratio of GFP intensity after acceptor bleach to before acceptor bleach (n = 20 cells).