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. 2013 Feb 5;104(3):727-36.
doi: 10.1016/j.bpj.2012.12.030.

Quantitative measurement of protein relocalization in live cells

Alan Bush  1 , Alejandro Colman-Lerner
Affiliations

Quantitative measurement of protein relocalization in live cells

Alan Bush et al. Biophys J. .

Abstract

Microscope cytometry provides a powerful means to study signaling in live cells. Here we present a quantitative method to measure protein relocalization over time, which reports the absolute fraction of a tagged protein in each compartment. Using this method, we studied an essential step in the early propagation of the pheromone signal in Saccharomyces cerevisiae: recruitment to the membrane of the scaffold Ste5 by activated Gβγ dimers. We found that the dose response of Ste5 recruitment is graded (EC50 = 0.44 ± 0.08 nM, Hill coefficient = 0.8 ± 0.1). Then, we determined the effective dissociation constant (K(de)) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first activated. K(de) changed during the first minutes from a high affinity of < 0.65 nM to a steady-state value of 17 ± 9 nM. During the same period, the total number of binding sites decreased slightly, from 1940 ± 150 to 1400 ± 200. This work shows how careful quantification of a protein relocalization dynamic can give insight into the regulation mechanisms of a biological system.

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Figures

Figure 1
Figure 1
(A) Schematic representation of the pheromone pathway. When α-factor binds its receptor Ste2, the GEF activity of this GPCR is activated, causing the dissociation of the heterotrimeric G protein. Free Gβγ can recruit the scaffold protein Ste5 to the membrane, initiating the MAPK cascade. Phosphorylated Fus3 and Kss1 can phosphorylate other targets that initiate the mating response. (B) Image montage of YFP-Ste5 membrane recruitment. Saturating amounts of α-factor was added at time zero (indicated by the arrow) to strain YAB3770, and changes in fluorescence distribution were followed by time-lapse confocal microscopy. BF, bright field.
Figure 2
Figure 2
Membrane recruitment statistic. (A) Purposely defocused BF images (left) were processed by Cell-ID for image segmentation. Boundaries and IDs are overlaid in white on the BF image (center) and the fluorescence image (right). (B) Total YFP-Ste5 fluorescence in the focal plane versus time for cells treated with 100 nM α-factor (αF, triangles and dashed line) or SC medium (circles and solid line). Arrows on the x axis indicate times of the images in D. (C) Volume fluorescence versus time for the same cells as in B. (D) Image montage shows a representative cell for each treatment at the indicated times. (E) Increase in membrane recruitment statistic calculated as the ratio of surface to volume fluorescence versus time, for α-factor stimulated cells and two laser intensities that result in different photobleaching rates and total fluorescence. The inset shows the evolution of the volume fluorescence for these same cells, along with the best fit to exponential decay functions. The photobleaching rates are 1.7 ± 0.1 10−3s−1 for 1%, and 6.3 ± 0.3 10−3s−1 for 3% laser power. (F) Calibration of the recruitment statistic using cells with cytoplasmic fluorescence distribution of YFP (Bmh2-YFP) and membrane distribution of FM4-64. Each dashed line is an independent repetition of the calibration. The solid line (shaded region) represents the mean (standard error (SE)) of the calibration y = 0.317 ± 0.013 + x∗(0.291 ± 0.016).
Figure 3
Figure 3
Time-dependent D-R curve for Ste5 membrane recruitment. (A) Fraction of membrane Ste5 versus time for the indicated doses of α-factor, in strain RY2013b (2x YFP-STE5, FUS3-Q93A) with (right) and without (left) 10 μM of the inhibitor 1-NM-PP1. Error bars of the data points indicate the 95% CI of the mean, and the error bars of the ticks of the y axis represent the uncertainty of the calibration (see details of error estimation in Supporting Material). (B) D-R curves of the fraction of membrane Ste5, 405 s after pheromone addition, with (dashed line, triangles) or without (solid line, circles) inhibitor. The curves represent the best fit to a Hill function by nonlinear least squares. Error bars as in A. (C) Time evolution of the parameters of the fits EC50 (inset: amplitude A and Hill coefficient n) for the curves with (dashed line, triangles) and without (solid line, circles) inhibitor. The shaded regions represent the SE of the estimated parameters.
Figure 4
Figure 4
Measurement of effective binding affinity. (A) Membrane recruitment dynamics for strains with the indicated integration number of the PSTE5-YFP-STE5 construct. Error bars represent the 95% CIs for the mean plus the calibration uncertainty (see Supporting Material for details). (B) Bound amount of YFP-Ste5 versus total amount of YFP-Ste5, assuming a concentration of 484 molecules of YFP-Ste5 per integration, considering the recruitment at 4 min (circles) or the maximal level (triangles). The solid line is the best fit of a bimolecular association model for the recruitment level at 5 min, resulting in Kd = 17 ± 9 nM. For the peak recruitment level, only an upper bound for Kd = 0.65 nM (gray dashed line) can be determined, because the best fit results in a Kd of zero (black pointed line). (C) Mean CFP-STE4 membrane fluorescence for different strains, ordered by YFP-STE5 integration number. For each strain, nine images were acquired. The box-whisker represents the distribution of the averages of all cells within an image, the thick black line represents the median, and box boundaries represent the first and third quantiles. Whiskers extend to the most extreme value within 1.5 times the interquantile range, measured from the box boundary. Shared letters (a–d) between two strains indicate nonsignificant differences as computed by Tukey’s honest significance difference test, with α = 0.05. The dashed gray line represents the autofluorescence level. (D) Time evolution of Kd. The best fit of the bimolecular association model at each time is marked with a dot. The horizontal lines correspond to the 95% CI of the Kd. The bars are shaded according to the χ2 cost of the fit for the corresponding value of Kd (see Supporting Material for details). (E) Evolution of the total amount of Ste5 binding sites. Dots, lines, and bars as in panel D. (F) Regions of acceptable model parameters (Kd and number of Ste5 binding sites), for the SS (solid line) and peak (dashed line) recruitment. The area within these regions is shaded according to the χ2 cost of the fit. The dots represent the best fits, and the error bars indicate the 95% CIs as presented in D and E.
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References

    1. Kholodenko B.N., Hoek J.B., Westerhoff H.V. Why cytoplasmic signalling proteins should be recruited to cell membranes. Trends Cell Biol. 2000;10:173–178. - PubMed
    1. Leonard W.J., O’Shea J.J. Jaks and STATs: biological implications. Annu. Rev. Immunol. 1998;16:293–322. - PubMed
    1. Fayard E., Tintignac L.A., Hemmings B.A. Protein kinase B/Akt at a glance. J. Cell Sci. 2005;118:5675–5678. - PubMed
    1. Sakai N., Sasaki K., Saito N. Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein. J. Cell Biol. 1997;139:1465–1476. - PMC - PubMed
    1. Oancea E., Meyer T. Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell. 1998;95:307–318. - PubMed

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