Rapid and sustained nuclear–cytoplasmic ERK oscillations induced by epidermal growth factor

Although the ERK pathway has a central role in the response of cells to growth factors, its regulatory structure and dynamics are incompletely understood. To investigate ERK activation in real time, we expressed an ERK–GFP fusion protein in human mammary epithelial cells. On EGF stimulation, we observed sustained oscillations of the ERK–GFP fusion protein between the nucleus and cytoplasm with a periodicity of ∼15 min. The oscillations were persistent (>45 cycles), independent of cell cycle phase, and were highly dependent on cell density, essentially disappearing at confluency. Oscillations occurred even at ligand doses that elicited very low levels of ERK phosphorylation, and could be detected biochemically in both transfected and nontransfected cells. Mathematical modeling revealed that negative feedback from phosphorylated ERK to the cascade input was necessary to match the robustness of the oscillation characteristics observed over a broad range of ligand concentrations. Our characterization of single-cell ERK dynamics provides a quantitative foundation for understanding the regulatory structure of this signaling cascade

Systems biology

ProMAT: protein microarray analysis too

Detection of metabolic fluxes of O and H atoms into intracellular water in mammalian cells.

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue

FTIR spectromicroscopy difference spectra of Rat-1 Fibroblasts.

<p>FTIR spectra from a cluster of Rat-1 cells grown on a CaF₂ optical slide (∼60% coverage). Spectra were recorded (25×25 µm² aperture) over 360 minutes after washing the cells with DMEM prepared with 100% ²H₂O. <i>A</i>, a two-dimensional plot depicting the absolute absorbance spectra after subtracting the background (an area with ²H-O-²H but no cells). <i>B</i>, the same data plotted in three dimensions in which the initial spectrum (defined as t  = 0) of a cell cluster was used as the reference. In both <i>A</i> and <i>B</i> the spectra are recorded every 5 minutes. The bands at ∼3400 cm⁻¹ (¹H-O-²H stretch) and ∼1450 cm⁻¹ (¹H-O-²H bend) increase over time while the bands at ∼2300 cm⁻¹ (shoulder of the ²H-O-²H stretching mode) and ∼1225 cm⁻¹ (²H-O-²H bend) decrease over time.</p

Regression coefficients of tissue culture “growth” and “wash” experiments, and estimations of metabolic flux.

<p>Regression coefficients of tissue culture “growth” and “wash” experiments, and estimations of metabolic flux.</p

FTIR spectromicroscopy difference spectra of Rat-1 Fibroblasts in lag phase.

<p>FTIR spectra from a cluster of lag phase Rat-1 cells grown on a CaF₂ optical slide. Spectra were recorded (25×25 µm² aperture) over 160 minutes after washing the cells with DMEM prepared with 100% ²H₂O The initial spectrum (defined as t  = 0) was used as the reference. The scale is the same as that used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039685#pone-0039685-g002" target="_blank">Figure 2<i>A</i></a>. The horizontal line indicates that there is essentially no difference between the various spectra.</p