Localization and Trafficking of Fluorescently Tagged ERK1 and ERK2
The action of ERK1 and ERK2 activity on the nuclear substrates requires crossing the nuclear envelope and the localization of phospho-ERK into the nucleus. The nucleo-cytoplasmic trafficking of ERK is therefore crucial for the correct functioning of the p
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1. Introduction The Extracellular Regulated Kinase 1 and 2 (ERK1 and 2) convert a variety of extracellular stimuli into a complex set of cellular responses, regulating processes as diverse as proliferation, differentiation, and synaptic plasticity (1–4). ERK1/2 acts on both cytosolic and nuclear targets, and the list of the downstream partners, nuclear and cytosolic, is ever expanding, underscoring the complexity and pervasiveness of their action. The phosphorylation of nuclear substrates is essential for the induction of specific programs
Matilde Marchi and Riccardo Parra have contributed equally to this chapter
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_17, © Springer Science+Business Media, LLC 2010
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of gene expression: this signalling requires that ERK1 and 2, upon stimulation, translocate into the nucleus to carry on the activation of the nuclear targets (5). Notwithstanding the importance of this process, little is known about the modalities, time course and regulation of ERK exchange between nucleus and cytoplasm in living cells. In the past few years, it has been demonstrated that this process is rapid and that it is regulated by activity (7–10). By expressing low levels ( 2.2, even in extremely bright cells. It is quite useful to refine the transfection protocol in order to avoid an excessive level of expression, and, to this effect, a plot as in Fig. 1b can result quite useful. 4.4.3. Calibration of the Imaging Setup
Although the above analysis does not require to know the relationship between measured fluorescence and concentration of ERK-GFP, this can be obtained by comparing the cell fluorescence with a known standard. Artificial cells are prepared by dispersing a water solution of recombinant GFP, at known concentration, in mineral oil, and a calibration curve can be obtained relating the measured fluorescence to the GFP concentration (Fig. 2). These artificial cells are imaged exactly in the same conditions of the experiments on living cells. That means objective, filter set, pinhole diameter, laser setting, and photomultiplier settings must be exactly matched. Furthermore, it would be useful
Fig. 2. Calibration of the imaging system. (a) Recombinant EGFP was diluted at decreasing concentrations in saline solution. The micelles were prepared by dispersing the solution in mineral oil. Imaging was performed on the confocal microscope in carefully controlled conditions to ensure applicability of the calibration to the live cell experiments. The three panels show micelles at three different concentrations; quantification has been performed in the central part of the droplet to avoid spurious effects due to lensing occurring nearby the edge. Bar 10 mm. (b) Calibration data: each point is the average of at least 10 measures (30 at the three lower concentrations) (9).
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to monitor the laser power at the focal plane of the lens with an appropriate laser meter. If the
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