Imaging Translation in Single Cells Using Fluorescent Microscopy.
ABSTRACT The regulation of translation provides a mechanism to control not only the abundance of proteins, but also the precise time and subcellular location that they are synthesized. Much of what is known concerning the molecular basis for translational control has been gleaned from experiments (e.g., luciferase assays and polysome analysis) that measure average changes in the protein synthesis of a population of cells, however, mechanistic insights can be obscured in ensemble measurements. The development of fluorescent microscopy techniques and reagents has allowed translation to be studied within its cellular context. Here we highlight recent methodologies that can be used to study global changes in protein synthesis or regulation of specific mRNAs in single cells. Imaging of translation has provided direct evidence for local translation of mRNAs at synapses in neurons and will become an important tool for studying translational control.
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ABSTRACT: To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks.Frontiers in Genetics 08/2014; 5:263. DOI:10.3389/fgene.2014.00263
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ABSTRACT: In this paper, laser beam resonant interaction with pendant microdroplets that are seeded with a laser dye (Rhodamine 6G (Rh6G)) water solution or oily Vitamin A emulsion with Rhodamine 6G solution in water is investigated through fluorescence spectra analysis. The excitation is made with the second harmonic generated beam emitted by a pulsed Nd:YAG laser system at 532 nm. The pendant microdroplets containing emulsion exhibit an enhanced fluorescence signal. This effect can be explained as being due to the scattering of light by the sub-micrometric drops of oily Vitamin A in emulsion and by the spherical geometry of the pendant droplet. The droplet acts as an optical resonator amplifying the fluorescence signal with the possibility of producing lasing effect. Here, we also investigate how Rhodamine 6G concentration, pumping laser beam energies and number of pumping laser pulses influence the fluorescence behavior. The results can be useful in optical imaging, since they can lead to the use of smaller quantities of fluorescent dyes to obtain results with the same quality.Biomicrofluidics 01/2015; 9(1):014126. DOI:10.1063/1.4913648 · 3.77 Impact Factor
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ABSTRACT: Translational control plays an essential role in the regulation of gene expression. It is especially important in defining the proteome, maintaining homeostasis, and controlling cell proliferation, growth, and development. Numerous disease states result from aberrant regulation of protein synthesis, so understanding the molecular basis and mechanisms of translational control is critical. Here we outline the pathway of protein synthesis, with special emphasis on the initiation phase, and identify areas needing further clarification. Features of translational control are described together with numerous specific examples, and we discuss prospects for future conceptual advances.Cold Spring Harbor perspectives in biology 12/2012; 4(12). DOI:10.1101/cshperspect.a011528 · 8.23 Impact Factor