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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- "This often involves several levels of control , including transcriptional regulation (e.g., control of gene expression), post-transcriptional regulation (e.g., regulation of mRNA stability) and modulation of protein levels (e.g., regulation of protein stability). Diverse and complementary experimental approaches have been developed to monitor these parameters, including reporter gene assays to track changes in transcriptional activation at a given promoter (Roura et al., 2013; van Rossum et al., 2013; Khan et al., 2014), quantitative real-time PCR (qPCR) and expression microarrays to measure the relative levels of mRNA transcripts (Skrzypski, 2008; Gorreta et al., 2012), and western blot analysis and fluorescent imaging techniques to measure changes in protein abundance over time (Wiechert et al., 2007; Chao et al., 2012). Though a comprehensive discussion of these techniques is beyond the scope of this review, the interested reader is referred to several recent reviews (Wilkins, 2009; van Rossum et al., 2013). "
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: 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
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ABSTRACT: Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology.Cell Communication and Signaling 04/2013; 11(1):24. DOI:10.1186/1478-811X-11-24 · 4.67 Impact Factor