Imaging Translation in Single Cells Using Fluorescent Microscopy

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Cold Spring Harbor perspectives in biology (Impact Factor: 8.68). 09/2012; 4(11). DOI: 10.1101/cshperspect.a012310
Source: PubMed


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). "
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