Abstract
Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.
Keywords: Caenorhabditis elegans, FRAP, Messenger RNA, Protein synthesis, Protein translation
Background
Proper regulation of protein synthesis is critical for cell homeostasis and growth. Deregulation of protein synthesis has been implicated in pathologies such as cancer and senescent decline (Bjornsti and Houghton, 2004; Syntichaki et al., 2007). Currently available biochemical methods for measuring general protein synthesis rate include metabolic labeling and polysomal profiling (Martin, 1998; Rennie et al., 1994). The applicability of these methodologies is limited due to poor intake and uncontrolled or unequal distribution of the label throughout the animal or tissue of interest. Also, these methods lack specificity and significant changes in specific cells or tissues of interest may be masked due to variability in intrinsic rates of translation of the bulk of the sample. In this protocol, we describe a method for monitoring protein synthesis rates in the nematode Caenorhabditis elegans, based on fluorescence recovery after photobleaching (FRAP). The experimental approach is based on the expression of fluorescent proteins, in cells and tissues of interest of transgenic animals. Fluorescence is then photobleached by irradiating cells, tissues or whole animals with a powerful light source. Recovery of fluorescence, indicative of new protein synthesis, is then monitored in cells or tissues of interest.
Materials and Reagents
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Procedure
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Acknowledgments
This work was funded by grants from the European Research Council (ERC), the European Commission 7th Framework Programme. The protocol has been adapted from Syntichaki et al. (2007), Nature 445, 922-926.
References
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