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. 2017 Jan 20;7(2):e2108.
doi: 10.21769/BioProtoc.2108.

P-body and Stress Granule Quantification in Caenorhabditis elegans

Matthias Rieckher  1 , Nektarios Tavernarakis  2
Affiliations

P-body and Stress Granule Quantification in Caenorhabditis elegans

Matthias Rieckher et al. Bio Protoc. .

Abstract

Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules are Processing bodies (P bodies), which majorly harbors translationally inactive mRNAs and an array of proteins mediating mRNA degradation, translational repression and cellular mRNA transport (Sheth and Parker, 2003). Another type of RNP granules, the stress granules (SGs), majorly contain mRNAs associated with translation initiation factors and are formed upon stress-induced translational stalling (Kedersha et al., 2000 and 1999). Multiple evidence obtained from studies in unicellular organisms supports a model in which P bodies and SGs physically interact during cellular stress to direct mRNAs for transport, decay, temporal storage or reentry into translation (Anderson and Kedersha, 2008; Decker and Parker, 2012). The quantification, distribution and colocalization of P bodies and/or SGs are essential tools to study the composition of RNP granules and their contribution to fundamental cellular processes, such as stress response and translational regulation. In this protocol we describe a method to quantify P bodies and SGs in somatic tissues of the nematode Caenorhabditis elegans.

Keywords: Caenorhabditis elegans; Processing bodies; Stress granules; Transgenesis; mRNP granules.

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Figures

Figure 1.
Figure 1.. Identification of C. elegans larval stages in a mixed population.
The image depicts all 4 larval stages, young adult and day 1 adult as seen under a dissecting stereomicroscope. The arrow indicates the half-moon shaped structure in the area of the vulva, which is indicative for the L4 larval stage.
Figure 2.
Figure 2.. Representative images of epifluorescence images of an adult transgenic animal expressing the P body reporter DCAP-1::dsRED.
Images were recorded with an Axio Imager Z2 through a 10x magnification objective. Size bars are 100 µm. A. DIC image. The inlay shows a digitally enhanced view on the pharynx. The arrow points out the grinder, which has to be in focus before switching to the fluorescent channel. B. Fluorescent image of the P body reporter DCAP-1::dsRED. C. Screenshot of image processing. Choose the polygon selection tool to surround the whole animal. Open Analyze > Measure to get area size and MPI. (Full genotype of transgenic animal: N2;Ex[pdcap-1DCAP-1::dsRED; pRF4] published in Rieckher et al., 2015 .)
Figure 4.
Figure 4.. Preparation of agarose pads for fluorescent imaging in C. elegans.
A. Two glass slides modified with tape flank an empty microscope slide. B. Place a drop of 50 µl 5% agarose solution in the middle of the microscope slide (arrow). C. Swiftly put another microscope slide on top and gently push it down to flatten the agarose drop.
See this image and copyright information in PMC

References

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