Background

Thus far, most protocols to study P bodies and SGs were developed for yeast or human cell lines (Buchan et al., 2010). Little is known about the function of somatic RNP granules in multicellular organisms. The simple model organism C. elegans has been extensively used to study germline-specific P granules, which are distinct from P bodies and SGs, and important structures for germline development and function (Updike and Strome, 2010). Although the principles of the presented procedure can be applied to count germline-specific P granules, the protocol focusses on the quantification of somatic RNP granules. Several studies have identified a conserved function of somatic P bodies in the translational deregulation via miRNA pathways in C. elegans (Ding et al., 2005; Zhang et al., 2007). More recently, various tools were created to study the involvement of cytoplasmic RNP granules in cellular and organismal stress response, development and ageing in the nematode (Cornes et al., 2015; Huelgas-Morales et al., 2016; Rieckher et al., 2015; Rousakis et al., 2014; Sun et al., 2011; Table 1).

Such studies take advantage of the comparatively easy implementation of transgenesis methods in C. elegans that allow to constitutively express fluorescent fusion proteins (e.g., green fluorescent protein [GFP]), endogenously or in specific tissues (Rieckher et al., 2009). A collection of fosmids carrying gfp-tagged P body- and SG-specific genes can be obtained at the ‘C. elegans TransGeneome’ project, a genome-scale transgenic project for fluorescent- and affinity-tagged proteins for expression in the nematode (Sarov et al., 2012; Table 1). C. elegans is transparent, which allows for efficient application of fluorescence microscopy methods that are easily combined with differential interference contrast (DIC) microscopy to reveal fluorescent protein expression in an anatomical context. Mounting transgenic animals for P body and SG imaging is based on a previously described method using nanoparticles for immobilization (Kim et al., 2013), since commonly applied anesthetics in C. elegans can induce stress, resulting in increased RNP granule formation. Fluorescence-tagged P body or SG-intensity can be imaged by epifluorescence light microscopy (see Procedure C), while fluorescence intensity, a detailed count and size measurements of P bodies and SGs can be obtained via confocal laser scanning microscopy (see Procedure D).

Table 1. Tools available for transgenic expression of P body/SG-factors in C. elegans

*available at CGC
⁺germline-specific promoter fusion
^$C. elegans TransGeneome project (Sarov et al., 2012)
^&can be found in both types of RNP granules