3.4 Nuage and endosomal trafficking In two recent works, AGO proteins AGO1 and AGO2, the RNA-binding protein GW182, and miRNA-repressible mRNAs are shown to accumulate in the late endosomes/MVBs (Gibbings et al., 2009; Lee et al., 2009). When the formation of MVBs or fusion of MVBs with lysosomes is compromised, silencing is misregulated. This suggests that the targeting and/or turnover of RISCs are critical to regulate gene silencing. To determine if the cytoplasmic nuage has biological relevance to endosomal trafficking, ovaries were stained for the pi-bodies (KRIMP and PCM as markers for the nuage and P-bodies, respectively) and different markers of the secretory or endosomal pathway. These include the transitional endoplasmic reticulum (tER) marker (TER94; Leon and McKearin, 1999; Ruden et al., 2000), the late endosome/lysosome marker Cluster of differentiation 63 (CD63; Hemler, 2003), the lysosomal marker Lysosomal-associated membrane protein (LAMP2; Akasaki et al., 1993), and Adenosine diphosphate (ADP)- ribosylation factor (ARF6), which is a Ras-related Guanosine triphosphate (GTP)-binding protein that controls membrane trafficking between the plasma membrane and early endosomes (reviewed in Chavrier and Goud, 1999; Donaldson, 2003). In the wild-type ovary, pi-bodies that were marked by KRIMP and PCM, overlapped with TER94, CD63, LAMP2, and ARF6 (Figure 3.4.1). The examination of the GFPtagged Protein disulfide isomerase (PDI), an enzyme that is involved in protein folding in the ER lumen, did not show any obvious overlaps with KRIMP (Figure 3.4.2), indicating that the pi-body/endosome overlaps are not artifactual. In the piRNA pathway mutants spn-E and aub where pi-body assembly is disrupted, KRIMP but not PCM, remained 104 overlapped with the tER and endosomal markers (Figure 3.4.1). This is consistent with the observation by Lee et al (2009) that AGO1 and AGO2 are associated with membranes in the cytoplasm in RNAi-defective mutant HPS4. Figure 3.4.1 Cytoplasmic nuage is tethered to ER/endosomal compartments. Immunostaining of the nuage component KRIMP (magenta), P-body protein PCM (red), and tER/endosomal markers TER94, CD63, LAMP2, and ARF6 (green). In the wild-type ovary, KRIMP, PCM, and TER94/CD63/LAMP2/ARF6 overlap. In the piRNA pathway mutants spn-E and aub, pi-body assembly is compromised but KRIMP remains tethered to the endosomal proteins. Bar is 10 μm. 105 . (LAMP2; Akasaki et al., 199 3), and Adenosine diphosphate (ADP)- ribosylation factor 6 (ARF6), which is a Ras-related Guanosine triphosphate (GTP)-binding protein that controls membrane trafficking. 3.4 Nuage and endosomal trafficking In two recent works, AGO proteins AGO1 and AGO2, the RNA-binding protein GW182, and miRNA-repressible mRNAs are shown to accumulate in the late endosomes/MVBs. and tER/endosomal markers TER94, CD63, LAMP2, and ARF6 (green). In the wild-type ovary, KRIMP, PCM, and TER94/CD63/LAMP2/ARF6 overlap. In the piRNA pathway mutants spn-E and aub, pi-body assembly