becomes apparent. We propose that the formation of a larger GLS-1/GLD-3 RNP complex positively influences the transla- tional activity of distinct mRNA(s), which encodes a germline survival factor (Figure 8B). Alternatively, it may repress a germline death factor. The recent discovery of the cytoplasmic poly(A) polymerase GLD-4, which physically binds to GLS-1, suggests the involvement of poly(A) tail length control [24]. Our model is strongly supported by our analysis of the GLS-1 interaction-deficient gld-3(ax562ts) mutation. At restrictive tem- perature gld-3(ax562) animals produce both predominant GLD-3 isoforms to wild-type levels but carry a single glycine-to-arginine (G/R) replacement in their fourth KH domain. The bulky, charged side chain of arginine is expected to have severe structural consequences on KH domain folds [28]. Consistent with this, we find that GLD-3ax562 does not form a complex with GLS-1 in vitro. Importantly, other known protein interactors of GLD-3, e.g. GLD-2, are not compromised in interacting with GLD-3ax562 and GLS-1 remains expressed in gld-3(ax562) embryos at restrictive temperature (Rybarska and Eckmann, unpublished results). Together this suggests that the G/R substitution disturbs GLD-3 folding locally to specifically inhibit GLS-1/GLD-3 complex in contrast to distorting global GLD-3 structure and compromising all GLD-3 functions. We have considered the possibility that the mutation in the KH domain of the gld-3(ax562) allele might affect the RNA-binding capacities of GLD-3, as KH domains are known to serve as RNA- binding surfaces as well as protein interaction platforms [29]. However, we found the general RNA-binding affinity of GLD- 3Lax562 in RNA homopolymer-binding assays unaffected when compared to GLD-3LWT (Jedamzik and Eckmann, unpublished results). Therefore, the G/R substitution seems more likely to compromise the formation of a GLS-1/GLD-3 complex rather than interfering with interactions with mRNA targets. Unfortu- nately, no target mRNAs required for germline survival have been identified to date. Nevertheless, our working model invokes the formation of a larger GLS-1/GLD-3 RNP complex that positively regulates a distinct mRNA target(s) (Figure 8B). We speculate that this type of regulation might involve poly(A) tail metabolism as GLS-1 is able to bind and stimulate the poly(A) polymerase activity of GLD-4 [24]. In the current work we find that gld- 4(RNAi) induces Gls animals at elevated temperatures. Hence, GLS-1 might recruit GLD-3 into a trimeric complex with GLD-4, and together they may be critical for germline survival. Additional unknown components may influence the structure/ stability and function of maternal GLS-1/GLD-3 RNP complexes in the early embryo. Analysis of the gld-3(ax562) temperature- sensitive Gls phenotype would support this. The GLS-1/GLD- 3ax562 complex is only severely compromised in vivo at higher temperatures. In contrast, in vitro or in yeast the GLS-1/GLD- 3ax562 complex does not form and we infer that other factors might stabilize the complex in vivo at permissive temperature. Consid- ering the expression and co-localization of GLS-1 and GLD-3 in early embryos, we propose that germline survival is initiated, as a function of the maternal GLS-1/GLD-3 complex, during early embryogenesis rather than early postembryonic development. This is also consistent with the temperature sensitive period of gld- 3(ax562). Homozygote gld-3(ax562) animals produce adult animals without germ lines when shifted during early embryogenesis. Later shifts have a less detrimental effect on germ cell loss even when maintained at high temperatures (Rybarska and Eckmann, unpublished results). In summary, GLS-1 is a master modulator of multiple GLD-3 functions throughout germline development. GLS-1 limits and extends GLD-3 availability by providing an interactive platform to GLS-1 and Germline Development bring in additional modulators to form new RNP complexes with varied activities. Proteins functionally analogous to GLS-1 are highly likely to exist in other organisms. Modulators of conserved RNPs are undoubtedly required to implement cell fate decisions during development across animals. As metazoan germ cells depend strongly on post-transcriptional regulatory mechanisms, these functional analogues may also be found in their germplasm granules. Materials and Methods Strains Worms were handled according to standard procedures and grown at 20uC unless otherwise stated [30]. Strains used in this study: LGI: gls-1(ef4), gls-1(ef8), nDf23/unc-13(e1091) lin-11(n566), glp-4(bn4ts); LGII: gld-3(q730), gld-3(ax562ts), fbf-1(ok91); LGIII: glp-1(q224ts), ced-4(n1162); LGV unc-51(e1189), fog-2(q71); the wild-type strain was bristol N2. The gls-1(ef4) and gls-1(ef8) deletion mutants were generated in an EMS based deletion screen [13] and are described further in Text S1. Adult germ line phenotypes were scored 24 hrs past midL4. Antibody Production and Immunocytochemistry Antibodies against the following proteins were used as described: anti-GLD-1 [31], anti-FOG-2 [32], SP56 [33], anti- RME-2 [34], anti-GLD-3 [20]; anti-PGL-1 [35], anti-GLH-2 [19]. A rabbit polyclonal antibody serum (C5C0) was generated against a GST::GLS-1 fusion comprising aa 249–576. Affinity purification was carried out using a maltose binding protein fusion of the identical GLS-1 piece immobilized on a HiTrap (Amersham) column and eluted at low pH. A monoclonal antibody was generated immunizing mice with a peptide corresponding to the very C-terminus of GLS-1 (aa 989–1011). The mouse anti-GLS-1 antibody (mo184C16) recognized in vitro produced GLS-1 protein (not shown) and is very specific for GLS- 1 in staining experiments. However, we found that it is also more sensitive to paraformaldehyde (PFA) fixation and thus stains less prominently granules. For some P granule double labelling experiments we used an anti-PGL-1 peptide antiserum of guinea pigs that stained P granules very similar to published anti-PGL-1 antibodies [35]; the affinity purified antibody is specific as no signal was observed in pgl-1(bn101) animals. Immunofluorescence on extruded and 1% PFA fixed germ lines was carried out in solution as described [5]. Embryo (Figure 2D) and whole worm immunocytochemistry (Figure 6) with methanol/acetone fixation was described elsewhere [36]. Images were taken on a Zeiss Imager M1 equipped with an Axiocam MRm (Zeiss) and processed with AxioVision (Zeiss) and Photoshop CS3 (Adobe). For optical sections we either generated images on an Imager Z1 with an Apotome (Zeiss) or on a confocal microscope (LSM Meta510, Zeiss). Secondary antibodies were coupled to fluoro- chromes FITC, CY3 and CY5 (Jackson Laboratories). Protein Biochemistry and Immunoblotting Recombinant GST fusions of GLD-3L fragments were produced in BL21(pRIL) E. coli. Recombinant C-terminally His- tagged protein fusions of GLS-1, GFP and GLD-3L (wt and ax562) were produced in SF+ insect cells with the help of baculoviruses, which were generated and tested for their protein expression levels in SF+ insect cells according to the manufacturers protocols (Invitrogen). Co-expression was performed by viral co- infection. MBP::GLD-3L and GST::GLD-3 fragments were purified on amylose resin (NEB) and glutathione beads (Sigma), respectively. PLoS Genetics | www.plosgenetics.org 13 May 2009 | Volume 5 | Issue 5 | e1000494