GLS-1 and Germline Development Figure 2. GLS-1 Expression in the Hermaphrodite Germ Line and Embryos. (A) GLS-1 profile in a wild-type adult germ line. Monoclonal anti- GLS-1 staining and epi-fluorescence microscopy. GLS-1 is expressed throughout the germ line and overlaps with GLD-3 expression. diplot., diplotene; TZ, transition zone. (B) Confocal images of a distal tip of a wild-type germ line. GLS-1 (green), detected with the polyclonal antibody. Its granular pattern overlaps (arrowheads) with P granules, marked by PGL-1 (red). Note, some P granules stain strongly for GLS-1 and weakly for PGL-1. (C) No immunoreactivity of the monoclonal GLS-1 antibody is seen in gls-1(ef8) mutant germ lines (outlined). The GLD-3 pattern appears unchanged in gls- 1(ef8). (D) In early wild-type embryos maternal GLS-1 localizes to P granules. Monoclonal anti-GLS-1 stainings. Optical sections of epi-fluorescence images taken with an Apotome (Zeiss). 2-cell embryo (two different focal planes given, top two rows) and 4-cell embryo (third row). 4-cell embryo of a gls-1(ef8) mother (bottom). Anterior, left; posterior, right. Note, granules strongly enriched for GLS-1 and GLD-3 and less so for GLH-2 (arrow), granules enriched for GLS-1, GLD-3 and GLH-2 (arrowhead). doi:10.1371/journal.pgen.1000494.g002 bottom row). Interestingly, GLD-3 localization to P granules was still observed in these embryos (Figure 2D, compare GLD-3 and GLH-2 images), thus GLS-1 is not required for GLD-3 localization to P granules. Taken together, GLS-1 is dynamically expressed in the embryo and is a component of P granules in germ cell precursors. GLS-1 co-localizes with GLD-3 in the early embryo and both are enriched in germplasm granules that contain very little or no PGL-1 or GLH-2. GLS-1 Interacts Specifically with GLD-3 To investigate the specificity of the GLS-1/GLD-3 interaction we performed further tests. GLS-1 interacted positively in yeast with both GLD-3 isoforms but not with other Bicaudal-C orthologs from C. elegans or Drosophila, e.g. ceBCC-1 or dmBic-C. No interaction was observed with the GLD-3 interactors FBF-1 or FBF-2 (Figure 3A). Next, we demonstrated a direct physical interaction between GLS-1 and GLD-3L with proteins expressed in insect cells (Figure 3B). All proteins carried a C-terminal His-tag and GLD-3L carried in addition an N-terminal Maltose Binding affinity tag (MBP). GLS-1 or GFP were co-expressed with GLD-3L by co- infection with baculoviruses encoding the individual fusion proteins. GLD-3L and associated proteins were captured on amylose resin and bead-bound proteins were analyzed by immunoblotting. GLS-1, but not GFP, was specifically pulled down by GLD-3L and not by MBP-coated beads alone (Figure 3B, lanes 4–6). To assess if the interactions were RNA mediated, we treated the extracts with RNAse A prior to the co-purification. PLoS Genetics | www.plosgenetics.org 4 May 2009 | Volume 5 | Issue 5 | e1000494