Fig. 3

SUP-46 is ubiquitously expressed and localizes to multiple nuclear and cytoplasmic compartments. a SUP-46 is nuclear in developing gametes in the germline and in somatic cells (head neurons indicated), but is not detected in spermatids. b SUP-46 is nuclear in non-dividing cells (asterisk), but localizes to the cytoplasm in dividing cells (arrows) in the mitotic region of the adult hermaphrodite gonad. The distal tip cell (DTC) at the distal end of the gonad is indicated. c SUP-46 is absent from the nucleolus in germ cells of the loop region of the hermaphrodite germline (white arrows left-hand image), but accumulates in the nucleolus of developing oocytes in the oogenesis region (white arrows, right-hand image). Nucleoli are indicated by hatched circles and were identified in corresponding light images, shown as enlarged insets with red arrows pointing to the nucleolus. Gonad is outlined by a hatched line. Orientation of the germline (distal or proximal) is indicated. See also Additional files 2 and 3 showing z series through the loop region or oogenesis region, respectively. d SUP-46 is associated with chromatin (marked by histone H2B::mCherry) until just before the meiotic divisions. In the top row, SUP-46 in the male germline is strongly associated with chromatin in primary spermatocytes (hatched circle), but accumulates in perichromatin granules (arrow) at meiosis anaphase I. SUP-46 is absent from spermatids (arrowhead). In row 2, SUP-46 in the hermaphrodite germline is strongly associated with chromatin in the “–2” oocyte but becomes diffusely nuclear and no longer chromatin-associated in the “–1” oocyte (oocyte closest to spermatheca). Row 3 is a higher magnification image of the “–2” oocyte nucleus from row 2 and shows a granular distribution of SUP-46 on chromatin. Bottom row shows SUP-46 is depleted from the nucleus and accumulates in the cytoplasm of the “–1” oocyte just before nuclear envelope breakdown and ovulation. Oocytes are outlined by solid lines, with nuclei outlined by hatched circles. All images are oriented with the distal region of the germline to the left. e Pachytene region (top row) and condensation zone (bottom row) of the male germline showing SUP-46 associated with active chromatin (marked by histone H3K4me2), but not inactive chromatin (X chromosome; marked by histone H3K9me2). f SUP-46 accumulates in the meiotic residual body (arrow) during spermatogenesis. Left image is a low magnification view of a dissected male gonad, with the meiotic zone on the right. Remaining images are a higher magnification view of the meiotic region. Wheat germ agglutinin lectin (WGA) marks spermatids. g In embryos, SUP-46 is primarily nuclear in interphase cells (asterisk) but accumulates in the cytoplasm of dividing cells (white circle), recognized by condensed chromatin marked by histone H2B. Embryo is outlined in blue. See also Additional file 4, a video showing SUP-46 re-accumulating in the nucleus after cell division. h SUP-46 accumulates in P granules (arrow) of the posterior P2 cell of the embryo (top row), but not in perinuclear P granules in the adult germline (bottom row). i P granule (arrow) accumulation of PGL-1,3 and SUP-46 is decreased in the deps-1(bn124) mutant raised at the non-permissive temperature (24 °C). (a, c–i) Strain expresses SUP-46::FLAG::GFP. b Strain expresses SUP-46::FLAG::mCherry. (d Rows 1–3) strain also expresses mCherry::H2B to visualize chromatin and GFP::PH(PLC1delta) to help visualize cell boundaries. (d bottom row, g) Strain also expresses HIS-24::mCherry and mCherry::H2B. (h, i) P granules in 4-cell embryos were detected with an antibody to (h) PGL-1 or (i) PGL-1 and PGL-3. Embryos are oriented with the P2 cell on the right. (a, c, d, g) Imaged live. (b, e, f, h, i) Fixed and stained with antibodies or WGA. (a–i) Scale bars represent 15 μm. With the exception of (g), which is a 14-μm thick confocal composite image, images are of a single confocal optical plane