Fig. 3. Immunofluorescence localization of SPE-4 in sperm cells. Bar in A1, 5 μm, applies to all panels. Each horizontal, lettered row contains a series of four or five corresponding images: Nomarski DIC to visualize cytology (#1 panel), DAPI staining to visualize nuclei (blue #2 panel), monoclonal antibody ICB4 staining to visualize the fibrous body-membranous organelle (FB- MO) complexes (RITC red #3 panel), affinity purified anti SPE-4 rabbit polyclonal antibody staining (FITC green #4 panel) and a photograph through a filter set that simultaneously passes the DAPI, RITC and FITC signals (yellow #5). The genotype of the cells depicted in each row is indicated at the bottom of the leftmost panel. All #4 panels were photographed and printed at the same exposure index except that E4 was printed at 50% exposure index relative to the other panels (see below). (A) Wild-type spermatids budding from the residual body (rb). Both 1CB4 staining (A3) and EU43 SPE-4 peptide antiserum staining (A4) occur in spermatids during their budding from the rb and these signals coincide to produce the yellow signal observed in A5. (B) A wild-type spermatid (lower right corner) and a spermatozoon with a single pseudopod (p at arrow) extending from its cell body. Staining with 1CB4 (B3) reveals that the punctate signal is mostly found in the cell body and a similar, overlapping signal (B4) is observed after staining with the 9910 SPE-4 antiserum. (C) spe-4
) null mutant arrested spermatocyte. Staining with 9910 SPE-4 fusion protein antiserum reveals that no SPE-4 protein is present, as for 9911 SPE-4 antiserum (which was also raised to the same fusion protein and is not shown for wild type). (D) spe-4
) mutant arrested spermatocyte. Staining with 9911 SPE-4 antiserum (D4) reveals that this spe- 4(hc78
) missense mutant synthesizes a small amount of mutant SPE-4. (E) spe-4
) mutant arrested spermatocyte. Staining with 9910 SPE-4 antiserum (E4) reveals that the spe-4
) missense mutant synthesizes a large amount of mutant SPE-4; this panel was printed at 50% of the exposure index relative to the other #4 panels in order to eliminate color over-saturation (compare to B4 and C4). (F) Wild-type spermatids budding from the rb. Staining with the EU20 SPE-4 antiserum (F4) shows segregation of SPE-4 (and probably at least one other protein; see the text for details) to spermatids during development. (G) Two spe-4
) null mutant spermatocytes. Staining with the EU20 SPE-4 antiserum (G4) reveals a fluorescent signal in this null mutant, suggesting this antiserum recognizes an epitope on a protein other than spe-4
(for further explanation, see the text).