Fig. 4. Peptide binding and competition experiments. Paired phase (A-D) and fluorescence (A'-D') micrographs of spermatids with attached ectoplasmic specializations that were mechanically isolated from rat seminiferous epithelium and treated for 30 minutes with buffer (A,A') or with buffer containing 20 µM synthetic peptides directly conjugated to rhodamine B. The peptides consisted of two control sequences (QRLFGKDEL (B,B') and FRVKLKQGQR (C,C') and the PtdIns(4,5)P₂ binding region of gelsolin (QRLFQVKGRR (D,D')). Note that the PtdIns(4,5)P₂ binding region of gelsolin labels regions surrounding the spermatid head more strongly than the control peptides or buffer alone. Bar, 5 µm. (E) Shown here are the results of a peptide competition experiment. From left to right, the lanes are of supernatants collected from spermatids with attached ectoplasmic specializations treated with buffer alone, sequence QRLFGKDEL (Control Peptide 1), sequence FRVKLKQGQR (Control Peptide 2), and sequence QRLFQVKGRR (Gelsolin Peptide). The blots were probed with antibodies to actin and gelsolin. Notice that the amount of actin and gelsolin present in the supernatant from material treated with the PtdIns(4,5)P₂ binding region of gelsolin (QRLFQVKGRR) is greater than in supernatants treated with control peptides or buffer alone.