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Gelsolin — evidence for a role in turnover of junction-related actin filaments in Sertoli cells

¹ Department of Anatomy, 2177 Wesbrook Mall, Faculty of Medicine, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada
² Department of Physiology, Institute of Medicine and Engineering, University of Pennsylvania, 3340 Smith Walk, 1010 Vagelos Labs, Philadelphia, PA 19104, USA

View larger version (106K): [in a new window]   Fig. 1. Position of ectoplasmic specializations in the seminiferous epithelium of the testis and immunolocalization of gelsolin to the actin component of the structures. (A) Ectoplasmic specializations are present only in Sertoli cells and occur apically at sites of adhesion to spermatids and basally at sites of adhesion to neighboring Sertoli cells. (B,C) Typical appearance of ectoplasmic specializations in transmission electron micrographs. The structures consist of the plasma membrane of the Sertoli cell, a layer of actin filaments and a cistern of endoplasmic reticulum. The junctions shown here are from sites of Sertoli cell attachment to spermatids in the ground squirrel testis. Bars, 100 nm. (D) Immunofluorescence localization of gelsolin to ectoplasmic specializations in frozen sections of perfusion fixed rat testis. Sections were treated with a primary monoclonal antibody to gelsolin and with a secondary antibody to conjugated to Texas Red. Actin filaments were labeled with fluorescent phalloidin. Within the seminiferous epithelium, gelsolin and actin are co-localized at ectoplasmic specializations. Sites of apical and basal ectoplasmic specializations are indicated by the `a' and `b', respectively, in the panel labeled for actin. Specific staining for gelsolin was not observed in any of the controls (not shown). Bar, 10 µm. (E) Immunoelectron microscopic localization of gelsolin to the actin zone of ectoplasmic specializations. Spermatids with attached ectoplasmic specializations were mechanically dissociated from perfusion-fixed testes and treated with a primary antibody to gelsolin and a secondary antibody conjugated to nanogold. The material was embedded and sectioned, and then the sections were silver enhanced and stained. Shown here is an ectoplasmic specialization attached to a spermatid head. Notice that silver grains (small arrows) are associated with the actin zone of the junction plaque. Bar, 500 nm.

View larger version (91K): [in a new window]   Fig. 2. Immunolocalization of gelsolin to ectoplasmic specializations in rabbit testis. (A) Fluorescence of fixed-frozen sections of rabbit testis labeled for actin and gelsolin. Apical and basal ectoplasmic specializations are indicated by the `a' and `b', respectively, in the actin panel. Bar, 10 µm. (B) Immunoelectron microscopic localization of gelsolin to the actin filament-containing region of an apical ectoplasmic specialization. The material was processed and labeled exactly as described in the legend to Fig. 1. As in the rat, the silver grains (small arrows) are associated with the actin zone of the junction plaque. Bar, 250 nm. (C) Immunoblot of rabbit testis and purified bovine gelsolin (Sigma). The antibodies from Sigma and from Transduction Laboratories reacted with a single band on immunoblots of rabbit testis, and this band migrated slightly ahead of purified bovine plasma gelsolin. A similar band was not present on control blots in which the primary antibody was replaced with a similar concentration of normal mouse IgG (not shown). The lines indicate the top and bottom of the gel. Loading densities were 0.05 µg of purified gelsolin and 100 µg of decapsulated testis homogenate.

View larger version (102K): [in a new window]   Fig. 3. Immunolocalization of PtdIns(4,5)P2 and PLC in rat ectoplasmic specializations. (A) Phase and fluorescence images of isolated spermatids with attached ectoplasmic specializations that have been labeled with an antibody raised against PtdIns(4,5)P2. The spermatids have also been treated with fluorescent phallotoxin to label actin. Notice that the probe for PtdIns(4,5)P2 stains the region surrounding the head containing an ectoplasmic specialization that labels with the probe for actin. Specific staining for PtdIns(4,5)P2 was not observed in any of the controls (not shown). Bar, 5 µm. (B,C) Immunofluorescence localization of PLC in fixed frozen sections of rat seminiferous epithelium at stage V (B) and stage VII (C) of spermatogenesis. The locations of apical and basal ectoplasmic specializations are indicated by the `a' and `b', respectively, in the actin panels. At stage V, the probe for PLC reacts weakly at ectoplasmic specializations. The situation is much different at stage VII when apical ectoplasmic specializations are disassembling as part of the sperm release process and basal ectoplasmic specializations are turning over to allow the next generation of spermatocytes through junction complexes between Sertoli cells. At this stage, apical and basal regions containing ectoplasmic specializations, indicated by the actin staining, also react with the probe for PLC. Specific staining was not observed in any of the controls (data not shown). The intense staining of the tubule wall (asterisk) is caused by nonspecific staining by the secondary antibody. Bar, 10 µm. (D) Immunoblot of rat testis and rat seminiferous epithelium. The PLC antibody reacts specifically with a single band in each lane (148 kDa). The minor bands indicated by the asterisk are nonspecific and are present in blots treated with normal mouse IgG instead of primary antibody. The lines indicate the top and bottom of the gel. Loading densities were 80 µg of testis homogenate and 80 µg of seminiferous epithelium.

View larger version (77K): [in a new window]   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.

View larger version (48K): [in a new window]   Fig. 5. PLC in the presence of Ca2+ results in the loss of filamentous actin from rat ectoplasmic specializations. Spermatids with attached ectoplasmic specializations were incubated in the presence or absence of either PLC in the presence and absence of Ca2+. Shown in panels A,A' to D,D' are paired phase and fluorescence micrographs of spermatids with attached adhesion complexes fixed and labeled with fluorescent phalloidin for filamentous actin immediately after isolation (A,A') or after incubation with or without PLC in the presence or absence of a calculated 11 µM free Ca2+ (B,B'-D,D') The least amount of filamentous actin is associated with cells treated with PLC in the presence of Ca2+. (E) Treatment of spermatid/adhesion complexes with PLC in the presence of Ca2+ resulted in increased levels of actin and gelsolin in supernatants, relative to controls, as assessed by immunoblot. In these experiments, spermatids with attached junction complexes were incubated in buffers with or without PLC in the presence or absence of a calculated 1.5 mM (upper two blots) or 11 µM free Ca2+ (lower two blots). Cells were removed from solution by centrifugation and equivalent volumes of supernatants assessed for actin and gelsolin on immunoblots. The amount of actin and gelsolin are greatest in supernatants from spermatid/junction complexes treated with both PLC and Ca2+. Significantly, treatment with PLC in the absence of Ca2+ results in increased gelsolin in supernatants, but not in increased actin.

View larger version (72K): [in a new window]   Fig. 6. Putative model for gelsolin activation at the time of ectoplasmic specialization disassembly in Sertoli cells. The model involves hydrolysis of PIP2 (phosphatidylinositol 4,5-bisphosphate) resulting both in the release of gelsolin and a surge in local levels of Ca2+. Although PIP2 is shown in association with the plasma membrane, the model is not meant to exclude the possibility that PIP2 is also present in the membrane of the ER. IP3, inositol (1,4,5)-trisphosphate; IP3R, inositol (1,4,5)-trisphosphate receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phosphoinositide-specific phospholipase C.