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
* Author for correspondence (e-mail: guttman{at}interchange.ubc.ca)
Accepted 26 October 2001
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Summary |
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Key words: Gelsolin, Adhesion junctions, Ectoplasmic specializations
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Introduction |
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Molecular components of ectoplasmic specializations include 6ß1
integrin (Palombi et al.,
1992
; Salanova et al.,
1995
), vinculin (Grove and
Vogl, 1989
), fimbrin (Grove and
Vogl, 1989
),
-actinin
(Franke et al., 1978
), espin
(Bartles et al., 1996
), and
myosin VIIa (Hasson et al.,
1997
). Integrin-linked kinase (ILK) also is present at the sites,
whereas focal adhesion kinase (FAK) is not
(Mulholland et al., 2001
), nor
is myosin II (Vogl and Soucy,
1985
).
Turnover of ectoplasmic specializations is related to two changes in
intercellular adhesion that are fundamental to the process of spermatogenesis.
At basal sites, turnover is correlated with the loss of attachment between
adjacent Sertoli cell plasma membranes and the movement of spermatocytes from
basal to adluminal compartments of the epithelium
(Russell, 1977). At apical
sites, disassembly is associated with sperm release
(Russell, 1984
). Little is
known about how the structures are regulated or how the three elements (plasma
membrane, actin filaments, endoplasmic reticulum) of the structures are
functionally interrelated.
Here we report that gelsolin is a component of ectoplasmic specializations.
In addition, we report that phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2) and phosphoinositide-specific
phospholipase C (PLC) are present in the structures. Treatment of
isolated ectoplasmic specializations with exogenous PLC
or with a
synthetic peptide of the PtdIns(4,5)P2 binding region of
gelsolin results in the release of gelsolin and loss of filamentous actin from
the adhesion junctions. Our results support a model for the disassembly of
junction-related actin filaments during sperm release and turnover of the
blood-testis barrier that involves the gelsolin-phosphoinositide pathway.
Moreover, we include in our model the possibility that the endoplasmic
reticulum component of ectoplasmic specializations may participate in
gelsolin-mediated filament disassembly by regulating Ca2+ levels
within the filament layer.
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Materials and Methods |
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Animals
Animals used in this study were reproductively active Sprague-Dawley rats
and New Zealand White rabbits. They were acquired and maintained in accordance
with guidelines established by the Canadian Council on Animal Care.
Immunofluorescence
For immunolocalization of gelsolin, testes were perfusion (rat) or
immersion (rabbit) fixed with 3% paraformaldehyde in PBS (150 mM NaCl, 5 mM
KCl, 0.8 mM KH2PO4, 3.2 mM
Na2HPO4, pH 7.3) and then cryosectioned. Sections were
single or double labeled with Alexa 488 phalloidin (Molecular Probes, Eugene,
OR) for filamentous actin and with mouse monoclonal antibodies generated
against gelsolin (0.0625 µg/ml Sigma antibody; 0.0049 µg/ml Transduction
Laboratories antibody) (Sigma; BD Transduction Laboratories, Mississauga, ON).
Secondary antibodies consisted of goat anti-mouse IgG conjugated to Texas Red.
Controls included replacing primary antibodies with equivalent concentrations
of normal mouse IgG, replacing primary antibody with buffer alone, and
replacing both primary and secondary antibodies with buffer alone.
For immunolocalization of PLC, fixed frozen sections were single or
double labeled with 50 µg/ml mouse anti-Phospholipase C
IgG
(Transduction Laboratories) and with Alexa 568 phalloidin (Molecular Probes,
Eugene, OR). Secondary antibodies consisted of goat anti-mouse IgG conjugated
to Alexa 488 (Molecular Probes). Controls were similar to those described for
gelsolin immunostaining.
To immunolocalize PtdIns(4,5)P2, rat testes were perfusion fixed with 3% paraformaldehyde in PBS containing 2 mM EGTA. The tissue was cut into small pieces and then spermatids with attached junction plaques were mechanically dissociated from the epithelium by asperating the pieces through a graded series of syringe needles. Large fragments were allowed to settle and spermatids with attached ectoplasmic specializations that were still in suspension were removed and attached to polylysine-coated slides. These spermatid/junction complexes were then single- or double-labeled with 10 µg/ml of purified mouse monoclonal anti-PtdIns(4,5)P2 IgM (Echelon Research Laboratories, Salt Lake City, UT) and with Alexa 568 phalloidin (Molecular Probes). Secondary antibodies consisted of goat anti-mouse IgM conjugated to Alexa 488 (Molecular Probes). Controls (not shown) included replacing the specific antibody with the equivalent concentration of normal mouse IgM, replacing the primary antibody with buffer alone, or replacing both primary and secondary antibodies with buffer alone.
Immunoelectron microscopic localization of Gelsolin
Testes were perfusion (rat) or immersion (rabbit) fixed with 3%
paraformaldehyde in PBS. The tissue was cut into small pieces and then
spermatids with attached junction plaques were mechanically dissociated from
the epithelium by asperating the pieces through a graded series of syringe
needles. Large fragments were allowed to settle and then were removed. Cells
remaining in solution were concentrated by centrifugation, treated with 50 mM
glycine, and then labeled first with a primary antibody to gelsolin (167
µg/ml Sigma antibody; 23 µg/ml Transduction Laboratories antibody) and
then with a secondary goat anti-mouse antibody (1:40 dilution) conjugated to
nanogold (1.4 nm) (Nanoprobes, Yaphank, NY). Controls were similar to those
for immunofluorescence. The material was embedded in Unicryl (British BioCell
International, Cardif, UK) and all sections treated with a silver enhancement
system (HQ SILVER, Nanoprobes).
Peptide competition experiments
Testicular fractions enriched for spermatids with attached ectoplasmic
specializations were isolated generally as described elsewhere
(Miller et al., 1999). The
method involved manually collecting epithelia from seminiferous tubules in PEM
buffer (80 mM Pipes, 1.0 mM EGTA, 1.0 mM MgCl2, pH 6.8) containing
250 mM sucrose, 10 µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin,
0.5 µg/ml pepstatin, 0.1 mM PMSF, and then mechanically fragmenting the
material by asperation through syringe needles. The fragments were loaded onto
three-step sucrose gradients (30-60% sucrose in PEM buffer) and then the
gradients were centrifuged. The fractions were resuspended in cold buffer (3
mM EGTA, 25 mM Hepes, 80 mM KCl, 0.5 mM DTT, pH 7.0) and then the suspension
was divided into four equal volumes that were incubated on ice for 10 minutes.
Following this, the cells were pelleted by centrifugation and then resupended
in carrier buffer alone, or carrier buffer containing 40 µM of control
peptides (QRLFGKDEL or FRVKLKQGQR) or the PtdIns(4,5)P2
binding region of gelsolin (QRLFQVKGRR) directly conjugated to rhodamine B
(Cunningham et al., 1996
). The
control peptide QRLFGKDEL consisted of the first four residues of the specific
peptide followed by residues thought to localize the peptide to the ER. The
other control peptide had the same sequence as the specific peptide, but in
random order. The specific peptide consisted of residues 160-169 of human
plasma gelsolin with rhodamine B conjugated to the N-terminus.
CaCl2 was adjusted in each tube to result in a calculated 10 µM
free Ca2+. The suspensions were incubated at 37°C for 15
minutes with gentle agitation every 30 seconds. Cells were pelleted by
centrifugation and equivalent volumes of supernatant collected from each tube
and relative actin concentrations compared qualitatively by immunoblots using
a monoclonal anti-actin antibody (Sigma) used at 0.01 mg/ml. To access
qualitatively the amount of gelsolin in the supernatants, we stripped the
blots probed for actin and re-probed them using a polyclonal rabbit anti-mouse
gelsolin antibody (gift from Dr Toshi Azuma, Brigham and Women's Hospital,
Boston, Massachusetts) used at 1:2000. Unlike the antibodies used for the
morphological work, this probe reacted with rat gelsolin on Westerns.
To determine whether the three synthetic peptides could bind to ectoplasmic specializations, spermatids with attached ectoplasmic specializations were treated for 30 minutes with buffer alone (3 mM EGTA, 25 mM Hepes, 80 mM KCl, 0.5 mM DTT, pH 6.5) or with buffer containing 20 µM synthetic peptides. The cells were washed and then examined with a fluorescence microscope.
PLC experiments
Testicular fractions enriched for spermatids with attached ectoplasmic
specializations were obtained as described for the peptide competition
experiments, pooled, and then diluted in 1 ml of buffer (3 mM EGTA, 25 mM
Hepes, 80 mM KCl, 0.5 mM DTT) not containing Ca2+. Equal volumes of
suspensions were added to the required number of treatment tubes and then the
cells pelleted by centrifugation. The supernatants were discarded and the
cells in each of four tubes were resuspended in 500 µl buffer containing a
calculated 11 µM free Ca2+ (3 mM EGTA, 25 mM Hepes, 80 mM KCl,
0.5 mM DTT, 2.92 mM CaCl2). Cells to be used in the no
Ca2+ control were resuspended in 500 µl buffer containing no
Ca2+ (3 mM EGTA, 25 mM Hepes, 80 mM KCl, 0.5 mM DTT). The cells
were allowed to sit on ice for 10 minutes. Following this, the tubes again
were centrifuged and the pellets resuspended in 100 µl of treatment buffers
containing the appropriate calculated amounts of Ca2+ (11 µM or
1.5 mM) and PLC or buffer alone. The reaction mixtures were incubated
for 15 minutes at 37°C with gentle agitation every 30 seconds. Following
incubation, cells were pelleted by centrifugation and equivalent volumes of
supernatant were removed from each tube and assayed, by immunoblot, for actin
and gelsolin as described above for the peptide competition experiments.
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Results |
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The presence of gelsolin in ectoplasmic specializations has significant
implications for the assembly and disassembly of the actin plaques. Gelsolin
is a potent Ca2+-dependent actin severing and capping protein
(Sun et al., 1999). The
presence of gelsolin within the actin containing region of ectoplasmic
specializations, structures that are stable during most of the long process of
spermatogenesis, indicates to us that much of the protein may be `inhibited'
until it is required for actin disassembly or reorganization. In addition to
the lack of Ca2+, the only known inhibitors of the severing and
capping functions of gelsolin are certain phospholipids
(Janmey and Stossel, 1987
;
Meerschaert et al., 1998
;
Sun et al., 1999
), the most
notable of which is PtdIns(4,5)P2.
To determine whether PtdIns(4,5)P2is present in
ectoplasmic specializations, we treated isolated spermatids, to which the
adhesion complexes remained attached, with an antibody to the phospholipid.
The antibody positively reacted with regions that also labeled with probes for
junction related actin filaments (Fig.
3A). Similar staining was not observed when normal mouse IgG was
substituted for primary antibody. We also labeled fixed sections of rat testis
with antibodies to phosphoinositide-specific phospholipase C (PLC).
This probe specifically labeled regions of the seminiferous epithelium known
to contain both basal and apical ectoplasmic specializations, and was stage
specific. At stages when the junctions are stable, staining at the junctions
was weak (Fig. 3B). Significantly, staining was most intense during the period of spermatogenesis
(stage VII in rat) when the adhesion complexes are disassembling apically and
turning over basally (Fig. 3C).
The antibody reacted specifically with one band on blots of rat testis and rat
seminiferous epithelium (Fig.
3D).
|
As an alternative approach to verifying the presence of
PtdIns(4,5)P2 at the junction plaque, we labeled unfixed
spermatid/junction complexes, in the absence of Ca2+, with a
synthetic peptide of the PtdIns(4,5)P2 binding domain of
gelsolin directly conjugated to rhodamine B
(Cunningham et al., 1996).
Staining of regions known to contain ectoplasmic specializations in controls
was weak or absent (Fig. 4A-C,
A'-C'), whereas staining with the specific peptide was
relatively intense (Fig.
4D,D').
|
To test the hypothesis that gelsolin in ectoplasmic specializations may be
bound to PtdIns(4,5)P2, we mechanically dissociated rat
spermatids, to which ectoplasmic specializations of Sertoli cells remained
attached, from the seminiferous epithelium and incubated the cells with a
synthetic peptide of the PtdIns(4,5)P2 binding region of
gelsolin or with PLC. In the first set of experiments, we predicted
that the specific peptide would compete with endogenous gelsolin for binding
to PtdIns(4,5)P2 and, in the presence of Ca2+,
would result in increased actin disassembly when compared with controls.
Relative to supernatants collected from cells treated with buffer alone or
with two control peptides, more actin was present in blots of supernatants
collected from cells treated with specific peptide
(Fig. 4E). Importantly, when
blots were re-probed with an antibody that recognizes rat gelsolin on western
blots, more gelsolin was detected in supernatants collected from cells treated
with specific peptide than in blots of supernatants treated with control
peptides or buffer alone.
In the second set of experiments, we predicted that exogenously added
PLC would hydrolyze PtdIns(4,5)P2 to inositol
(1,4,5)-trisphosphate (Ins(1,4,5)P3) and diacylglycerol,
thereby releasing gelsolin. In the presence of Ca2+, actin in the
adhesion complexes associated with spermatid heads should disassemble and the
amount of actin in solution should increase relative to controls. The buffer
systems used contained calculated micromolar and millimolar levels of
Ca2+. Millimolar levels were included in the design to swamp any
effect of PLC
treatment on Ca2+ release from
junction-related ER that would indirectly activate any gelsolin not bound to
PtdIns(4,5)P2. Following incubation, spermatid/junction
complexes were pelletted by centrifugation and equivalent volumes of
supernatants from experimental and control cells collected and analyzed, by
immunoblotting, for actin. In some experiments, an equivalent volume of cells
was removed from each tube prior to centrifugation and stained with
fluorescent phallotoxin to label filamentous actin. Generally, less actin was
visible in the adhesion complexes associated with cells treated with
PLC
in the presence of Ca2+ than in control cells. At mM
levels, Ca2+ alone increased actin in supernatants relative to
other controls. Significantly, treatment with PLC
in the presence of
µM and mM Ca2+ increased the amount of actin in supernatants
collected from spermatid/junction complexes relative to supernatants collected
from all control cells (Fig.
5E). When the same blots of supernatants were re-probed for
gelsolin, PLC
in the absence of Ca2+ increased the level of
gelsolin in supernatants without a corresponding increase in the amount of
actin. At mM levels, Ca2+ alone increased the amount of gelsolin in
supernatants. At µM levels, the effect was reduced and the amount of
gelsolin present in supernatants was less than when samples were treated with
PLC
in the presence or absence of Ca2+.
|
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Discussion |
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The fact that the severing and capping functions of gelsolin are
Ca2+ dependent (Yin and
Stossel, 1979), together with the general finding that
Ins(1,4,5)P3 stimulates the release of Ca2+
from intracellular stores (Berridge,
1993
), may account for the presence of a cistern of endoplasmic
reticulum as an integral part of the adhesion complex. The endoplasmic
reticulum of this complex is suspected to regulate Ca2+ levels
(Franchi and Camatini, 1985
;
Pelletier et al., 1999
);
however, strong experimental evidence that the cistern actually can sequester
and release the cation locally is still lacking. It is possible that, in
stable plaques, the ER may function to maintain low levels of local
Ca2+ within the actin layer, thereby inhibiting the severing
function of gelsolin, and perhaps of related proteins. The capping function of
gelsolin can occur at lower Ca2+ concentrations than the severing
function (Janmey et al., 1985
).
This observation may account for the presence of gelsolin among actin
filaments in stable actin plaques, in addition to being located on either side
of the actin layer where presumably the gelsolin could be bound to
PtdIns(4,5)P2 in adjacent membranes. Gelsolin caps on
actin filaments may account for the effect of increased Ca2+ noted
in our experiments. During plaque disassembly, hydrolysis of
PtdIns(4,5)P2 by PLC
would not only release
gelsolin bound to PtdIns(4,5)P2, but may generate a local
surge in Ca2+ release from the ER, through the action of
Ins(1,4,5)P3 (Fig.
6). This Ca2+ surge would stimulate the severing
function of gelsolin within the actin plaque.
|
How actin filament disassembly, involving gelsolin, is initiated and coupled to signaling pathways associated directly with intercellular adhesion molecules in the plasma membrane remains to be determined. In addition to a role in filament disassembly, it is possible that gelsolin is involved with nucleation and elongation of actin filaments during junction assembly and during the changes in actin filament rearrangement that occur within ectoplasmic specializations during spermatogenesis. The finding that gelsolin is a molecular component of structures related to sites of intercellular adhesion in Sertoli cells may have general implications for the molecular mechanisms underlying assembly and disassembly of actin complexes associated with intercellular adhesion junctions generally in cells.
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Acknowledgments |
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