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From the * Department of Pathology and Laboratory
Medicine, Brown University, Providence, Rhode Island; and the
Division of Biological Sciences, CIIT Centers
for Health Research, Research Triangle Park, North Carolina.
| Correspondence to: Dr Kamin Johnson, CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, NC 27709 (e-mail: kjohnson{at}ciit.org). |
| Received for publication December 28, 2004; accepted for publication February 26, 2005. |
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Abstract |
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Key words: Flamingo, spermatid, seminiferous epithelium
Transmembrane cadherin superfamily proteins are important mediators of intercellular adhesion. The testis expresses numerous structurally diverse cadherins in a cell type–specific manner (Johnson et al, 2000, 2004; Johnson and Boekelheide, 2002). Classic cadherins are one family of the cadherin superfamily and have been localized to inter-Sertoli cell and Sertoli cell–germ cell adhesion junctions (Cyr et al, 1992; Byers et al, 1994; Mulholland et al, 2001; Johnson et al, 2002; NP Lee et al, 2003). In addition to classic cadherins, the cadherin superfamily contains at least 6 subfamilies of protocadherins, including celsr cadherins (celsr: cadherin, EGF-like, LAG-like, seven pass receptor)(Hadjantonakis et al, 1998; Nollet et al, 2000). One celsr homolog, flamingo (fmi), has been identified in Drosophila, and 3 celsr paralogs (celsr1, celsr2, and celsr3) have been identified in mammals, including humans (Wu and Maniatis, 2000; Tissir et al, 2002).
Structural aspects of celsr proteins make them particularly interesting cadherins. Celsr proteins are members of a novel group of putative cell adhesion proteins that contain a 7-transmembrane spanning motif that can potentially interact with G proteins (Stacey et al, 2000). The extracellular region is composed of 9 cadherin repeat domains, indicating a cell adhesion function. Celsr2 can bind to itself (a homophilic interaction) to mediate cell adhesion, although extracellular interactions with other proteins also are likely (Usui et al, 1999; Y Shima et al, 2004). The celsr transmembrane region is homologous to the secretin family of G protein–coupled receptors (GPCRs). Although this receptor family has been shown to generate intracellular signals via cAMP and/or inositol triphosphate (Nussenzveig et al, 1994; Nowak et al, 2000), there are no reports of G protein coupling to any celsr homolog. The cytoplasmic domains of celsr paralogs vary considerably, which may be important in determining the biological function of each member.
Functional studies of fmi and celsr indicate roles in planar cell polarity (PCP) and neurogenesis. In Drosophila, fmi interacts genetically with other receptors and adaptor/signaling molecules (including frizzled, disheveled, strabismus, prickle, and diego) to generate and maintain planar polarity of sensory hair bristle cells, inner ear cells, and eye photoreceptor cells (Usui et al 1999; Das et al, 2002, 2004). During PCP development, fmi is believed to anchor signaling molecules at sites of cell-cell contact via a homophilic interaction. The role of fmi in neuronal development is distinct from PCP. In this process, fmi interacts with unknown factors to control dendritic arborization and to guide axons to their correct locations (Gao et al, 1999; RC Lee et al, 2003; Senti et al, 2003). In mammals, celsr2 performs a similar role in neurogenesis (Y Shima et al, 2004), while celsr1 is required for proper development of inner ear PCP and the neural tube (Curtin et al, 2003).
In mice, the 3 celsr paralogs display distinct spatiotemporal expression patterns during development within the central nervous system and urogenital systems (Shima et al, 2002; Tissir et al, 2002). Since celsr is highly conserved and its expression is tightly regulated, it is assumed that celsr performs specialized tissue-specific functions and is essential for survival and reproduction. Previously, we described the presence of celsr2 mRNA in rat testis, suggesting a potential role in spermatogenesis (Johnson et al, 2000). In this article, the hypothesis tested was that celsr paralogs have unique expression patterns and Sertoli cell–germ cell adhesion activity in the postnatal testis. Our data support this hypothesis by demonstrating the following: 1) testicular expression of the 3 celsr paralogs is cell type specific; and 2) celsr2 and celsr3 show differential Sertoli cell–germ cell adhesion activity in a primary cell culture model.
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Materials and Methods |
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Polymerase Chain Reaction Cloning and Semiquantitative Reverse Transcription–Polymerase Chain Reaction
To clone the intervening sequence between 2 previously published rat celsr2
fragments (accession numbers AF177695 and AF177697), a polymerase chain
reaction (PCR)–based approach using Expand Hi Fidelity polymerase (Roche
Diagnostics, Indianapolis, Ind) was used with a primer specific to each
fragment: forward primer 5'-CGGATCCCCTCCACTTTCCAACGTCTCC-3' and
reverse primer 5'-CGAATTCCTCCAGGTAGGTTGTGTCCGA-3'. For the
template, cDNA was prepared from adult testis as previously described
(Johnson et al, 2000). The
resulting approximately 1-kb PCR product was cloned into p-BluescriptII SK
(Stratagene, La Jolla, Calif) and both strands sequenced. A partial cDNA
fragment of rat celsr1 was cloned by PCR using degenerate primers against
amino acids in extracellular cadherin repeats 6 (NGIPQK) and 8 (MYQIVE) that
are conserved in celsr proteins: forward primer
5'-AAYGGIATHCCICARAA-3' and reverse primer
5'-YTCIACDATYTGRTACAT-3'. For this reaction, RedTaq polymerase and
cDNA prepared from postnatal day (PND) 7 testes were used. The resulting
approximately 500-bp PCR product was cloned into pCR4-TOPO vector (Invitrogen
Co, Carlsbad, Calif) and sequenced. Sequences were submitted to GenBank
(accession numbers AY212289 and AY212290).
Testis semiquantitative reverse transcription (RT)–PCR was performed using a "primer-dropping" method, as previously described (Wong et al, 1994; Johnson et al, 2000). As template, cDNA prepared from detunicated testes at PND 7, 21, 28, and 40 was used. Primers were as follows for the mRNA quantitation: celsr1 forward 5'-CTCTTATTCTTGCCACCACT-3'; celsr1 reverse 5'-GATTTCTACATTGAGCCCAC-3' celsr3 forward 5'-GAGGATCCCGACCCCGTTACTACTGCT-3'; celsr3 reverse 5'-GGAATTCGCGTTTTGCCTACCTACTC-3'; hypoxanthine phosphoribosyltransferase (HPRT) forward 5'-CCTGCTGGATTACATTAAAGCGCTG-3'; and HPRT reverse 5'-GTCAAGGGCATATCCAACAACAAAC-3'. During PCR, HPRT primers were added after 4 and 8 cycles for the celsr1 and celsr3 reactions, respectively. Images of the ethidium bromide–stained agarose gels were obtained and PCR products quantified using NIH Image 1.61. For RT-PCR of the 93RS2 rat Sertoli cell line (Jiang et al, 1997), cDNA was prepared from semiconfluent cultures grown at 40°C. celsr1 and celsr3 primers were those used for testis postnatal expression analysis, while the primers for celsr2 were as follows: forward 5'-GCCATCACCGCTCGGGACA-3' and reverse 5'-CGTCATTTACCAGGATCTCCAGGT-3'.
Antibody Generation
Celsr2 and celsr3 antibodies were generated in chickens against
extracellular cadherin repeat domains 4 to 8, expressed in bacteria as
histidine-tagged proteins. Celsr coding regions were amplified using Expand Hi
Fidelity polymerase (Roche Diagnostics), testis cDNA template, and the
following primers: celsr2 forward 5'-GGATCCGTCAGCACCCCTTTCCAG-3';
celsr2 reverse 5'-GGTACCTTAGCGGTCGAGGAGGCGGACA-3'; celsr3 forward
5'-GGATCCGTCAGCACACCCTTTCAG-3'; celsr3 reverse
5'-GGTACCTTAGTCGACCAGGCGTACATGC-3'. PCR products were restricted
with BamHI and KpnI and cloned into pQE30 bacterial expression vector
(Qiagen Inc, Valencia, Calif). The resulting plasmids were sequenced to verify
the insertion integrity. Recombinant proteins were expressed in M15 (pREP4)
bacteria (Qiagen) by induction with 1 mM isopropyl
ß-D-thiogalactopyranoside for 4 hours. After pelleting, bacteria were
resuspended in native lysis buffer (50 mM NaH2PO4, 300
mM NaCl, 10 mM imidazole, pH 8.0) and lysed by sonication on ice. After
centrifugation at 10 000 x g for 20 minutes, the resulting
pellet (containing insoluble celsr recombinant proteins) was solubilized in 50
mM NaH2PO4, 10 mM Tris, 6 M guanidine-HCl, pH 8.0. Celsr
recombinant proteins were purified from this solution using a Ni-NTA agarose
column (Qiagen), with elution by low pH, according to the manufacturer's
instructions. Peak fractions were pooled and dialyzed against decreasing
concentrations of urea (starting at 4 M) in renaturation buffer (25 mM HEPES,
pH 7.6; 100 mM KCl; 12.5 mM MgCl2; 0.1 mM EDTA; and 0.1% IGEPAL
CA-630). Finally, purified celsr recombinant proteins were dialyzed into PBS
and polyclonal chicken antibodies generated in cooperation with Pocono Rabbit
Farm and Laboratory (Canadensis, Pa). From either serum or total egg
immunoglobulin Y (IgY), celsr2 and celsr3 antibodies were affinity purified
against antigen coupled to Affi-Gel 15 (Bio-Rad, Hercules, Calif). Antibodies
were eluted from the affinity column with 100 mM glycine, pH 2.5.
Isoform-specific antibodies were generated by passing the affinity-purified
antibodies over the heterologous affinity column and collecting the
flow-through.
Immunostaining and Western Blotting
Using liquid nitrogen, testes were flash-frozen in OCT compound (Sekura
Finetek Inc, Torrance, Calif) and 8-µm cryosections fixed with -20°C
acetone for 5 minutes. After blocking for 30 minutes with PBS+ (5% normal goat
serum, 0.1% bovine serum albumin in phosphate-buffered saline [PBS], pH 7.4),
sections were incubated for 1 hour at room temperature or overnight at 4°C
with primary antibody diluted in PBS+. The final concentration of primary
antibodies used are as follows: 1:20 anti-celsr2, 1:100 anti-celsr3, 1:200
rabbit anti-rab7 (Santa Cruz Biotechnology, Santa Cruz, Calif), 1:50 mouse
anti-Golgi 58-kd protein (clone 58k9), 1:250 rabbit anti-
-mannosidase
II (gift of K. Moremen; University of Georgia, Athens, Ga), and 1:100 rabbit
anti-protocadherin 3 (Johnson et
al, 2004). After washing with PBS, sections were incubated for 45
minutes at room temperature with goat anti-chicken IgG coupled to Alexa594
(Molecular Probes, Eugene, Oreg) diluted 1:500 in PBS+ and either goat
anti-mouse IgG or goat anti-rabbit IgG coupled to Alexa 488 (Molecular Probes)
diluted 1:1000 in PBS+. After washing with PBS and mounting with Gel/Mount
(Biomeda Corp, Foster City, Calif), sections were viewed using a Ziess
Axiovert 35 or Nikon Eclipse E800 microscope and images were captured with a
Spot RT digital camera (Diagnostic Instruments Inc, Sterling Heights, Mich).
Colocalization was determined using a fluorescein/Texas red dual filter
set.
For immunostaining of celsr2 transfected Hela cells, a C-terminally FLAG tagged mouse celsr2 expression vector was generated in pIRES2-EGFP (BD Biosciences Clontech, Palo Alto, Calif). The full-length celsr2 coding sequence was amplified using Expand Hi-Fidelity polymerase (Roche Applied Science) from a celsr2 expression plasmid (kindly provided by Dr Tadashi Uemura). The final construct was validated by DNA sequencing and Western blotting of lysates from transfected cells, which showed the expected size protein fragments (data not shown). After transfection of Hela cells grown on glass coverslips for 48 hours, cells were processed in a manner similar to testis cryosections and immunostained with the celsr2 antibody and an antibody against the FLAG tag (clone M2). Images were taken using a Zeiss LSM510 confocal microscope (Carl Ziess Inc, Thornwood, NY).
For Western blot analysis, all testicular lysate preparation steps were performed at 4°C. Triton X-100 soluble fraction was prepared according to Maekawa et al (2002). Testes from PND 7, 21, 28, and 40 animals and adult (7 animals at day 7 and 2 animals at all other time points) were decapsulated, pooled, weighed, and homogenized with 3 volumes of Triton X-100–containing lysis buffer (50 mM Tris, pH 7.5; 1% Triton X-100; 5 mM EDTA; and 1 mM MgCl2) containing complete protease inhibitor cocktail (Roche Diagnostics) by 10 strokes of a glass Dounce homogenizer. Lysates were centrifuged at 1000 x g for 5 minutes to pellet cellular debris, and supernatants were centrifuged at 10 000 x g for 10 minutes. Protein concentration in the final supernatant was determined via Bio-Rad DC protein assay (Bio-Rad), and 150 µg was run on 5% SDS-PAGE. After transferring to Immobilon-P membrane (Millipore Corp, Bedford, Mass) using a semidry transfer apparatus (Hoefer Semiphor; Hoefer Scientific Instruments, San Francisco, Calif), the membrane was blocked for 30 minutes at room temperature with blocking solution (20 mM Tris, pH 7.4; 137 mM NaCl; and 5% nonfat dry milk). Celsr2 antibody diluted 1:10 in TBST (20 mM Tris, pH 7.4; 137 mM NaCl; and 0.1% Tween-20) was incubated with the membrane for 1 hour at room temperature or overnight at 4°C. After washing in TBST, rabbit anti-chicken IgG coupled to horseradish peroxidase (A-9046; Sigma) diluted 1:2000 in TBST was added to the membrane and incubated for 1 hour at room temperature. Secondary antibody was visualized with enhanced chemiluminescence according to the manufacturer's instructions (Amersham Biosciences Corp, Piscataway, NJ).
Sertoli Cell–Germ Cell Co-Cultures
Sertoli cell–germ cell co-cultures were prepared following the
co-culture method of Gray and Beamand
(1984). All steps were
performed under sterile conditions. In brief, detunicated testes from PND 21
rats were minced with a razor blade, incubated with 0.24 U trypsin (T-4799;
Sigma) and 210 U DNaseI (DN-25; Sigma) in HBSS (Invitrogen) for 30 minutes at
35°C. After settling at unit gravity, seminiferous tubules were incubated
with 15 615 U collagenase (17100-017; Invitrogen), 9600 U hyaluronidase
(H-3506; Sigma), and 252 U DNaseI in HBSS for 1 hour at 35°C. Following
digestion, the cellular mixture was centrifuged for 5 minutes at 800 x
g, and the pellet was resuspended in Sertoli cell–germ cell
medium (Dulbecco modified Eagle media mixed 1:1 with Hams F12 media
(Invitrogen) plus 1 ng/mg epidermal growth factor, 10 µl/mL ITS+ premix
(containing insulin, transferrin, selenous acid, bovine serum albumin, and
linoleic acid; Collaborative Research, Bedford, Mass), 50 mg/mL gentamicin
(Invitrogen) containing 0.1% trypsin inhibitor (Invitrogen). Sertoli cells and
germ cells were plated at 5 x 106 cells per 35-mm well on
6-well plastic culture dishes coated with 3 µg/mL laminin (Invitrogen).
Co-cultures were incubated at 32°C in 5% CO2 for at least 24
hours before use.
All in vitro experiments were performed in 6-well (35-mm) plastic culture dishes. All wells of each plate were pooled before counting detached cells. Co-cultures were exposed to either 26 µg/mL celsr2 extracellular cadherin domains 4–8 antigen (for 12 or 24 hours [n = 6/time point]), 26 µg/mL celsr3 extracellular cadherin domains 4–8 antigen (for 12 or 24 hours [n = 3/timepoint]) suspended in PBS (0.2% vol/vol in culture medium), or 100 µM mono-(2-ethylhexyl)phthalate (MEHP; TCI America, Portland, Oreg) (for 12 or 24 hours [n = 5/time point]) dissolved in dimethylsulfoxide (DMS0; 0.5% vol/vol in culture medium). Addition of PBS or DMSO vehicles served as negative controls; the number of control replicates mirrored those of the treated groups. After 12- and 24-hour incubations, the medium was collected, centrifuged at 800 x g for 5 minutes, and the cellular pellet resuspended in 1 mL culture medium. Detached germ cells were counted with a hematocytometer. Data from treatment groups were expressed as a ratio to control.
Statistical Analysis
Statistical significance was determined by differences (P <
.05) between the groups detected using a 1-way analysis of variance followed
by a Tukey's multiple comparison test. Statistical analysis was performed
using GraphPad Prism version 4.0 software (San Diego, Calif).
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Results |
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Unique developmental patterns were observed for all 3 celsr paralogs, indicating expression in different testicular cell types. Celsr1 and celsr2 expression was highest at PND 7. During postnatal development, celsr2 mRNA reached a steady level of expression at PND 21, while celsr1 mRNA became nearly undetectable at PND 21 and subsequent time points. Celsr3 mRNA showed an inverse relationship to celsr1 and celsr2 expression, with mRNA levels increasing throughout postnatal development coincident with increasing germ cell numbers.
To determine if celsr paralogs are expressed in immortalized Sertoli cells, a rat Sertoli cell line (93RS2) was analyzed by RT-PCR (Figure 2). Celsr1 and celsr2 but not celsr3 were detected in 93RS2 cells. A negative result when no reverse transcriptase was used and observation of the expected amplicon size confirmed RT-PCR specificity.
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Western blotting of detunicated rat testis homogenates throughout postnatal development with the celsr2 antibodies identified 2 specific bands migrating at approximately 340 kd and 250 kd (Figure 3C). The expected molecular weight of full-length celsr2 based upon amino acid sequence is approximately 320 kd; however, celsr proteins contain a consensus proteolytic sequence in the extracellular juxtamembrane region termed the G protein–coupled receptor proteolysis site (GPS), which would cleave the full-length protein into an approximately 260-kd extracellular fragment and a 60-kd transmembrane/cytoplasmic fragment (Usui et al, 1999; Krasnoperov et al, 2002). Therefore, the celsr2 antibodies detected proteins of the expected size by Western blot analysis. Like the celsr2 mRNA pattern, celsr2 protein levels declined throughout postnatal testicular development as germ cells became the predominant testicular cell type (Figure 3D). Despite repeated attempts, no specific bands were detected by Western blot analysis of testicular homogenates with the celsr3 antibodies (data not shown). Because celsr3 antibodies specifically detected the recombinant celsr3 protein by Western blot and ELISA, the negative result using testicular homogenates may be due to a relatively low level of celsr3 in the homogenates.
Celsr Testicular Immunostaining
Using the affinity-purified celsr antibodies, the cellular localization of
celsr2 and celsr3 was determined in rat testis cryosections. With celsr2
antibodies, two prominent immunostaining patterns were observed that
fluctuated in staining intensity during the immunization period. Celsr2
antibodies from early bleeds displayed a consistent pattern throughout
postnatal testicular development. Immunostaining was predominant at the base
of the seminiferous tubule, with a regular pattern consistent with
localization to Sertoli cells (Figure 4A
through D). In PND 28 and 40 testes, an obvious spokelike pattern
was observed, indicative of Sertoli cell expression. Occasionally, punctate
celsr2 immunostaining within the epithelium was seen. With later bleeds, the
celsr2 punctate staining was more prominent at the expense of the Sertoli cell
pattern. Celsr2 punctate staining in adult epithelium was stage-specific,
being observed in all stages except approximately stages IX and X (data not
shown). Unlike the celsr2 pattern, celsr3 antibodies did not produce any
specific immunostaining from PND 7 through PND 28
(Figure 4E and F). Specific
punctate celsr3 immunostaining was observed in the apical seminiferous
epithelium of PND 40 (Figure
4G) and in adult testis cryosections (data not shown). Together
with the mRNA expression pattern, these data are consistent with celsr3
protein being expressed in and localized to elongating spermatids. Celsr3
antibodies produced punctate immunostaining during all stages of
spermatogenesis except stages VII–VIII, just before elongate spermatid
detachment from the apical epithelium (data not shown). Preabsorption of both
the celsr2 (Figure 4H) and
celsr3 (data not shown) antibodies with antigen abolished immunostaining.
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3,
although this colocalization was not absolute
(Figure 5A through C). In
addition, incomplete colocalization of the celsr2 punctate signal was also
observed with rab7 (Figure 5D through
F), a marker for late endocytic vesicles, and 58k9, a germ cell
Golgi complex marker (Figure 5G through
I) (Chavrier et al,
1990; Bashour and Bloom,
1998). The celsr2 spokelike Sertoli cell pattern colocalized with
-mannosidase II, a Sertoli cell Golgi complex marker
(Figure 5J through
L)(Johnson et al,
1996; Igdoura et al,
1999).
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Celsr2-Induced Germ Cell Detachment In Vitro
A role for celsr2 in Sertoli cell–germ cell adhesion was investigated
using co-cultures of primary Sertoli cells and germ cells. Sertoli
cell–germ cell co-cultures are composed of a monolayer of Sertoli cells,
to which clusters of spermatocytes and spermatogonia adhere. Celsr2
immunostaining demonstrated a punctate pattern in areas in which clusters of
germ cells (spermatocytes and/or spermatogonia) adhered to the Sertoli cell
monolayer (Figure 6A). This
immunostaining was blocked by antigen preabsorption of the antibody
(Figure 6B). As a positive
control for disruption of germ cell adhesion, 100 µM MEHP was added to the
culture medium, resulting in a progressive detachment of germ cells from
Sertoli cells, as previously reported
(Figure
6C)(Gray and Beamond,
1984). Addition of celsr2 protein fragment consisting of
extracellular cadherin domains 4–8 resulted in a significant increase in
germ cell detachment from Sertoli cells, when compared to the negative
control, at 12 hours and 24 hours following exposure
(Figure 6C). At 12 hours and 24
hours following exposure to celsr2 protein fragment, germ cell detachment was
similar to that observed after 12 hours of MEHP exposure
(Figure 6C). Importantly, the
homologous celsr3 protein fragment consisting of extracellular cadherin
domains 4 through 8 did not induce germ cell detachment
(Figure 6C).
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Discussion |
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During postnatal development, dynamic changes in mRNA and protein expression levels occur as a result of dramatic changes in relative testicular cell populations. After the initiation of meiosis, Sertoli cell products appear to decrease, and many germ cell products appear to increase because of the relatively large increase in germ cell numbers (JE Shima et al, 2004). Celsr1 mRNA is abundant in the fetal testis (Hadjantonakis et al, 1998). In addition, both celsr1 and celsr2 transcripts are highly expressed at early postnatal time points, and levels decline between PND 7 and 21, when germ cell meiosis is initiated. Celsr3 shows the converse testicular expression pattern, with undetectable levels at PND 7 and increasing levels observed as germ cells exit the mitotic cycle and mature into haploid spermatids. These data indicated that celsr1 and celsr2 were expressed in Sertoli cells (and potentially in early germ cells), while celsr3 was a germ cell product.
Such a conclusion was corroborated by analysis of celsr protein expression via Western blot and/or immunolocalization. Celsr2 antibodies from early bleeds during the immunization process clearly stained Sertoli cells, identified as a spokelike pattern on testis cryosections; from later bleeds, celsr2 antibodies detected structures associated with germ cells. We conclude that celsr2 is produced in both Sertoli cells and germ cells. In contrast, celsr3 antibodies gave a positive immunostaining reaction on testis cryosections only when elongating spermatids were present. Although the celsr3-positive structure was not defined, it was associated with elongate spermatids. A hallmark of many germ cell–specific proteins functioning during spermiogenesis is mRNA expression at an earlier developmental point followed by translation repression (Eddy et al, 1993). Celsr3 mRNA expression was observed at PND 21, but a positive immunostaining signal was absent until elongating spermatids populated the testis. It may be that celsr3 expression is translationally regulated. In addition, celsr3 mRNA was not detected in a Sertoli cell line. Although the possibility cannot be excluded that celsr3 is a Sertoli cell product induced by germ cells, these data indicate that celsr3 is produced exclusively in germ cells.
With regard to celsr2, the spokelike immunostaining pattern in
Figure 4 is consistent with
localization to either the Sertoli cell plasma membrane or Golgi complex. This
pattern is distinct from Sertoli cell adhesion junctions detected with classic
cadherin antibodies; in the seminiferous epithelium, antibodies against
classic cadherins or cadherin binding proteins (catenins) are associated with
basal inter-Sertoli cell junctions, puncta that are likely desmosome-like
junctions, and an area juxtaposed to the elongate spermatid head
(Mulholland et al, 2001; Johnson and Boekelheide,
2002). Colocalization of celsr2 with -mannosidase II, a
marker of Sertoli cell Golgi complexes
(Johnson and Boekelheide,
1993), demonstrated ceslr2 localization to the Sertoli cell Golgi
complex. However, areas of celsr2 immunostaining were distinct from the
Sertoli cell Golgi complex; therefore, celsr2 may be localized to both Sertoli
cell Golgi complex and other Sertoli cell membranes, perhaps the plasma
membrane. It is unknown if celsr2 has a function within the Golgi complex or
if this localization reflects celsr2 trafficking through the Golgi complex to
the plasma membrane. In Hela cells overexpressing celsr2, abundant plasma
membrane staining was noted, indicating the potential of celsr2 plasma
membrane localization. By Western blot analysis of testis homogenates, 2
celsr2 fragments were detected with sizes appropriate for the full-length
protein and a proteolyzed extracellular fragment cleaved at a conserved
juxtamembrane sequence termed the GPS
(Ponting et al, 1999). In
other GPS-containing GPCRs, processing at the GPS is required for protein
trafficking through the Golgi system to the plasma membrane
(Krasnoperov et al, 2002).
These data support the hypothesis that testicular celsr2 is
post-translationally processed within the Golgi complex and transported to the
plasma membrane.
To examine celsr2 localization to germ cells, colocalization with proteins
previously described in germ cells was investigated. Celsr2 frequently
colocalized with protocadherin 3 at punctate structures in the
centrisomal region of spermatocytes
(Johnson et al, 2004). Endocytic vesicles and the Golgi complex are located in the centrisomal region
of spermatocytes and round spermatids. Celsr2 colocalized with the germ cell
Golgi complex marker 58k9 and rab7, a protein marker of late endocytic
vesicles (Dong et al, 2004).
These data indicate that celsr2 may be associated with the Golgi complex
and/or late endocytic vesicles in the centrisomal region. Although celsr2
immunostaining was not apparent along the plasma membrane of germ cells,
association with a putative endocytic compartment indicates that celsr2 may be
expressed at the germ cell plasma membrane but at sufficient concentration for
detection by immunostaining only within intracellular vesicles.
Regarding the function of celsr cadherins in testis, our data support the
hypothesis that this cadherin family performs both an adhesion and a signaling
function; this hypothesis also is supported by the protein structure of celsr
cadherins. In testis cryosections, celsr2 in germ cells codistributes with
rab7, perhaps in an endocytic compartment. A growing body of evidence
indicates that following phosphorylation and internalization of a
ligand-activated GPCR, an -arrestin–mediated signaling event is
propagated from within endocytic vesicles (reviewed by
Lefkowitz and Whalen, 2004).
Therefore, the punctate celsr2-positive structure in germ cells may represent
an endocytic compartment involved in regulating celsr2 signaling.
Concerning an adhesion function of celsr cadherins in testis, we were unable to detect celsr2 or celsr3 immunostaining at typical adhesion junctions (such as ectoplasmic specializations or desmosome-like junctions) in testis cryosections. Nevertheless, addition of celsr2 extracellular fragment containing 4 cadherin repeats (EC4–EC8) resulted in increased germ cell detachment from Sertoli cells in a co-culture model of primary cells. Addition of the homologous extracellular region of celsr3 did not induce germ cell detachment from Sertoli cells. This indicates that celsr2 fragment–induced germ cell detachment was specific to celsr2. Both the Drosophila celsr homolog (fmi) and mammalian celsr2 can produce cell adhesion via homophilic interactions (Usui et al, 1999; Y Shima et al, 2004). In testis, celsr2 is expressed in both Sertoli cells and germ cells. Collectively, these data indicate that celsr2 mediates Sertoli cell–germ cell adhesion in the testis via homophilic celsr2 interactions but not at typical adhesion junctions.
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Acknowledgments |
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Footnotes |
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