Cell Tissue Res (1999) 296:499–510
© Springer-Verlag 1999
REGULAR ARTICLE
Reinhard Windoffer · Monika Borchert-Stuhlträger
Nikolas K. Haass · Sabine Thomas · Michaela Hergt
Clemens J. Bulitta · Rudolf E. Leube
Tissue expression of the vesicle protein pantophysin
Received: 20 October 1998 / Accepted: 8 January 1999
Abstract The cell-type restricted expression of cytoplasmic microvesicle membrane protein isoforms may be
a consequence of the functional adaptation of these vesicles to the execution of specialized processes in cells of
different specialization. To characterize the expression of
the vesicle protein pantophysin, an isoform of the synaptic vesicle proteins synaptophysin and synaptoporin, we
have prepared and characterized antibodies useful for the
immunological detection of pantophysin in vitro and in
situ. Using these reagents, we show by immunoblot analyses that pantophysin expression is not homogeneous
but differs significantly between various bovine tissues.
Furthermore, these differences are not exactly paralleled
by the expression of other vesicle proteins of the
SCAMP (secretory carrier-associated membrane protein)
and VAMP (vesicle-associated membrane protein) types
that have previously been localized to pantophysin vesicles in cultured cells. By immunofluorescence microscopy, pantophysin expression is seen predominantly in
non-neuroendocrine cells with pronounced membrane
traffic. Pantophysin staining codistributes with SCAMP
and VAMP immunoreactivities in most instances but differs in some. Remarkably, pantophysin staining in liver
is restricted to cells surrounding sinusoids and is not detectable in hepatocytes, similar to that of the SCAMP
This work was supported by the German Research Council
(Le566/3–1)
R. Windoffer · M. Borchert-Stuhlträger · S. Thomas
R.E. Leube (✉)
Department of Anatomy, Johannes Gutenberg-University Mainz,
Becherweg 13, D-55099 Mainz, Germany
e-mail: [email protected];
Tel.: +49 6131 392731; Fax: +49 6131 394615
N.K. Haass · M. Hergt
Division of Cell Biology, German Cancer Research Center,
Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
C.J. Bulitta
Klinik für Allgemein- und Abdominalchirurgie,
Universitätsklinik Mainz, Langenbeckstraße 1,
D-55101 Mainz, Germany
and VAMP isoforms as detected by our reagents in tissue
sections.
Key words Vesicle protein · Physin · Secretory
carrier-associated membrane protein · Vesicle-associated
membrane protein · Synaptobrevin · Expression · Liver ·
Bovine · Human
Introduction
Research over the last few years has revealed that basic
molecular mechanisms are shared during the regulation
of vesicle formation and fusion in many different pathways of membrane trafficking in diverse cells (for reviews, see Ferro-Novick and Jahn 1994; Rothman 1994;
Pfeffer 1996). Especially intriguing are the remarkable
similarities between the highly specialized secretory
transmitter-containing vesicles in neurons and constitutive vesicles participating in the ubiquitous house-keeping functions of intracellular membrane movement in all
eukaryotic cells (for reviews, see Bennett and Scheller
1993; Ferro-Novick and Jahn 1994; Rothman 1994; Südhof 1995; Calakos and Scheller 1996; Linial and Parnas
1996). These similarities are reflected by the presence, in
constitutive vesicles, of molecules that share significant
sequence features with specific synaptic vesicle proteins
(Simons and Zerial 1993; McMahon et al. 1993; Haass et
al. 1996; Südhof and Rizo 1996; Advani et al. 1998; Galli et al. 1998; Janz and Südhof 1998; Shimuta et al.
1998; Takeshima et al. 1998; Wong et al. 1998). The
characterization of the expression patterns of individual
members of these polypeptide groups may therefore help
to elucidate their functions in a given cellular context.
A particularly abundant type of integral membrane
protein of synaptic vesicles is characterized by four
transmembrane domains and cytoplasmic ends. Three
distinct gene families encoding such polypeptides have
been identified to date: (1) a group that we shall refer to
as physins, encompassing the two neuronal isoforms
synaptophysin and synaptoporin (also termed syn-
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aptophysin II), the broadly distributed pantophysin, and
mitsugumin, an isoform recently identified in striated
skeletal muscle (Leube et al. 1987; Südhof et al. 1987;
Knaus et al. 1990; Fykse et al. 1993; Leube 1994; Haass
et al. 1996; Shimuta et al. 1998; Takeshima et al. 1998);
(2) gyrins comprising the neuronal synaptogyrin and
ubiquitous cellugyrin (Stenius et al. 1995; Janz and Südhof 1998); and (3) SCAMPs (secretory carrier-associated
membrane proteins) with at least three related isoforms
that were originally identified in secretory vesicles in
glandular tissues but that are also present in constitutive
vesicle types and in synaptic vesicles (Brand et al. 1991;
Brand and Castle 1993; Singleton et al. 1997).
Of these three families, the physins have clearly received most attention. Synaptophysin as the most prominent member of this group has been characterized as one
of the major polypeptide components in synaptic vesicles, and its cell-type restricted protein detection in neurons and neuroendocrine cells has found wide application in tumor diagnosis and histodiagnosis (for a review,
see Wiedenmann and Huttner 1989). Several functions
have been ascribed to this molecule ranging from the
formation of a fusion pore (Thomas et al. 1988), to the
regulation of transmitter release by interaction with components of the docking complex (Calakos and Scheller
1994; Edelmann et al. 1995; Washbourne et al. 1995),
and to participation in vesicle biogenesis (Leube et al.
1989, 1994; Leimer et al. 1996). On the other hand, the
complete deletion of synaptophysin expression by gene
inactivation through homologous recombination does not
result in detectable functional impairments in mice (Eshkind and Leube 1995; McMahon et al. 1996). The identification of pantophysin as a widely distributed member
of the physin family, however, has suggested that these
molecules perform, either alone or in cooperation with
other similarly tailored polypeptides, basic cellular functions (Leube 1994; Haass et al. 1996). Pantophysin has
been localized to cytoplasmic vesicles that participate in
intracellular traffic between various membrane compartments and contain the ubiquitous vesicle proteins, vesicle-associated membrane protein 3 (VAMP3)/cellubrevin
and SCAMPs (Haass et al. 1996). In an attempt to gain
further insight into potential functions of pantophysin,
we have prepared specific antibodies to examine its expression in human and bovine tissues in comparison with
that of other ubiquitous vesicle membrane proteins of the
VAMP and SCAMP types.
Materials and methods
Antibodies
For the production of monoclonal antibodies against defined epitopes of human pantophysin mixtures of peptides corresponding to
the aminoterminus (peptide P-PHN1; APNIYLVRQRISRLGQRMSGFQINLC; see also Haass et al. 1996), parts of the first intravesicular domain (P-PHIV1; FGYPFRLNEASFQPPPGVNIC)
and the carboxyterminus (peptide P-PHC3; CKETSLHSPSNTSAPHSQGGIPPPTGI; see also Haass et al. 1996) were used
(underlined cysteines were added for coupling). The immunization
of mice, hybridoma formation, and subcloning were performed according to standard protocols (cf. Harlow and Lane 1988). Immunoreactivity of hybridomas was first screened by enzyme-linked
immunosorbent assays (ELISAs; cf. Leube et al. 1994) with serial
dilutions of peptides alone, peptides coupled to ovalbumin, or recombinant fusion proteins from Escherichia coli (see Haass et al.
1996) and HRP-conjugated goat anti-mouse antibodies (Promega
Biotec, Madison, Wis.) for detection with the chromogen 2, 2’azinobis(3-ethylbenzthiazoline-sulfonic acid; Boehringer, Mannheim, Germany) measuring the absorbance at 405 nm. Selected
antibodies were further tested by dot blot assays with the same antigens, by immunoblotting of SDS-polyacrylamide gel electrophoresis (SDS-PAGE)-separated recombinant proteins, and by immunofluorescence microscopy of methanol/acetone-fixed human hepatocellular carcinoma PLC cells previously shown to express
pantophysin (Haass et al. 1996). The IgG subtype of each hybridoma was determined by a stick test (Sigma, St. Louis, Mo.), and by
ELISA with subtype-specific antibodies (Caltag Laboratories, San
Francisco, Calif.). Ascites was prepared by intraperitoneal injection into mice.
In addition, the following recently described antibodies were
used: affinity-purified chicken antibodies raised against the synthetic carboxyterminal peptide P-PHC3 of human pantophysin
(Haass et al. 1996; referred to as ch1); affinity-purified rabbit antibodies against a carboxyterminal peptide of human synaptophysin
(Dako Diagnostika, Hamburg, Germany; referred to as rb4); affinity-purified rabbit antibodies rb9 against the cytoplasmic domain
of rat VAMP3/cellubrevin also reacting with VAMPs 1 and 2 (synaptobrevins 1 and 2; see Haass et al. 1996); monoclonal antibody
SG7C12 reacting with SCAMPs 1–3, albeit with different affinity
to individual isoforms (kindly provided by Prof. D. Castle, University of Virginia, Charlottesville, Va.; see Brand et al. 1991;
Brand and Castle 1993; Singleton et al. 1997); monoclonal antibody Ks18.174 against human cytokeratin 18 (Progen, Heidelberg,
Germany); and rabbit antibodies raised against recombinant nidogen (serum 1046+; kindly provided by Prof. R. Timpl, MPI, Martinsried, Germany). Secondary fluorochrome-conjugated antibodies were: cy3-conjugated donkey anti-chicken IgY (Jackson ImmunoResearch Laboratories, West Grove, Pa.), cy2-conjugated
goat anti-mouse IgG (Rockland, Gilbertsville, Pa.), and fluorescein (DTAF)-conjugated goat anti-rabbit IgG (Rockland).
Immunohistology
Bovine tissues were obtained from a local slaughterhouse and
were frozen, immediately after removal, in isopentane that had
been precooled in liquid nitrogen. Human tissues were snap-frozen, after surgical removal, in liquid nitrogen directly in the operating room. Frozen tissue samples were cut on a cryostat, and
dried sections (5–10 µm) were stored at –70°C until use. After being thawed, sections were either fixed in methanol/acetone and
processed as recently described (Haass et al. 1996) or fixed in 3%
formaldehyde for 20 min at 4°C, permeabilized with 0.01% digitonin for 10 min, and treated with 5% bovine serum albumin for 1 h
(all solutions in phosphate-buffered saline; PBS). Sections were
then successively incubated with primary antibody for 1 h, washed
in PBS for 15 min, incubated with secondary antibody for 1 h, and
washed again with PBS for 15 min. To test the specificity of pantophysin antibodies, cryosections were incubated in the same way as
described above, but primary antibodies (at their final dilution)
were preabsorbed with peptides used for immunization (1 µg/ml)
for 30 min at room temperature. Fluorescence was routinely
viewed on an epifluorescence microscope (Axiophot, Carl Zeiss,
Jena, Germany) and was recorded either by photography or by direct input via a Hamamatsu C4742–95 digital camera (Hamamatsu, Herrsching, Germany). Fluorescence was monitored in
some instances with a confocal laser-scan microscope (Leica,
Bensheim, Germany). Adobe Photoshop software (version 4.0)
was used to arrange the figures.
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Cell fractionation and immunoblotting
Frozen tissue samples were pulverized in liquid nitrogen in a porcelain mortar by means of a pestle. The tissue powder was added
to hypotonic H buffer, consisting of 10 mM TRIS-HCl (pH 7.4),
1 mM EGTA, 1 mM EDTA, 2 mM DTT, and 0.1 mg/ml PMSF,
0.2 mg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride), and homogenized in a tight-fitting Dounce homogenizer with 30 up and
down strokes on ice. All further steps were performed at 4°C.
Centrifugation at 1 000 g for 15 min resulted in pellet fraction P
1 000 and supernatant S 1 000. Further centrifugation at 100 000 g
for 1 h yielded pellet P 100 000 and supernatant S 100 000. Protein
concentrations were determined by means of the Bradford reagent
(protein assay from Bio-Rad Laboratories, Hercules, Calif.). Equal
amounts of polypeptides were dissolved in loading buffer, separated by SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with primary and HRP-coupled secondary antibodies (all
from Jackson ImmunoResearch Laboratories) following standard
protocols (cf. Leube 1995). Detection of bound secondary antibodies was by enhanced chemiluminescence (Amersham International, Buckinghamshire, UK).
Results
Generation and characterization of pantophysin
antibodies
We have recently described antibodies raised against the
amino- and carboxyterminus of human pantophysin by
using synthetic peptides as antigens (Haass et al. 1996).
These antibodies were shown to react with their cognate
epitopes when present in fusion proteins produced in E.
coli. However, only affinity-purified antibodies against
the carboxyterminus were of sufficient quality for routine immunohistology of tissue sections and immunoblotting of complex cell fractions. One of these antibod-
Fig. 1 Immunoblot detection of pantophysin in 100 000 g pellets
obtained from cell lysates of bovine testis; 100 µg polypeptides
were loaded in each lane, separated by 15% SDS-PAGE, blotted
onto nitrocellulose, and reacted with either affinity-purified antibodies ch1 that were raised in chicken against a carboxyterminal
peptide of human pantophysin, or murine monoclonal antibody
mc50 generated against the same carboxyterminal peptide of human pantophysin (detection of bound antibodies with enhanced
chemiluminescence and HRP-coupled secondary antibodies). The
relative positions and molecular weights of co-electrophoresed
marker proteins are given left in units of 1000, from top to bottom
ovalbumin (Mr 45 000), glyceraldehyde-3-phosphate dehydrogenase (Mr 36 000), carbonic anhydrase (Mr 29 000), trypsinogen
(Mr 24 000), trypsin inhibitor (Mr 20 000)
Fig. 2A–C Photomontage of immunoblots (lanes 1–5 the same
gel, lanes 6 separate gels) detecting ubiquitous vesicle proteins in
bovine tissues. Aliquots of 100 µg polypeptides contained in
100 000 g pellets of bovine epididymis, parotid gland, muscle
(striated skeletal muscle), small intestine (mucosa-enriched), testis, and liver were loaded in each lane, separated by SDS-PAGE
(15% in A, B; 18% in C) and reacted with primary antibodies.
Bound antibodies were detected with HRP-coupled secondary antibodies in combination with an enhanced chemiluminescense
system. The blots in A were reacted with murine monoclonal antibody mc50 recognizing a carboxyterminal epitope of human pantophyisn (PPH; long exposure of lane liver to show very weak reactivity). The blots in B were reacted with murine monoclonal antibody SG7C12 detecting secretory carrier-associated membrane
proteins (SCAMP; major 37-kDa and minor 35-kDa polypeptide).
The blots in C were reacted (long exposure of lane liver to show
weak reaction) with antibody rb9 raised against an epitope present
in several VAMP isoforms (VAMP). The position and Mr (in units
of 1 000) of co-electrophoresed marker proteins are shown left:
ovalbumin (Mr 45 000), glyceraldehyde-3-phosphate dehydrogenase (Mr 36 000), carbonic anhydrase (Mr 29 000), trypsinogen
(Mr 24 000), trypsin inhibitor (Mr 20 000), α-lactalbumin (Mr
14 000)
ies from chicken (ch1; Haass et al. 1996) reacted specifically with two polypeptide bands of approximately 30
and 32 kDa in 100,000 g pellet fractions of bovine testis
(Fig. 1), these molecular weights (MW) being only
slightly larger than the calculated MW of 28 565 Da of
the polypeptide found in human (Leube 1994). Multiple
polypeptide bands of even higher apparent molecular
weight had previously been detected in cultured human
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Fig. 3 Indirect immunofluorescence microscopy of formaldehydefixed tissue cryosections (10 µm thick) of bovine testis detecting
pantophysin (PPH) with affinity-purified chicken antibodies ch1
(A; controls in C, D), and SCAMPs (SCAMP) with monoclonal
antibody SG7C12 (B) in seminiferous tubules (st) and surrounding
tissue of the lamina propria (lp). A Note that all cells of the seminiferous tubules are strongly positive for pantophysin and that the
immunofluorescence in the surrounding connective tissue of the
lamina propria (lp) is restricted to the cytoplasm of fibroblasts and
is absent in the extracellular matrix. B Note the different distribution pattern of SCAMP immunoreactivity in comparison to that of
pantoysin. C Pantophysin immunoreactivity (corresponding Nomarsky contrast micrograph in D) after preabsorption of ch1 antibodies with the pantophysin carboxyterminal peptide used for immunization (1 µg/ml). Note the absence of significant fluorescence
(lu position of the lumen of a seminiferous tubule). Bars
50 µm
cells with the same antibodies and were thought to represent the glycosylated form (Haass et al. 1996). The
reaction could be negated by preincubation of the antibodies with the peptide used for immunization (not
shown).
To circumvent the problems of tedious affinity purification and to avoid the limitations imposed by small antibody amounts, we immunized mice with a combination
of various peptides taken from different parts of human
pantophysin to prepare monoclonal antibodies. Of the
hybridomas tested, only those reacting with the cytoplas-
mically exposed end-domain gave satisfactory signals
both in immunofluorescence microscopy and immunoblotting. The immunoblot reaction of one of these antibodies (mc50; IgG1 antibody) is shown in Fig. 1 and
demonstrates an identical reaction pattern in bovine testis as ch1. This antibody was used for further experiments.
With this antibody, we detected the accumulation of
pantophysin in a high-speed pellet fraction from which it
could be extracted with the nonionic detergent Triton X100 (not shown) indicating that the polypeptide was
present in a light membrane compartment similar to that
previously shown to contain pantophysin in a cultured
human hepatocellular-carcinoma-derived PLC subclone
by using antibody ch1 (Haass et al. 1996). Furthermore,
when the 100 000 g pellet fractions from bovine testis
were loaded onto linear sucrose gradients and subjected
to equilibrium centrifugation, pantophysin could only be
detected in a few light fractions (not shown; compare
Haass et al. 1996).
Immunoblot detection of pantophysin in bovine tissues
Various tissues were analyzed for the presence of pantophysin by using monoclonal pantophysin antibody mc50
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Fig. 5 Double-label immunofluorescence microscopy of formaldehyde-fixed sections of bovine epididymis by using primary antibodies ch1 detecting pantophysin (PPH; A) and SG7C12 reacting
with SCAMPs (SCAMP; B). Note the strong adluminal labeling of
SCAMP antibodies in contrast to the more homogeneous labeling
of all epithelial cells (ep) with pantophysin antibodies and the
bright pantophysin staining of spermatids present in the lumen
(lu). Bar 50 µm
Fig. 4 Double-label immunofluorescence localization of pantophysin (PPH; A) with affinity-purified chicken antibodies ch1 and
of VAMPs (VAMP; B) with affinity-purified rabbit antibodies rb9
in a methanol/acetone-treated 5-µm-thick cryosection of human
testis (corresponding phase micrograph in C). Note the similar
pattern of multipunctate immunofluorescence in all cellular elements. Bar 50 µm
(Fig. 2A shows examples of pantophysin-immunoreactivity in 100 000 g pellets of bovine tissues). Expression
was clearly detectable not only in testis as expected (see
Fig. 1), but also in parotid gland, epididymis, and small
intestine, whereas only trace amounts of pantophysin
were visible in striated skeletal muscle and liver. The apparent molecular weight and the number of reactive
polypeptide bands varied between these tissues, probably
indicating different patterns of polypeptide modification.
Since we had previously shown that pantophysin colocalizes with the ubiquitous vesicle proteins SCAMP
and VAMP3/cellubrevin in cultured cells (Haass et al.
1996), we wanted to compare their expression in bovine
tissues. The immunoblot results for an antibody reacting
with different SCAMP isoforms (Singleton et al. 1997)
is depicted in Fig. 2B, demonstrating the strongest reactivity in epididymis, parotid gland, and liver, weaker reactivity in testis, a low signal in small intestine, and none
in striated skeletal muscle. To identify VAMP isoforms,
we used an antibody that was originally designed to de-
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Fig. 6 Fluorescence micrographs depicting immunoreactivities
for pantophysin (PPH; affinity-purified antibodies ch1), synaptophysin (SPH; rabbit antibodies rb4) and SCAMPs (SCAMP;
monoclonal antibody SG7C12) in formaldehyde-fixed sections of
bovine pancreas (A–F) and bovine parotid gland (G). A Strong
pantophysin-immunoreactivity is visible in glandular cells (gl) of
exocrine pancreas. B/C, D/E Co-incubations of tissue sections
with antibodies detecting either pantophysin (B, D) or synaptophysin (C, E) demonstrate differences and similarities in pantophysin
expression in endocrine islets of Langerhans (en), which are always strongly positive for synaptophysin. Note the absence of
synaptophysin reactivity in the exocrine part of the pancreas (ex).
D, E were generated with the help of a confocal laser-scan microscope to demonstrate immunoreactivity with both reagents in the
same focal plane (e.g., cell denoted by arrow). F Note the labeling
of glandular cells (gl) in exocrine pancreas with antibodies against
SCAMPs. G Strong pantophysin immunoreactivity is detectable
in the glandular cells (gl) of the parotid gland. Bars
50 µm in A, C, 25 µm in E, 20 µm in F, G
tect VAMP3/cellubrevin but also reacts with VAMPs 1
and 2 (synaptobrevins 1 and 2) and possibly other VAMP
isoforms in immunoblots (Haass et al. 1996). This antibody recognized two polypeptide bands in the tissue
blots of fractions from epididymis, parotid gland, and
testis. Very weak detection of these polypeptides was
noted in liver, and barely detectable levels were seen in
muscle and small intestine. Taken together, the immuno-
blot results demonstrated differences in the expression of
pantophysin, SCAMPs, and VAMPs as visualized by our
antibodies.
Immunofluorescence microscopy of bovine
and human tissues
Immunofluorescence microscopy was performed to extend the expression studies to the cellular level. First, bovine testis was analyzed as it contained considerable
amounts of immunoreactivities for all examined polypeptides (see Fig. 2). The cytoplasm of practically all
cells showed strong multipunctate labeling with pantophysin antibodies, whereas the surrounding extracellular matrix of the lamina propria was negative (Fig. 3A).
This immunoreactivity was completely abolished after
preincubation of the antibody with the immunogen (Fig.
3C, D). The staining pattern in cow and human exhibited
a strong resemblance to that obtained with VAMP antibodies as demonstrated by colocalization (Fig. 4). In
contrast, SCAMP immunoreactivity was more concentrated toward the lumen of the seminiferous tubules and
appeared to be much coarser (Fig. 3B). Strong pantophysin immunoreactivity was also seen in the ductus epidid-
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Fig. 7 Photomicrographs of pantophysin immunofluorescence
(Nomarsky contrast in B and E corresponding to regions shown in
A and D, respectively) in formaldehyde-fixed sections of human
gastrointestinal tissues. Pantophysin was detected with affinity-purified antibodies ch1 together with cy3-labeled secondary antibodies. A, B Colon: epithelial cells (ec) are strongly positive, whereas
cells in the lamina propria (lp) show slightly weaker immunoreactivity (gl gut lumen). C Stomach: cells in the foveola gastrica (fg)
exhibit strong immunofluorescence, with weaker reactivity being
found in the gastric glands (gg). D, E Small intestine: epithelial
cells (ec) are strongly positive; cells in the lamina propria (lp)
show barely detectable immunoreactivity in this section. Bars
50 µm
ymidis, again only partially overlapping with the staining for SCAMPs (Fig. 5). Furthermore, the competable
pantophysin immunofluorescence in the epididymis was
not restricted to epithelial cells but also included spermatids (see the lumen in Fig. 5A).
Expression of pantophysin was further examined in
exocrine glands. In both the exocrine pancreas and the
parotid gland, a strong multipunctate immunofluorescence was seen with antibodies directed toward pantophysin (Fig. 6A, B, D, G; compare with human pancreas in Haass et al. 1996), SCAMPs (Fig. 6F; for expression in parotid gland, see Brand et al. 1991; Brand and
Castle 1993; Wu and Castle 1997), and VAMPs (not
shown; for expression in pancreas and parotid gland, see
Braun et al. 1994; Regazzi et al. 1995; Fujita-Yoshigaki
et al. 1996; Gaisano et al. 1996; Rossetto et al. 1996;
Sengupta et al. 1996). On the other hand, pantophysin
antibody labeling of the endocrine islets of Langerhans
was variable (cf. Fig. 6B, D), in contrast to the consistently strong staining with synaptophysin antibodies
(Fig. 6C, E).
Next, pantophysin expression was studied in tissue
sections of the gastrointestinal tract (Fig. 7). In each
case, the strongest reactivity was observed in epithelial
cells, including practically all cell types lining the surface and glands of the stomach (Fig. 7C), small intestine
(Fig. 7D, E), and large intestine (Fig. 7A, B). The reaction in fibroblasts of the lamina propria, however, was
weaker and varied from clearly punctate (Fig. 7A) to
near background levels (Fig. 7D).
Lack of pantophysin expression in bovine hepatocytes
The low level of pantophysin- and VAMP-epitope expression in liver prompted us to investigate their distribution pattern in more detail by immunofluorescence microscopy. Surprisingly, the large majority of cells
showed no significant immunolabeling with various
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Fig. 8 Immunofluorescence microscopy of formaldehyde-fixed
sections of bovine liver after reaction with antibodies to pantophysin (PPH; affinity-purified antibodies ch1 in A, C; monoclonal antibody mc50 in E), a common VAMP epitope (VAMP; affinity-purified antibodies rb9 in B) and SCAMPs (SCAMP; monoclonal antibody SG7C12 in D) demonstrating, in all instances, a lack of significant labeling in hepatocytes (asterisks) and multipunctate
staining in cells bordering sinusoids (arrows). C, D Colocalization
of pantophysin (C) and SCAMPs (D) in endothelial-like cells in
the same tissue section. Bars 25 µm
pantophysin antibodies (Fig. 8A, C, E). The typical
multipunctate fluorescence was restricted to elongated
cells that were located around sinusoids. A similar pattern was also observed with antibodies against the common VAMP epitope and SCAMPs (Fig. 8B, D), which
codistributed in the same cells together with pantophysin
(Fig. 8C, D). To establish that hepatocytes did not express significant amounts of pantophysin, double-label
immunofluorescence microscopy was performed with
cytokeratin 18 antibodies that stain exclusively hepatocytes but do not react with endothelial and fibroblastic
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cells (cf. Moll et al. 1982). There was practically no
overlap of immunoreactivity to pantophysin and cytokeratin 18 (Fig. 9A–D). In contrast, pantophysin-positive
cells were next to areas stained with antibodies to nidogen, a prominent basal membrane component (Timpl
1996). Partial overlap of both immunoreactivities can be
seen in Fig. 9E–G, and the enlargement in Fig. 9H depicts a region in which the cytoplasmically restricted
punctate pantophysin staining can be distinguished from
the even fluorescence for nidogen in the surrounding extracellular matrix.
Discussion
We have examined pantophysin expression in bovine and
human tissues with new immunological reagents. Enrichment of pantophysin in a high-speed pellet, its extractability from this fraction by Triton X-100, and its
accumulation in distinct sucrose gradient fractions all
correspond well to the properties previously described
for pantophysin expressed in cultured cell lines, which,
in some instances, had also been transfected with antibody-tagged versions of the molecule (Haass et al.
1996). Using these specific pantophysin antibodies, we
have now extended earlier analyses in which pantophysin mRNA had been shown to be expressed in many different tissues and cultured cell lines (Zhong et al. 1992;
Leube 1994; Haass et al. 1996). Our immunoblot results
portray a heterogeneous expression pattern, in contrast to
the ubiquitous detection of pantophysin mRNA by reverse transcription/polymerase chain reaction (Leube
1994) and the identification of pantophysin mRNA by
Northern blot hybridizations, albeit at different levels in
non-neuroendocrine and neuroendocrine cells (Zhong et
al. 1992; Leube 1994; Haass et al. 1996). Furthermore,
this heterogeneity of protein expression is not paralleled
by the expression of SCAMPs and VAMPs, both of
which have been shown to colocalize in the same cytoplasmic microvesicles in cultured human cell lines
Fig. 9A–H Double-label immunofluorescence microscopy of bovine liver (formaldehyde fixation) showing reaction of antibodies
against pantophysin (PPH; antibody ch1) together with either antibodies against cytokeratin 18 (CK) or nidogen (NID). Images
where retrieved with a black and white digital camera (A, B, E, F)
and photoshop software was used to produce false colors and
overlay pictures (C, D, G, H). A–D Note the differences between
pantophysin immunoreactivity in A, which is limited to cells located around sinusoids (arrows), and the staining for cytokeratin
18 in B, which is restricted to hepatocytes (asterisks). The overlay
colored pictures in C (obtained from A, B) and D show the lack of
significant overlap between the labeling for pantophysin (green)
and cytokeratin (red). E–H Note the absence of both pantophysin
and nidogen staining in hepatocytes (asterisks) but the labeling of
regions directly surrounding sinusoids (arrows). Compare the similar fluorescence distribution in E and F and the partial overlap
(yellow) in the corresponding overlay picture in G. The highpower view in H shows a region in which the intracellular labeling
with pantophysin antibodies (red) can be separated from the mostly extracellular staining with antibodies against nidogen (green).
Bars 50 µm in A, E, 25 µm in D, H
(Haass et al. 1996) and both of which exhibit
considerade but not the same differences in protein expression levels in bovine tissues. This is in accordance
with other investigations that have so far failed to identify a linked expression of individual members of the different multigene families encoding specific integral vesicle proteins in different neurons, even including examples in which a molecular association had been shown
(compare synaptophysin and VAMP2/synaptobrevin 2
expression, e.g., Trimble et al. 1990; Marquèze-Pouey et
al. 1991; Fykse et al. 1993). Therefore, different mixtures of these polypeptide types could define the specific
microvesicle composition in a given tissue, thereby allowing alternative molecular interactions. Furthermore,
the expression of the individual members of the multigene families is not exclusive but overlaps in certain cell
types (Fig. 6) where they may colocalize in the same
vesicles (Fykse et al. 1993; Chilcote et al. 1995; Regazzi
et al. 1995; Gaisano et al. 1996; Haass et al. 1996; Wu
and Castle 1997). The coexpression of several isoforms
of the same polypeptide type in the same cell may ensure
functional redundancy and could explain why the deletion of synaptophysin does not result in major phenotypic alterations in knock-out mice (Eshkind and Leube
1995; McMahon et al. 1996). The observed differences
in the distribution of the immunoreactivities of the three
investigated polypeptide types in some tissues (e.g., Fig.
4, Fig. 5) add another degree of complexity to their expression, indicating alternative modes of protein distribution in a given cellular context.
An intriguing observation of the presented study is
the lack of pantophysin, VAMP, or SCAMP eptiopes in
hepatocytes, which are known to be highly active cells in
terms of membrane trafficking and which are widely
used as paradigms for the investigation of directed vesicle movement (e.g., Ihrke and Hubbard 1995). This is
somewhat surprising, since we have recently shown that
pantophysin is expressed in hepatocellular carcinomaderived PLC cells (Haass et al. 1996). It is unlikely,
however, that the lack of immunoreactivity of all antibodies in normal hepatocytes is attributable to epitopemasking, as cells surrounding sinusoids are clearly positive in all instances. Therefore, hepatocytes may either
express other members of the multigene families not detectable with the antibodies used for this study or (less
likely) use completely different mechanisms of membrane trafficking. Indeed, for all three multigene families, new members have been identified recently (Singleton et al. 1997; Advani et al. 1998; Galli et al. 1998;
Shimuta et al. 1998; Takeshima et al. 1998; Wong et al.
1998) and more may be characterized soon (Bock and
Scheller 1997). Some of these recently identified polypeptides show highly restricted expression, e.g., mitsugumin, a physin isoform so far only detected in significant amounts in skeletal muscle and kidney (Shimuta et
al. 1998; Takeshima et al. 1998). Others, such as various
VAMP isoforms that are expressed at higher levels in
liver (e.g., VAMPs 4 and 7; Advani et al. 1998) may indeed be expressed in hepatocytes. Our results also indi-
509
cate that there should be a reexamination of the results
described for VAMP3/cellubrevin and SCAMPs in vesicle fractions obtained from liver (McMahon et al. 1993;
Pol et al. 1997; Brand et al. 1991; Thoides et al. 1993;
Singleton et al. 1997), as it is possible that they reflect
the distribution of these molecules in the vesicles of endothelial cells but not, or only in part, of hepatocytes.
In conclusion, the newly characterized antibodies described here should be useful tools for the examination
of pantophysin expression in human and bovine cells and
tissues. Our results lay the foundation for further studies
of the differential synthesis of members of the physin
gene family, and it is expected that, in addition to the recently identified physin isoform mitsugumin29, which is
present in significant amounts in striated skeletal muscle
and kidney (Shimuta et al. 1998; Takeshima et al. 1998),
other related polypeptides will be identified that are
present in the vesicles of specific cells such as hepatocytes.
Acknowledgements We gratefully acknowledge the generous
gifts of antibodies against SCAMPs by Prof. David Castle (University of Virginia, Charlottesville, Va., USA) and against nidogen
by Prof. Rupert Timpl (MPI, Martinsried, Germany). We also
thank Anke Marschall and Stephanie Heupel for expert technical
help, Dr. Hans-Richard Rackwitz for synthesis and purification of
synthetic peptides, and Prof. Werner W. Franke for his support
during the initial stages of this project at the German Cancer Research Center (Heidelberg, Germany).
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