* Department of Anatomy and Cell Biology, University of Heidelberg, D-69120 Heidelberg, Germany; and Division of Cell
Biology, German Cancer Research Center, D-69120 Heidelberg, Germany
Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). Synaptopodin contains a high amount of proline (~20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level.
In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.
THE postsynaptic segment of neuronal dendrites consists of the postsynaptic densities (PSD)1 and the associated dendritic shaft. In the dendritic shaft, an actin-based cytoskeleton is found, which anchors in the
PSD (Matus, 1982). It is generally accepted that learning
and memory require morphological changes in neurons
(for review see Wallace et al., 1991 Despite the recent progress of the study of the presynaptic proteins, the composition of the PSD and the associated dendritic shaft are still largely elusive (Kennedy,
1993 Podocytes of the renal glomerulus are unique cells with
a complex cellular organization. With respect to their cytoarchitecture, podocytes may be divided into three structurally and functionally different segments: cell body, major processes, and foot processes. In the foot processes a
complete microfilament-based contractile apparatus is
present, which is composed of actin, myosin II, In a previous study, we described the association of a
protein defined by mAb G1 with the actin system of
podocyte foot processes (Mundel et al., 1991 Tissues for Immunohistochemistry
Female adult Sprague-Dawley rats (BW 200g) were perfused via the abdominal aorta with 2% paraformaldehyde in PBS for 3 min at 220 mm Hg
followed by cryoprotectant sucrose-PBS solution (800 mOsmol) for 5 min
at 220 mm Hg. Organs were harvested and slices of tissues were frozen in
isopentane cooled by liquid nitrogen to Hippocampal Cultures
Cultures of hippocampal cells were prepared from the brains of 18-d-old
fetal Sprague-Dawley rats exactly as described (Cid-Arregui et al., 1995 Podocyte Cultures
Adult podocyte cell lines were cloned by limiting dilution from glomerular
cultures of the "immortomouse" (Jat et al., 1991 Depolymerization Studies
These experiments were carried out with differentiated arborized
podocytes after 7 to 14 d at 37°C. Cells were treated for different time intervals (3, 6, and 24 h) with actin-depolymerizing cytochalasin B (0.4-10
µm) and microtubule-depolymerizing colcemid (0.5 µM). At different
time points after drug treatment, cells were prepared for immunofluorescence labeling. To study the reversibility of alterations, the drugs were
removed, and cells were washed twice with RPMI 1640 and allowed to recover for different time intervals before preparation for immunocyto-chemistry.
Antibodies
Synaptopodin-specific Antibodies.
The generation and initial characterization of anti-synaptopodin mAb G1 has been described previously (Mundel et al., 1991 Additional Primary Antibodies.
Rabbit anti-synaptophysin (DAKO, Hamburg, Germany), rabbit anti-MAP2 (Boehringer Mannheim), rabbit anti- Immunofluorescence Microscopy
Immunofluorescence staining was routinely performed on 4-µm, frozen
sections from rat kidney and 8-µm, frozen sections from rat brain as well
as with cultured rat hippocampal neurons growing on poly-lysine-coated
coverslips after 25 d in vitro and with cloned mouse podocytes growing on
collagen type I-coated glass cover slips. To test for additional expression
of synaptopodin, sections from the following organs were analyzed by immunohistochemistry: liver, heart, lung, skeletal muscle, adrenal gland, testis, salivary gland, stomach, pancreas, spleen, small intestine, and colon
(cytosolic extracts from these organs were also analyzed by immunoblotting; see below). Cultured cells were either fixed with ice-cold acetone at
Immunoelectron Microscopy
For preembedding immunoperoxidase labeling, cryostat sections of perfusion-fixed tissues from rat brain were cut at 20 µm and processed as free-floating sections. After preincubation in PBS containing 10% normal
swine serum, synaptopodin mAb G1 (prediluted 1:16 in PBS containing
10% normal swine serum) was applied overnight at room temperature,
followed by biotinylated donkey anti-mouse IgG (1:50) for 1 h. After
washing with PBS, sections were placed in streptavidin-conjugated peroxidase complexes (1:200). Peroxidase activity was detected using 3,3 Tissue Fractionation and Protein Extraction for
Immunoblotting and Protein Purification
Brains and kidneys from 20 adult Sprague-Dawley rats (body mass 200 g)
were harvested. Dissected forebrains and cerebella were immediately frozen in liquid nitrogen. Isolation of glomeruli from kidneys was done by
passing tissue through a series of steel sieves exactly as described (Mundel
et al., 1991 Protein extraction was carried out at 4°C in a tight-fitting Potter homogenizer with 15 strokes at 1,300 rpm in 10 vol of homogenization buffer
(20 mM Tris, 500 mM NaCl, pH 7.5) supplemented with 0.5% Chaps
(Sigma Chemical Co.), 5 mM EDTA, and the following protease inhibitors (all from Serva, Heidelberg, Germany): 2 µm Pepstatin, 2 µm Leupeptin, and 200 µm Pefabloc. Insoluble material was pelleted at 50,000 g
for 30 min at 4°C. The resulting crude supernatants (C-Sup) were stored at
For the analysis of synaptopodin expression during postnatal maturation of the brain, C-Sups were prepared from rat forebrains and cerebella
harvested at day 5, 10, 15, 20, and 50 postnatal. At each time point material from five animals was pooled.
Gel Electrophoresis, Immunoblotting, and Protein
Sequence Analysis
C-Sups and HS-Sups from isolated glomeruli, forebrain, and cerebellum
were diluted with SDS sample buffer, boiled for 5 min, and separated on
8% SDS polyacrylamide gels. Proteins were transferred to Immobilon P
membranes (Millipore Corp., Eschborn, Germany) by semidry blotting
and stained with 0.05% Coomassie brilliant blue in 40% methanol and 7%
acetic acid. Before immunodetection, the membranes were destained with
50% methanol and 2% acetic acid. Primary antibodies (either hybridoma
supernatant of mAb G1 or affinity-purified polyclonal antibodies 26-1E
and NT-61) were used at 1:500 to 1:2,000, and horseradish peroxidase-conjugated secondary antibodies (Promega, Heidelberg, Germany) were used at 1:20,000. The immunoreaction was visualized by chemiluminescence (Amersham Intl., Braunschweig, Germany) and film exposure. To test for
possible expression of synaptopodin in other organs than brain and kidney, cytosolic extracts from a variety of organs (liver, heart, lung, skeletal
muscle, adrenal gland, testis, salivary glands, stomach, pancreas, spleen,
small intestine, and colon) were analyzed by immunoblotting.
For protein sequencing, synaptopodin was enriched from rat forebrain
and from isolated glomeruli by preparative HPLC-reversed phase chromatography, and purification steps were monitored by Western blotting
using mAb G1 to detect the protein. Per run, 5 ml of HS-Sup (see above)
were loaded onto a Brownlee 4.6-mm C8 column (Applied Biosystems,
Weiterstadt, Germany) and developed with a linear gradient of 10 to 80%
acetonitrile during 45 min. To avoid contaminations, different columns
were used for the separation of brain and glomerular extracts. 1-ml fractions were collected; 100-µl aliquots were lyophylized, resuspended in
SDS sample buffer, and analyzed by immunoblotting. Positive fractions
were lyophylized, resuspended in 100 µl of lysis buffer, and separated by
two-dimensional gel electrophoresis with NEPHGE (non-equilibrium pH
gradient gel electrophoresis) in the first dimension, followed by separation in 8% SDS gels in the second dimension as described (Heid et al., 1994 cDNA Cloning and Sequencing
Data base searches identified three expressed sequence tag (EST) clones
obtained from Research Genetics (Huntsville, AL) termed sp17 (these sequence data available from EMBL/GenBank/DDBJ under accession
number H49442), sp47 (accession number R88417), and sp91 (accession
number R90893) orientated from 5 The mouse homologue of the synaptopodin gene product was cloned
by a combination of cDNA library screening and RT-PCR cloning. To this
end, a mouse brain cDNA library ( Sequence alignments, analyses, and data base searches were done with
the software program package HUSAR (Heidelberg Unix Sequence Analysis Resources).
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from human brain cortex and hippocampus, from
rat brain forebrain and cerebellum, and from rat kidney cortex using the
RNeasy total RNA kit (Quiagen, Hilden, Germany) according to the
manufacturer's instructions. Northern blot analyses were done as described (Schäfer et al., 1994 In Situ Hybridization Studies
Radioactive in situ hybridization of sagittal and coronal frozen sections
from adult mouse brain was carried out as described by Monyer et al.
(1992) Biochemical Analysis of Synaptopodin
Using mAb G1, a heat-stable protein with an apparent
molecular mass of 100 kD was recognized by immunoblotting in cytosolic extracts from rat brain (Fig. 1). In cytosolic
fractions from isolated rat kidney glomeruli, a heat-stable
band with an apparent molecular mass of 110 kD was shown
(Fig. 1). The originally described 44-kD band (Mundel et
al., 1991
A polyclonal antiserum (26-1E), directed against an internal aa sequence corresponding to rat brain synaptopodin peptide fragment No. 26 (which is identical to rat kidney peptide fragment No. 25; see below), recognized the
110-kD band in glomerular extracts and the 100-kD band
in cytosolic extracts from rat and human brain as originally
described mAb G1. A second polyclonal antibody (NT-61) directed against a peptide sequence located in the 5 To examine the solubility of synaptopodin, fractionation
experiments of rat forebrain were carried out. In low salt
(150 mM NaCl) buffer, only a partial extraction of synaptopodin was achieved, whereas homogenization with high
salt (500 mM NaCl) buffer supplemented with 0.5% Chaps
resulted in complete extraction of the protein (data not
shown).
Purification and Peptide Sequencing of Rat Brain and
Kidney Synaptopodin
Synaptopodin was purified from rat kidney glomeruli and
forebrain. Protein bands were cut from preparative two-dimensional blots and subjected to tryptic digestion. 20 of
the resulting internal peptides of the brain protein were
analyzed by Edmann degradation. Data base comparison
revealed that five peptides matched with two EST clones
(clones sp47 and sp91) from a human brain cDNA library.
A third EST clone from the same library (clone sp17) was
identified by overlapping with the 5
Cloning and Sequencing of Synaptopodin cDNA
The complete cDNA sequence of human brain synaptopodin was established from three EST clones (termed sp17,
sp47, and sp91) and an additional fragment that covered a
250-b gap between clones sp47 and sp91, which was obtained by RT-PCR cloning. While clone sp17 contained a
consensus sequence for initiation of translation, clone sp91
contained ~2.1-kB 3 We performed Northern blots with RNA from human
cerebral cortex, rat forebrain, and cerebellum as well as
from rat kidney cortex, using a riboprobe derived from
clone sp47. While in rat cerebellum no signal was obtained, in each of the other tissues, a single 4.4-kB transcript was detected after overnight exposure (Fig. 3 c),
with this confirming the immunoblotting and immunohistochemical results.
On sequence comparison, synaptopodin shows no significant homology to any published proteins. Due to its high
content of proline (~20%) evenly distributed along the
protein, no formation of any globular domain is possible.
In addition to several potential phosphorylation sites spread
throughout the molecule, synaptopodin contains two
PPXY motifs (corresponding to aa 318-321 and 337-340 of the human protein; Fig. 3 a), which are also found in the
mouse protein (aa 310-313 and 329-332; Fig. 3 b). These
PPXY motifs are involved in protein-protein interactions
between proteins bearing proline-rich peptide stretches
and the WW domain of a variety of proteins (Einbond and
Sudol, 1996 Synaptopodin Expression in Kidney and Brain
To see whether synaptopodin is expressed by other organs
than kidney and brain, we performed immunohistochemical stainings as well as Western blot analyses of a variety
of organs (for details see Materials and Methods). All tissues examined did not show any synaptopodin reactivity
with either technique, i.e., synaptopodin is restricted to
kidney and brain (data not shown).
Distribution of Synaptopodin in the Brain
To determine the distribution of synaptopodin in the
brain, we performed immunofluorescence stainings of sagittal and coronal serial sections of the adult rat brain. Synaptopodin was found in the olfactory bulb, the cerebral
cortex, the striatum, and the hippocampus (Fig. 4). The
immunoreactivity was observed in the dendritic layers,
whereas the perikarya were free of labeling. No other regions of the central nervous system (CNS) showed any expression of synaptopodin as confirmed by additional immunohistochemical mapping of serial coronal sections
from a complete rat brain using the preembedding peroxidase technique (data not shown). Applying the polyclonal
antisera NT-61 and 26-1E, the same pattern of immunoreactivity was observed as with the original mAb G1 (data
not shown). Within the striatum, synaptopodin expression
was restricted to the telencephalic part, i.e., the putamen,
whereas the globus pallidum, which belongs to the diencephalon, was not reactive with anti-synaptopodin antibodies (Fig. 5). To reveal the subcellular localization of
synaptopodin, we performed preembedding PAP labeling
experiments of telencephalic regions that had been found
to express synaptopodin. The electron-dense reaction product was restricted to postsynaptic densites and associated dendritic spines of a subset of synapses (Fig. 6). While the
PSD itself was homogeneously decorated with the DAB
reaction product (Fig. 6, a and b), in the adjacent dendritic
shaft a clustered arrangement of the immunoreactivity was
observed (Fig. 6, c and d). The restriction of synaptopodin
expression to a subset of synapses was confirmed by a double immunofluorescence labeling approach with mAb G1
for synaptopodin and polyclonal anti-synaptophysin antiserum as a marker of all synapses (Fig. 7).
The distribution pattern of synaptopodin was corroborated by in situ hybridization studies showing that synaptopodin mRNA was present in the olfactory bulb, cerebral
cortex, striatum, and hippocampus; expression was only
observed in perikarya but not in dendritic layers (Fig. 8).
Thus, as shown before by immunohistochemistry, within
the CNS, synaptopodin indeed is restricted to the telencephalon.
Expression of Synaptopodin during Postnatal
Maturation of Rat Brain
To examine the maturation-dependent expression of synaptopodin during brain development, we analyzed cytosolic extract from rat forebrains and cerebella at postnatal
days 5, 10, 15, 20, and 50 by Western blotting. Synaptopodin was first expressed around day 15, increased thereafter, and reached the maximum level of expression in the
adult brain (Fig. 9). Like in the adult brain, no expression
of synaptopodin was observed in the cerebellum during postnatal maturation. To prove equal protein loading, blot
membranes were probed with anti-tubulin antibody and
showed comparable signal intensity in all lanes (data not
shown).
Immunofluorescence Microscopy of Cultured
Hippocampal Neurons
We next analyzed the expression of synaptopodin in cultured neurons derived from embryonic 18-d-old rat hippocampi. The expression started at day 12 in vitro, increased thereafter in parallel with process and spine
formation, and reached its maximum expression after 25 d
in vitro (Fig. 10 a). Like in vivo, synaptopodin was only
found in dendrites as revealed in double labeling experiments with anti-MAP2 (Fig. 10 c). The immunofluorescence signal was arranged in a dotted pattern along the
dendrites, with the dots corresponding to synapses as demonstrated in double labeling experiments with anti-synaptophysin (Fig. 10 d).
Immunofluorescence Microscopy of Cultured Podocytes
The association of synaptopodin with actin was further analyzed in a conditionally immortal podocyte cell line recently established in our laboratory (Fig 11; Mundel et al.,
1997
In cultured podocytes, synaptopodin was not found in
undifferentiated cobblestones lacking processes (Fig. 11 c)
but was induced during process formation and showed
strongest labeling in differentiated, arborized cells, where
it was found in a dotted pattern along the actin microfilaments and in focal contacts (Fig. 11 d). The presence in focal contacts was confirmed in double labeling experiments with vinculin (data not shown).
The association with actin (Fig. 11 e) was confirmed in
double labeling experiments with phalloidin (Fig. 11 f).
Depolymerization of F-actin with cytochalasin B resulted
in a redistribution of synaptopodin in a coarse perinuclear
pattern being reversible after washing out of cytochalasin
B (Fig. 11 g). Depolymerization of the microtubular network with colcemid did not significantly affect the distribution pattern of synaptopodin (Fig. 11 h). These results
demonstrate that synaptopodin is intimately associated with actin, corroborating the biochemical findings that
complete solubilization of synaptopodin requires high salt
(500 mM NaCl) buffer.
In the present study we have cloned and characterized
synaptopodin, which constitutes a novel class of polypeptides in renal podocytes and telencephalic dendrites. Based
on its aa composition and tissue distribution, synaptopodin
is different from all other actin-associated proteins described so far. Synaptopodin is a rather basic protein with
a calculated molecular mass of 73.7 kD (human)/74.0 kD
(mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). The difference between calculated mass and the
molecular weight determined by SDS-PAGE and Western
blot as 100 kD may be attributed to either posttranslational
modifications or may be the consequence of the high content of proline leading to a relative mobility shift in the gel.
Synaptopodin extracted from kidney glomeruli shows a
slightly higher Mr of 110 kD than the 100-kD protein from
brain, as revealed by Western blot analysis. The reason for
this difference remains to be established. However, for
several reasons this difference appears to be due to posttranslational modifications, most likely the consequence of
differential phosphorylation (as is the case for VASP [vasodilator-stimulated phosphoprotein], another proline-rich, actin-associated protein; see below). First, by Northern
blot analysis, one single band with an approximate size of
4.4 kb, was found in rat kidney cortex as well as in rat forebrain and human brain cortex, ruling out the generation of
different isoforms by alternative splicing. Second, the internal peptide pattern obtained by tryptic digestion of
both the renal and the neuronal protein, resulted in very
similar HPLC profiles; all peptide sequences determined
from the glomerular protein were identical to corresponding sequences obtained from the brain protein. Third, two
polyclonal antisera directed against two internal peptide
sequences determined from brain synaptopodin recognized the 110-kD protein in kidney and the 100-kD protein in brain. One of these antibodies, 26-1E, was generated against an aa sequence in the central part of the neuronal form that is identical to the corresponding peptide fragment of the renal protein. The other antibody,
NT-61, was generated against an aa sequence deduced
from the NH2-terminal portion of the mouse brain ORF.
In contrast to the brain, in kidney, high concentrations
of proteolytic enzymes are present, and like proline-rich
proteins in general, synaptopodin is very susceptible to
proteolytic degradation. In an extract containing high concentrations of several protease inhibitors (for details see
Materials and Methods) that had been kept for 24 h at 4°C
before boiling with SDS sample buffer, several proteolytic
fragments appeared; one of the major fragments represents the originally described 44-kD protein (Mundel et
al., 1991 Due to its high content of proline evenly distributed
along the entire molecule, synaptopodin appears virtually
as a linear protein without any globular domain structure.
This linear conformation may result in a side to side arrangement along the actin microfilaments similar to that
recently described for dystrophin (Rybakova et al., 1996 Synaptopodin shares some interesting properties with
VASP (Haffner et al., 1995 Synaptopodin also shares some striking similarities with
dendrin, another proline-rich protein whose expression is
restricted to dendrites of the forebrain (Herb et al., 1997 The distribution of synaptopodin at postsynaptic densities and in dendritc spines is exactly the same as that described for actin in these areas (Matus et al., 1982 Recently, a novel protein termed striatin was described
that, within the telencephalon, also has a prominent expression in dendritic spines (Castets et al., 1996 Podocytes of the renal glomerulus are unique cells with
a complex cellular organization, consisting of cell body,
major processes, and foot processes. From the cell body,
major processes arise that directly or after additional
branching split into foot processes that interdigitate with
the foot processes of neighboring podocytes, leaving in between the filtration slits covered by the slit diaphragm. Like the PSD, the sole plate of podocyte foot processes
contains an electron-dense matrix of largely elusive composition (for review see Mundel and Kriz, 1995 Because of the restricted distribution of synaptopodin,
the question arises: which properties are shared by podocyte
foot processes and dendritic spines? Podocyte foot processes are able to retract (in extreme forms the result is
"foot process effacement") and to spread out again (Shirato
et al., 1996) including remodeling
of synaptic contacts. While substantial knowledge about
dynamics of axonal filopodia and growth cones in enabling
the axon to find its approriate target has accumulated (for
review see Bentley and O'Connor, 1994
), much less is
known about the dynamics of postsynaptic dendritic structures during synaptogenesis and synaptic remodeling. However, recent studies with developing hippocampal slices
(Dailey and Smith, 1996
) and cultured hippocampal neurons (Papa et al., 1995
; Ziv and Smith, 1996
) provided evidence for a role of dendritic filopodia in synaptogenesis and spine formation. As in axonal filopodia, the dynamics
of dendritic filopodia and spines appear to be mediated, at
least in part, by the actin-based cytoskeleton of the dendrites.
; Garner and Kindler, 1996
). However, advances in
protein microsequencing and yeast two hybrid system
have led to the identification of proteins interacting with
the cytoskeleton of the postsynaptic segment. Recently,
several members of a novel family of synapse-associated
proteins were identified that share the so-called PDZ domain in their NH2-terminal region, exhibit a guanylate kinase
activity, and are homologous to the tight junction-associated protein ZO-1 (for review see Garner and Kindler,
1996
; Sheng 1996
). These proteins are involved in mediating the clustering of postsynaptic receptors, e.g., of the
NMDA receptor as shown by a yeast two hybrid approach
(Kornau et al., 1995
). On the other hand it has been
found that NCAM-180, ankyrin, and a membrane-bound
ankyrin-binding protein are involved in linking spectrin
and
adducin to the microfilament system of the postsynaptic segment (summarized in a model by Garner and Kindler, 1996
). One possible candidate for providing the missing link between the receptor(s) and the actin filament
system (the activity of the NMDA receptor is dependent
on the integrity of actin [Rosenmund and Westbrook,
1993
]) appears to be
actinin-2, which was shown to bind
the NMDA receptor in competition with calmodulin
(Wyszynski et al., 1997
).
actinin,
talin, and vinculin (Drenckhahn and Franke, 1988
). The linkage of this system to the glomerular basement membrane at focal contacts is mediated by an
3
1 integrin
complex (Adler, 1992
). Like the PSD, the sole plate of
podocyte foot processes contains an amorphous density,
which in its molecular composition is largely unknown (for
review see Mundel and Kriz, 1995
). Podocytes are known
to respond to a variety of vasoactive substances with changes
of their cytoskeleton (Mundel and Kriz, 1995
) and are able
to perform slow and directed movements of their foot processes (Shirato et al., 1996
).
). We now report on the molecular characterization of this protein,
which we termed synaptopodin, since, in addition to renal
podocytes, it is expressed in the brain, where it associates with the actin cytoskeleton of PSD and dendritic spines in
a subset of telencephalic synapses. We discuss the potential role of synatopodin in modulating the actin-based motility of podocyte foot processes and telencephalic dendritic spines.
MATERIALS AND METHODS
160°C. In addition to kidney and
brain the following organs were analyzed: liver, heart, lung, skeletal muscle, adrenal gland, testis, salivary glands, stomach, pancreas, spleen, small
intestine, and colon.
).
Briefly, after chemical and mechanical dissociation of the hippocampi,
cells were plated onto polylysine-coated glass cover slips (15-mm diam).
These neurons survive and undergo full polarization when cultured in serum-free medium in the presence of a supporting layer of astrocytes (Goslin and Banker, 1991
).
) harboring a transgene
for a thermosensible variant of the SV-40-T-antigen, which is under the
control of the H-2Kb-promotor, whose activity can be increased by
interferon. The cellular origin of the cloned cell lines from the podocyte lineage was proven by the detection of the Wilm's tumor gene product (WT-1), which in the adult kidney is an exclusive marker of podocytes (Mundlos et
al., 1993
). Cells were propagated at 33°C in RPMI 1640 containing 10%
FCS (Boehringer Mannheim, Mannheim, Germany), 100 U/ml penicillin
(GIBCO BRL, Karlsruhe, Germany), 100 µg/ml streptomycin (GIBCO
BRL), and 10 U/ml recombinant mouse
interferon (Sigma Chemical Co.,
Munich, Germany) to induce synthesis of the immortalizing T antigen.
Subcultivation was done with trypsin at 37°C after cells had reached confluence. Cells were passaged after 1:5 dilution. To initiate differentiation,
cells were thermoshifted to 37°C and maintained in medium without
interferon (Mundel et al., 1997
).
). Polyclonal antisera were obtained by immunization of
rabbits with keyhole limpet hemocyanin-conjugated peptides. The antisera were affinity purified with the corresponding peptides linked to Ultralink (Pierce, Sankt Augustin, Germany) according to the manufacturer's instructions. In the experiments described in this manuscript, antiserum 26-1E directed against the sequence of amino acid (aa) 441-455
corresponding to rat brain synaptopodin peptide fragment No. 26 (which
is identical to rat glomerular synaptopodin peptide fragment No. 25; single
letter code SPGAAAEETVPEWASC) and antiserum NT-61 directed
against aa 181-195 of mouse brain synaptopodin corresponding to aa 188-
202 of human brain synaptopodin (single letter code MSSLLIDMQPSTLV) were routinely used.
tubulin (Sigma Chemical Co.), rabbit anti-WT-1 (C19; Santa Cruz Biotechnology, Heidelberg, Germany), monoclonal anti-vinculin (Sigma
Chemical Co.), and rhodamine-conjugated phalloidin (Sigma Chemical
Co.) to detect F-actin.
20°C for 5 min or with 2% paraformaldehyde and 4% sucrose in PBS for
5 min followed by permeabilization with 0.3% Triton X-100 in PBS for 5 min at room temperature. After rinsing with PBS, unspecific binding sites
were blocked with 2% FCS, 2% BSA, and 0.2% fish gelatine in PBS for at
least 30 min. Primary antibodies (prediluted in blocking solution) were
applied for 60 min at room temperature. Antigen-antibody complexes
were visualized using fluorochrome (Cy2 or Cy3)-conjugated secondary
antibodies (Biotrend, Cologne, Germany). Sections were washed with
PBS, rinsed with H2O, and mounted in 15% Mowiol (Calbiochem, Bad
Soden, Germany) and 50% glycerol in PBS. After overnight drying, specimens were analyzed and documented with a photomicroscope (Polyvar 2;
Leica, Bensheim, Germany). Simultaneous confocal double fluorescence microscopy was done with a laser scanning microscope (410 UV; Carl
Zeiss, Oberkochen, Germany) with appropriate filters. Micrographs were
taken with an imagecoder (Focus Graphics, Foster City, CA) or printed
with a sublimation video printer.
-diaminobenzidine as chromogen. The sections were stained en bloc with uranyl
acetate in maleate buffer, osmicated in 1% OsO4 for 1 h, dehydrated in
graded series of acetone, and flat embedded in epon between teflon-coated slide and coverglass. Areas of interest were selected under a light microscope, excised with a razor blade, and mounted onto preformed epon blocks. Thin sections were cut with an ultramicrotome E (Leica) and
examined under a Philips EM 301.
). Samples were stored at
80°C until used.
20°C or further processed to obtain heat-stable protein fractions (Herzog and Weber, 1978
). To this end, the concentration of NaCl was adjusted to a final concentration of 800 mM, and 5% 2-mercaptoethanol was
added. The samples were boiled for 5 min and insoluble material was pelleted at 50,000 g for 30 min at 4°C. The resulting heat-stable supernatants (HS-Sup) were stored at
20°C until used.
).
After transfer to Immobilon P membranes, protein spots were excised
from Coomassie brilliant blue-stained membranes. Tryptic digestions of
excised spots, HPLC separations of resulting internal peptide fragments,
and peptide microsequencing were done as described (Heid et al., 1994
),
except that Edman degradation was carried out on a protein sequencing
apparatus (Procise 494; Applied Biosystems).
to 3
, with clone sp17 containing a
"Kozak sequence" (Kozak, 1989
) before the start codon and clone sp91
ending with a polyA-tail (from a human brain cDNA library that covered
the open reading frame [ORF] of human synaptopodin, except for a gap
of 250 bases between clone sp47 and sp91). This gap was filled after reverse transcription of human hippocampal RNA into cDNA by PCR cloning using specific primers deduced from the sequences of clones sp47 and
sp91, respectively. The region spanning the ORF was sequenced in both
directions, using internal oligonucleotide primers.
TriplEx; Clontech, Heidelberg, Germany) was screened under low stringency conditions using clones sp17
and sp47 as probes. Among several shorter clones, one clone (termed 7A-1)
was obtained, which covers the region between bp 490 and the poly A tail
of mouse brain synaptopodin cDNA. The first 489 bp of the ORF were
cloned by nested RT-PCR from random-primed mouse brain cDNA using
two specific 3
primers derived from the 5
region of clone 7A-1 and one
degenerate primer corresponding to bp 1-25 of the human synaptopodin
ORF. With this approach, the complete ORF of mouse brain synaptopodin was established.
), with 32P-labeled antisense riboprobes using
clone sp47 as a template. Equal RNA loading was confimed by hybridization with a GAPDH-riboprobe.
using 35S-labeled antisense oligonucleotides derived from the region between bp 600 and 800 of mouse brain synaptopodin cDNA. Exposure time to Kodak XAR-5 film was 24 d.
RESULTS
) represents a proteolytic fragment of the 110-kD
protein (Fig. 1). On two-dimensional gel electrophoresis, synaptopodin appeared as a very basic protein with an isoelectric point around 9.4 (Fig. 2).
Fig. 1.
Western blot analysis of synaptopodin with
mAb G1. Lanes 1-4 show the
immunodetection; lanes 1-4
show staining of the identical membrane with Coomassie
brilliant blue to demonstrate
protein loading and separation. Cytosolic extracts from
rat forebrain (lane 1), cerebellum (lane 2), and isolated glomeruli (lanes 3 and 4) were analyzed in parallel. While in the forebrain a protein of ~100 kD is
present, no protein expression is seen in cerebellum. In kidney a
110-kD band is observed. The extract in lane 4 had been kept at
4°C for 24 h before separation. Several proteolytic fragments appear; the band indicated by an arrowhead represents the originally described 44-kD protein.
[View Larger Version of this Image (64K GIF file)]
Fig. 2.
Two-dimensional analysis of rat brain synaptopodin.
(a) Coomassie brilliant blue-stained two-dimensional Western
blot, using NEPHGE in the first dimension and SDS-PAGE in
the second, of a fraction enriched for synaptopodin (arrow) obtained after reversed-phase HPLC chromatography of a heat-
stable supernatant from rat forebrain cytosolic extracts. (b) ECL
detection of synaptopodin using mAb G1.
[View Larger Version of this Image (41K GIF file)]
region of mouse brain synaptopodin identifies the same 110/
100-kD bands in kidney/brain cytosolic extracts (data not
shown).
-end of clone sp47. To
prove the identity of the renal and the brain protein, six peptide fragments of the purified glomerular protein were
subjected to Edmann degradation. All of these peptide
fragments revealed identical aa composition as the correponding peptide fragments of the brain protein (Fig. 3).
Fig. 3.
(a) Nucleotide sequence (upper lines and numbers) and
deduced amino acid sequence (one letter code, lower lines, and
numbers) of human brain synaptopodin cDNA. Taking the first
possible start codon of the open reading frame, synaptopodin
consists of 685 aa. aa sequences determined by Edman degradation of proteolytically derived internal peptides of rat brain synaptopodin are underlined. (b) Comparison of the aa sequences
between mouse (upper lines) and human (lower lines) synaptopodin. At the protein level an 84% identity between the two species
is observed. aa sequences determined from internal peptides of
rat kidney synaptopodin are underlined. The sequence data of
human synaptopodin are available from EMBL/GenBank/DDBJ
under accession number Y11072. (c) Northern blot analysis of
synaptopodin. A single 4.4-kbp mRNA is detected in human
brain cortex (1), in rat kidney cortex (2), and rat forebrain (3). No
signal is obtained in rat cerebellum (4).
[View Larger Version of this Image (90K GIF file)]
-untranslated sequence and ended with a consensus site for polyadenylation (data not shown).
The deduced protein sequence codes for a 685-aa polypeptide with a calculated Mr of 73.7 kD and an isoelectric
point of 9.38 (Fig. 3 a). Molecular cloning of the mouse homologue of synaptopodin revealed an ORF of 2,071 bp
and additional 1,500-bp 3
-untranslated region. The ORF
codes for a 690-aa protein with a calculated Mr of 74.0 kD
and an isoelectric point of 9.27 (Fig. 3 b). At the protein
level an identity of 84% was found between human and
mouse.
).
Fig. 4.
Overview of synaptopodin staining in rat forebrain. The distribution of
synaptopodin in adult rat
brain was analyzed by indirect immunofluorescence labeling of 8-µm-thick frozen
sections from perfusion-fixed
tissue. This frontal section
through the forebrain reveals
expression in the cerebral
cortex (C), striatum (S), and hippocampus (H). The protein was also found in the olfactory bulb (not shown). No
other areas of the central
nervous system show any
synaptopodin expression.
[View Larger Version of this Image (140K GIF file)]
Fig. 5.
Immunofluorescence microcopy of synaptopodin in striatum. This micrograph shows the transition between the telencephalic
putamen (P) and the diencephalic globus pallidum (G). (a) Synaptopodin staining is restricted to the putamen and ends at the border to the diencephalon (arrows). (b) Staining with anti-synaptophysin to detect all synapses. Bar, 1.5 µm.
[View Larger Version of this Image (132K GIF file)]
Fig. 6.
Immunoelectron microscopic analysis of synaptopodin.
The subcellular localization of synaptopodin in the telencephalon
was analyzed by preembeding peroxidase labeling. (a) The electron-dense reaction product localizes to the postsynaptic densities and dendritic spines of distinct synapses. (b) At a higher magnification the strongest labeling is found at the PSD (arrows). (c
and d) Extension of synaptopodin expression into dendritic shaft.
This distribution of synaptopodin in the postsynaptic segment of
dendrites corresponds exactly to the distribution of actin at this
site. Bars, 0.2 µm.
[View Larger Version of this Image (83K GIF file)]
Fig. 7.
Confocal laser scanning double fluorescence analysis of
synaptopodin and synaptophysin in rat brain striatum. In contrast to synaptophysin (green), which is expressed in all synapses, synaptopodin (red) is only expressed by a subpopulation of synapses. It becomes evident that axosomatic synapses (arrows) are virtually free of synaptopodin immunoreactivity. Bar, 10 µm.
[View Larger Version of this Image (52K GIF file)]
Fig. 8.
Regional distribution of synaptopodin mRNA in mouse
brain. The signal is found in the olfactory bulb (O), cortex cerebri
(C), striatum (S), and hippocampus (H). This expression pattern
matches the immunohistochemical distribution of the protein.
[View Larger Version of this Image (88K GIF file)]
Fig. 9.
Maturation-dependent expression of synaptopodin during postnatal development of rat brain. (a)
Cytosolic extracts from cerebellum and forebrain harvested at day 5, 10, 15, and 20 postnatal and from adult rats
were analyzed by Western
blotting. In the cerebellum, synaptopodin was never expressed. In the forebrain,
synaptopodin first appeared
around day 15, increased
thereafter, and reached the
maximum level of expression
in the adult animal. (b) The
identical membrane was probed with anti-tubulin to prove equal
protein loading in all lanes.
[View Larger Version of this Image (37K GIF file)]
Fig. 10.
Expression of synaptopodin in cultured hippocampal neurons. (a) In 25-d-old cultured hippocampal neurons, a dotted pattern of immunoreactivity in dendrites is seen, whereas axons (arrows) are not labeled. (b) Phase contrast microscopy. (c) Confocal laser scanning double fluorescence analysis of synaptopodin (red) and MAP2 (green) to demonstrate the exclusive expression of synaptopodin in dendrites. (d) Confocal laser scanning double fluorescence analysis of synaptopodin and synaptophysin. The yellow signal results
from a complete overlap of the immunoreactivity and proves the synaptic localization of synaptopodin in cultured neurons. Bar, 10 µm.
[View Larger Version of this Image (88K GIF file)]
). This cell line can be maintained in two different
phenotypes, as undifferentiated cobblestones growing as
an epithelial monolayer (Fig. 11 a) and as differentiated, arborized cells equipped with processes (Fig. 11 b); these arborized podocytes always arise by conversion from cobblestones.
Fig. 11.
Expression of synaptopodin
in a cultured mouse podocyte cell line.
(a) Phase contrast morphology of undifferentiated podocytes. The cobblestone
morphology of undifferentiated podocytes
growing under permissive conditions is
shown. The cells form a monolayer as
they reach confluence. (b) Arborized
podocytes maintained under nonpermissive conditions are very large and flat.
Development of branched processes is
obvious. (c) Induction of synaptopodin after 4 d at nonpermissive temperature.
While all cells express the podocyte-specific transcription factor WT-1 (green),
cobblestones do not express synaptopodin (red). Currently differentiating cells show an induction of synaptopodin (arrows). In the center, a differentiated, binucleated, arborized cell is encountered
that expresses high levels of synaptopodin. (d) Expression of synaptopodin in a
differentiated, arborized cell with well
developed processes after 14 d at 37°C. Synaptopodin is found along the actin
filaments and in focal contacts (arrows).
The association of synaptopodin with actin was confirmed in double labeling experiments (e and f) with rhodamin-conjugated phalloidin. (e) A linear staining
of actin filaments with phalloidin is observed. f shows the punctated distribution of synaptopodin along the same actin
filaments as in e (arrows). (g) Depolymerization of actin filaments with cytochalasin B abolished the linear staining pattern, and synaptopodin clustered in
the perinuclear cytoplasm. (h) Depolymerization of microtubules with colcemid did not significantly affect the staining
pattern of synaptopodin. Bars, 5 µm.
[View Larger Version of this Image (60K GIF file)]
DISCUSSION
). However, this degradation can effectively be
prevented by boiling with SDS sample buffer immediately
after protein extraction.
).
The association of synaptopodin with actin was confirmed in cultured podocytes where the protein colocalized with
the microfilaments in a punctate pattern and was also detected in focal contacts. Moreover, after treatment with
the actin-depolymerizing drug cytochalasin B, this pattern
was abolished. The association with the microfilaments appears to be rather tight, since complete solubilization of
synaptopodin can only be achieved by extraction with high
salt buffer.
), another proline-rich, actin-associated protein, originally identified due to its phosphorylation upon stimulation of human platelets with cAMP- and
cGMP-elevating substances. Like platelets, podocytes also
respond to vasodilating substances with alterations of their
actin cytoskeleton (for review see Mundel and Kriz, 1995
).
Like synaptopodin, VASP appears as an elongated, rather
linear protein (Haffner et al., 1995
). Similar to synaptopodin, which in Western blots shows two bands of 100 and
110 kD, VASP occurs in Western blots as two bands of 46 and 50 kD, with the difference being due to differential phosphorylation (Reinhard et al., 1992
). The location at focal
adhesions of VASP places it at a site where multiple signals are integrated. This may also be true for synaptopodin, which is found in focal contacts of podocytes. The interactions of VASP with the microfilament system is mediated
by profilin (Reinhard et al., 1995
). It will be interesting to
see whether synaptopodin is a ligand for profilin as well.
).
While dendrin contains three PPXY motifs, synaptopodin
contains two of these PPXY motifs. These motifs are found
in several proline-rich proteins and are involved in protein-protein interactions between proline-rich stretches of
different proteins and the WW domain (Einbond and Sudol, 1996
) as well as the Abl SH3 domain (Bedford et al.,
1997
) of a variety of proteins. Among others, the WW domain is present in the postsynaptic cytoskeletal proteins
dystrophin and utrophin (Bork and Sudol, 1994
). Thus, it
appears possible that synaptopodin is involved in mediating interactions between actin and associated proteins of
the dendritic cytoskeleton.
). Thus,
at synapses, as in renal podocytes, synaptopodin colocalizes with actin. One of the most exciting features of synaptopodin is that within the CNS, its expression is restricted
to exclusively telencephalic PSD and associated dendritic
spines of the olfactory bulb, cerebral cortex, striatum, and
hippocampus. In situ hybridization studies, in addition to
confirming the exclusive telencephalic expression of synaptopodin, revealed that in contrast to other dendritic proteins like MAP2 (Garner et al., 1988
), synaptopodin
mRNA is not found in dendrites but in the perikarya only,
which may indicidate that synaptopodin synthesis is not
controlled locally in the dendritic cytoplasm. As the protein contains potential sites for phosphorylation by protein
kinase C, the activity of synaptopodin may be regulated by
protein kinase C, which is highly expressed in postsynaptic densities (Cheng et al., 1994
) and glomerular podocytes
(Cybulsky et al., 1990
).
). While
both synaptopodin and striatin show similar staining in
hippocampus and striatum, in the cerebral cortex, striatin
is restricted to the motorcortex, whereas synaptopodin is
present in all areas of the cortex. In contrast to synaptopodin, striatin is also found in the cerebellum and the spinal
cord; at the subcellular level, synaptopodin is only found
in dendrites, whereas striatin is also found in the cell bodies of telencephalic neurons. Since both proteins are only
expressed by a subset of dendrites within the hippocampus and striatum, it will be interesting to see whether they define the same synapses in these regions of the brain.
). The foot
processes are equipped with a microfilament-based contractile apparatus composed of actin, myosin II,
actinin,
talin, and vinculin, which is linked to the glomerular basement membrane at focal contacts by an
3
1 complex. This
contractile apparatus can respond to vasoactive substances
with alteration of its actin cytoskeleton and may provide the basis of foot processes motility. In a previous paper,
we demonstrated that synaptopodin was not expressed by
podocyte precursor cells during nephrogenesis but was
first seen as podocytes started to differentiate and develop
their typical process architecture (Mundel et al., 1991
). At
the ultrastructural level, synaptopodin was found to be associated with the actin microfilaments of podocyte foot
processes. This association is also seen in vitro, when
podocytes transform from simple cobblestone cells into an
arborized phenotype equipped with processes similar to
those seen in vivo. The late appearance of synaptopodin
during postnatal brain development and during differentiation of cultured hippocampal neurons appears to correlate with the maturation of synaptic formations on dendritic spines (Papa et al., 1995
; Ziv and Smith, 1996
). Thus,
at both sites the appearance of synaptopodin appears to be
correlated with the formation of cell "processes," which
are essential for the specific function of each cell type.
), i.e., an individual foot process may decrease
or increase in length. Dendritic spine formation and remodelling are crucially involved in what is known as plasticity of the receptor apparatus in the telencephalon (Wallace et al., 1991
). Recent work with cultured hippocampal
slices (Dailey and Smith, 1996
) as well as studies with cultured hippocampal neurons (Papa et al., 1995
; Ziv and
Smith, 1996
) revealed that dendritic filopodia and their
protrusive motility may play important roles in initiation
and elimination of synaptic contacts, both during development and during postdevelopmental synaptic remodelling processes, such as those representing the morphological basis of long-term memory. Dendritic filopodia seem not only
to protrude towards axons but also appear to be able to retract from their axonal counterparts (Ziv and Smith, 1996
).
Thus, at both sites, in podocyte foot processes as well as in
dendritic filopodia and spines, this motility appears to be
part of the formation and retraction of cell processes,
which may be achieved via the microtubular and actin cytoskeletons (Quinlan and Halpain, 1996
). One may speculate that synaptopodin may play a similar function associated
with the remodeling of cell processes in the kidney and the
CNS. At both sites, these movements and their regulation
are poorly understood. Synaptopodin may be a key protein
allowing future avenues for the study of these functions.
Received for publication 7 March 1997 and in revised form 10 July 1997.
Address all correspondence to Dr. Peter Mundel, Department of Anatomy and Cell Biology, University of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany. Tel.: (49) 6221-548687. Fax: (49) 6221-544951. e-mail: peter.mundel{at}urz.uni-heidelberg.deThis work was supported by a grant from the Deutsche Forschungsgemeinschaft (Kr 546/9-2).
aa, amino acid; CNS, central nervous system; MAP, microtubule-associated protein; ORF, open reading frame; PSD, postsynaptic densities.
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