* Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany; Institut für Biochemie I, Medizinische Fakultät, Universität
zu Köln, 50931 Köln, Germany; and § Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030
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Abstract |
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In a search for novel members of the -actinin superfamily, a Dictyostelium discoideum genomic library in yeast artificial chromosomes (YAC) was
screened under low stringency conditions using the acting-binding domain of the gelation factor as probe. A
new locus was identified and 8.6 kb of genomic DNA
were sequenced that encompassed the whole abpD
gene. The DNA sequence predicts a protein, interaptin,
with a calculated molecular mass of 204,300 D that is
constituted by an actin-binding domain, a central
coiled-coil rod domain and a membrane-associated domain. In Northern blot analyses a cAMP-stimulated
transcript of 5.8 kb is expressed at the stage when cell
differentiation occurs. Monoclonal antibodies raised
against bacterially expressed interaptin polypeptides
recognized a 200-kD developmentally and cAMP-regulated protein and a 160-kD constitutively expressed
protein in Western blots. In multicellular structures, interaptin appears to be enriched in anterior-like cells
which sort to the upper and lower cups during culmination. The protein is located at the nuclear envelope and
ER. In mutants deficient in interaptin development is
delayed, but the morphology of the mature fruiting
bodies appears normal. When starved in suspension
abpD
cells form EDTA-stable aggregates, which, in
contrast to wild type, dissociate. Based on its domains
and location, interaptin constitutes a potential link between intracellular membrane compartments and the
actin cytoskeleton.
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Introduction |
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THE cytoskeletal proteins that share the actin-binding domain (ABD)1 of -actinin constitute an expanding superfamily. In Dictyostelium discoideum,
this superfamily comprises
-actinin (Noegel et al., 1987
),
gelation factor (ABP-120; Noegel et al., 1989
), cortexillins I and II (Faix et al., 1996
), fimbrin (Prassler et al., 1997
), a
filamin-like protein (Hock and Condeelis, 1987
), and a
spectrin-like protein (Bennett and Condeelis, 1988
). Additional members have been described in mammalian cells,
like dystrophin (Hammonds, 1987
), plectin (Liu et al.,
1996
), and dystonin (Brown et al., 1995
). All these proteins are characterized by a modular organization (Matsudaira, 1991
), and share a conserved 250-amino acid F-actin- binding domain usually located at the NH2-terminal end.
The three-dimensional structure of an ABD has been resolved recently, and was shown to consist of two
-helical
subdomains connected by a long central
-helix (Goldsmith et al., 1997
). Members of this superfamily differ in
their rod and regulatory domains, which are responsible for oligomerization, calcium regulation, membrane association, or interaction with other proteins. For example, fimbrin,
-actinin, spectrin, and dystrophin possess calmodulin-like calcium binding domains that allow for calcium
regulation of their actin-binding activity. Thus, modular
organization allows for an ample degree of local and functional specialization of each member of the
-actinin superfamily.
Members of the -actinin superfamily are also involved
in the pathogenesis of disease. Spectrin is required for the
stability of many membrane skeletons and defects in
-spectrin result in hemolytic anemia (Gallagher and Forget, 1993
). Dystrophin associates with a membrane glycoprotein complex through its COOH-terminal domain, thus
anchoring the subsarcolemmal actin cytoskeleton to the
extracellular matrix in skeletal muscle (Campbell, 1995
). Mutations in the gene that encodes dystrophin are responsible for the Duchenne and Becker muscular dystrophies,
characterized by skeletal muscle degeneration (Hoffman
et al., 1987
). Plectin and dystonin possess a COOH-terminal domain responsible for binding to intermediate filaments. These two proteins localize to hemidesmosomes and play an important role connecting actin microfilaments to intermediate filaments. In humans, mutations in
the plectin gene are responsible for epidermolysis bullosa
with muscular dystrophy. In mice, mutations in the dystonin gene lead to a neurodegenerative disease accompanied
by epidermolysis bullosa (reviewed in Fuchs and Cleveland, 1998).
Dictyostelium has emerged as a useful model for studying the actin cytoskeleton. Dictyostelium amebae possess a
cytoskeleton comparable in its complexity to that of polymorphonuclear leukocytes (Noegel et al., 1997). Furthermore, the ease with which a variety of genetic approaches
can be used to generate mutants has made possible the
creation of cells with mutations in one or two actin cross-linking proteins, with the result that in most cases single mutants are either normal or display only moderate defects, whereas more profound defects are apparent in double mutants lacking certain combinations of actin cross-linkers (Witke et al., 1992
; Faix et al., 1996
; Rivero et al.,
1996a
,b). In the mouse, an analogous example has been recently reported. Mice lacking either dystrophin or utrophin (the autosomal homologue of dystrophin), display
only mild phenotypes, whereas double knockout mutants
show severe progressive muscular dystrophy (Grady et al.,
1997
). This has led to the view that the actin cross-linkers
constitute a functional network in which distinct components preferentially perform distinct functions and additionally overlap with each other in a cooperative manner (Rivero et al., 1996
b).
To have a definitive picture of the events that take place
during rearrangement of the actin cytoskeleton we must
identify all the components involved. Therefore, we
searched for novel members of the -actinin superfamily
in Dictyostelium. To this end, a genomic library in yeast artificial chromosomes (YAC; Kuspa et al., 1992
) was
screened under low stringency conditions using a probe
that encodes the ABD of the gelation factor. The new locus identified, abpD, encodes a protein of 204.3 kD constituted by an NH2-terminal ABD, a central coiled-coil rod
domain, and a COOH-terminal membrane-associated domain (MAD). Accumulation of abpD mRNA is developmentally regulated and stimulated by cAMP, which is unusual for a cytoskeletal protein gene. Immunofluorescence
and biochemical studies indicate an association with membranes of intracellular compartments like the nuclear envelope and the ER. Homologous recombination was used
to inactivate the abpD gene. When starved in suspension
abpD
cells are able to build multicellular aggregates,
which later dissociate into single cells probably due to a
defect in the processing and/or secretion of late adhesion
molecules. We have named this protein interaptin (Latin
interaptus, bound to each other), since it may constitute a
link between intracellular membrane compartments and
the actin cytoskeleton, and may thus be involved in the
trafficking of secretory vesicles among different intracellular compartments.
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Materials and Methods |
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Cloning of abpD
A cDNA fragment of the gelation factor that corresponds to the ABD
(amino acids 1-221) was used to probe an ordered YAC library under
low-stringency hybridization conditions as described previously (Titus et al.,
1994). In brief, the DNA fragment was 32P-labeled by randomly primed
DNA synthesis and hybridized to Southern blots of a set of 1,016 YAC
clones representing >99% of the Dictyostelium genome (Kuspa et al.,
1992
). The hybridization was carried out for 36 h at 37°C in 10 mM Tris-HCl, pH 8.0, 1 M NaCl, 5× Denhardt's solution, 0.1 mg/ml sheared
salmon sperm DNA, 0.5% SDS, 30% formamide, and 106 cpm/ml labeled
DNA. The blots were washed twice for 30 min each in 6× SSC and 0.5%
SDS at 58°C and exposed to film for 4 d. After the assignment of hybridization signals to specific YAC clones, overlapping clusters of clones were
assigned to specific chromosomal loci including the previously identified
abpA (
-actinin) and abpC (gelation factor) loci, as well as two new loci
(abpD and abpE) to which actin-binding protein genes had not been previously mapped. A YAC clone from locus abpD was purified from the endogenous yeast chromosomes by pulsed field gel electrophoresis (Kuspa
and Loomis, 1996a
) and used as a PCR template using degenerated
primers abs-1 (5'-CAAMAAAAWACTTTYACTMRWTGG-3') and
abs-2 (5'-TAAAATWAWAKTCCAAATTAAACC-3') corresponding
to highly conserved regions of the ABD of proteins of the
-actinin superfamily (see Fig. 3 A). A 300-bp PCR fragment was obtained that identified
a single locus in the middle of chromosome 4 (Kuspa and Loomis, 1996b
)
when hybridized to the same YAC set under standard high stringency
conditions (Kuspa et al., 1992
). Fig. 1 summarizes the strategy of cloning
of the abpD gene. The 300-bp product was used as a probe to screen a genomic DNA library containing EcoRI fragments. A 2.9-kb fragment (G1)
was isolated and used as starting point for isolation of four additional
clones (G2 to G5) that encompassed the whole abpD sequence by genomic walking in the 3' direction. In addition, screening of a
ZAP cDNA
library allowed the isolation of clone C5.
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The DNA clones were sequenced with gene specific primers using an automated sequencer (ABI 377 PRISM, Perkin Elmer, Norwalk, CO). The Wisconsin Package Version 9.0 of the Genetics Computer Group (University of Wisconsin, Madison, WI) was used for sequence analysis.
Protein Expression and Generation of Monoclonal Antibodies
A DNA fragment encoding Met-1 to Ser-137, corresponding to part of the
ABD of interaptin, was obtained by PCR using primers designed to introduce a NdeI site at the 5' end and a BamHI site at the 3' end. The amplified fragment was cloned into expression vector pT7-7 (Tabor and Richardson, 1992), and the recombinant protein expressed in Escherichia coli
BL21. After lysis of the cells the recombinant protein remained in the pellet. The pellet was sequentially extracted with 2 M and 4 M urea in TEDA
buffer (10 mM Tris-HCl, pH 7.8, 1 mM EGTA, 1 mM DTT, and 0.02%
NaN3). After the 4 M urea extraction the pellet was washed with PBS and used for immunization of mice.
A DNA fragment encoding Asn-954 to Gln-1158 of the rod domain, which includes one of the repetitive stretches underlined in Fig. 2, was obtained by PCR using primers designed to introduce a NdeI site and a start codon at the 5' end and a BamHI site at the 3' end. The amplified fragment was cloned into expression vector pET15b (Novagen, Madison, WI), and the recombinant His-tagged protein expressed in E. coli BL21. The product was purified from the soluble fraction of bacterial extracts on Ni2+-NTA agarose (QIAGEN GmbH, Hilden, Germany) and used to immunize mice.
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For mAb production BALB/c mice were immunized as described
(Schleicher et al., 1984). Spleen cells were fused with PAIB3AG8I myeloma cells two days after the last boost. Hybridomas were screened for
their ability to recognize the antigen on Western blots. mAb 234-151-9
recognized the ABD of interaptin, and mAb 260-60-10 recognized an
epitope in the rod domain.
Strains, Growth Conditions, Development, and cAMP Stimulation of D. discoideum
Cells of D. discoideum strain AX2-214 (referred to as wild-type), an axenically growing derivative of wild strain NC4, and transformants were
grown either in liquid nutrient medium at 21°C with shaking at 160 rpm
(Claviez et al., 1982), or on SM agar plates with Klebsiella aerogenes (Williams and Newell, 1976
). For development cells were grown to a density of
2 to 3 × 106 cells/ml, washed in 17 mM Soerensen phosphate buffer, pH
6.0, and 0.8 × 108 cells were deposited on nitrocellulose filters (Millipore
type HA; Millipore, Molsheim, France) and allowed to develop at 21°C as
described (Newell et al., 1969
). For development in shaking suspension
cells were washed as above, resuspended at 1 × 107 cells/ml in Soerensen
phosphate buffer and shaken at 160 rpm at 21°C.
The effect of cAMP on abpD expression was analyzed either in suspension or on aggregated cells. For analysis in suspension, cells were starved
under shaking and stimulated with 2 × 108 M cAMP pulses using a syringe attached to a perfusion pump. Cell samples were collected at regular
intervals for RNA extraction and Western blot analysis. For analysis of
aggregates, cells were allowed to develop on agar to the stage of tight
mounds. Cells were then harvested, disaggregated by five passages
through a 23-G needle and resuspended in Soerensen phosphate buffer at
a density of 2 × 107 cells/ml. After 2 h incubation with shaking in the presence of 5 mM cAMP cells were collected for RNA extraction and Western blot analysis.
Construction of lacZ Reporter Vectors and
-Galactosidase Staining
Two promoter::lacZ fusions were constructed using pDdGal (Harwood
and Drury, 1990) as expression vector (see Fig. 1). A 2-kb fragment was
excised from genomic clone G1 by digestion with EcoRI and MunI. This
fragment (L1) contained 1.8-kb of 5' flanking sequences and 225 bp of
coding sequence. Additionally, a 1.8-kb PCR fragment (L2) was obtained
using clone G1 as template, and primers designed to introduce a XbaI site
at the 5' end and a EcoRI site at the 3' end. This fragment contained 800 bp of 5' flanking sequence and 1 kb of sequence downstream of the ATG
codon, and included the first intron. Both fragments were cloned in frame
with the lacZ gene of pDdGal and the resulting vectors were introduced into AX2 cells by electroporation (Mann et al., 1994
). After selection for
growth in the presence of G418 (Sigma Chemical Co., St. Louis, MO),
transformants were confirmed by
-galactosidase staining. Cells were allowed to develop on nitrocellulose filters placed on 2% phosphate agar.
At appropriate times filters were stained for
-galactosidase activity according to the method of Dingermann et al. (1989)
.
Construction of a Vector Allowing Expression of a GFP Fusion Protein
A vector was constructed that allowed expression of green fluorescent
protein (GFP) fused to a COOH-terminal fragment of interaptin in Dictyostelium cells under the control of the actin-15 promoter and the actin-8
terminator. The cDNA fragment C5 (see Fig. 1) was cloned in frame at its
5' end to the coding region of the red shifted S65T mutant of Aequoria
victoria GFP in the transformation vector pDEX-GFP (Westphal et al.,
1997). The continuous reading frame was composed of GFP, the heptapeptide linker KLEFGTR resulting from the cloning strategy, and an
interaptin fragment from Glu-1441 to the end. The resulting vector was
introduced into AX2 cells by electroporation. After selection for growth
in the presence of G418 (Sigma Chemical Co.), GFP-expressing transformants were confirmed by visual inspection under a fluorescence microscope.
Disruption of the abpD Gene
For construction of an interaptin targeting vector a 0.9-kb DNA fragment
was excised from clone G2 by digestion with EcoRI and SpeI, blunted
with Klenow fragment and ligated into the HindIII site of pBsrBam
(Adachi et al., 1994
), previously blunted with Klenow. This plasmid was
linearized with EcoRI and the 3-kb EcoRI fragment G1 was cloned. The
resulting vector (see Fig. 10 A) was introduced into AX2 cells by electroporation. Since mAb specific for interaptin were not available at the
time the mutant was generated, a PCR approach using Dictyostelium
amoebae DNA as template (Rivero et al., 1996
b) was used for screening
after selection for growth in the presence of blasticidin (ICN Biomedicals
Inc., Aurora, OH). Primers were designed that allowed amplification of a
1.1-kb DNA fragment in wild-type cells (Fig 10 A). Due to the insertion of
the Bsr cassette, the fragment size increased to 2.5 kb, whereas the endogenous 1.1-kb fragment was absent. In ~35% of the transformants tested
the abpD gene was knocked out.
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Western, Southern, and Northern Blotting
SDS-PAGE and Western blotting were performed essentially as described previously (Laemmli, 1970; Towbin et al., 1979
). Polyvinylidene difluoride (PVDF; Immobilon-P; Millipore Corp., Bedford, MA) was used
as blotting matrix. Iodinated sheep anti-mouse antibodies or the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Freiburg, Germany) were used.
DNA and RNA were isolated as described (Noegel et al., 1985), transferred onto nylon membranes (Biodyne B; Pall Filtron, Dreieich, Germany) and incubated with 32P-labeled probes generated using a random
prime labeling kit (Stratagene, La Jolla, CA). Hybridization was performed at 37°C for 12-16 h in hybridization buffer containing 50% formamide and 2× SSC. The blots were washed twice for 5 min in 2× SSC containing 0.1% SDS at room temperature and for 60 min in a buffer
containing 50% formamide and 2× SSC at 37°C.
Fluorescence Microscopy
Cells were fixed either in cold methanol (20°C) or at room temperature
with picric acid/paraformaldehyde (15% vol/vol of a saturated aqueous
solution of picric acid/2% paraformaldehyde, pH 6.0) followed by 70%
ethanol. For studies on developed cells, multicellular structures were disaggregated by passage through a 23-G needle before fixation. For studies
on whole mounts, multicellular structures were transferred onto glass
slides and fixed for 5-10 min in methanol at room temperature. Interaptin
was detected using mAb 260-60-10, protein disulfide isomerase (PDI) using mAb 221-135-1 (Monnat et al., 1997
), the A subunit of the V/H+-ATPase using mAb 221-35-2 (Jenne et al., 1998
) and contact site A glycoprotein using mAb 33-249-17 (Berthold et al., 1985
), followed by incubation
with Cy3-labeled anti-mouse IgG (Weiner et al., 1993
). Nuclei were
stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.).
Confocal images were taken with an inverted Zeiss LSM 410 laser scanning microscope with a 40× Neofluar 1.3 oil immersion objective. For excitation, the 488-nm argon-ion laser line was used, and the emission collected with a 510-525-nm band-pass filter. Conditions for simultaneous
acquisition of GFP and Cy3 fluorescence and for image processing were as
previously described (Maniak et al., 1995). Confocal images of multicellular structures were taken with an inverted Leica TCS-SP laser scanning
microscope with a 16× PL Fluotar 0.5 oil immersion objective. The 568-nm krypton ion laser line was used for excitation. Images were processed using the accompanying software.
Cell Fractionation Experiments
Cells were allowed to develop for 15-18 h on nitrocellulose filters. Multicellular structures were washed off the filters, disaggregated and lysed in
cold TMKS buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 25 mM KCl,
250 mM sucrose) with a protease inhibitor cocktail (Boehringer Mannheim GmbH, Mannheim, Germany) by repeated passage through 5-µm
Nuclepore filters (Corning Costar Corp., Cambridge, MA). Cytosolic and
membrane (including nuclei) fractions were separated by ultracentrifugation at 120,000 g for 1 h at 4°C. For extraction experiments the pellet was
further treated by gentle homogenization with either 1 M KCl or 0.1 M
NaOH in TMKS buffer followed by ultracentrifugation. For fractionation experiments membrane fractions prepared as above were separated in a
discontinuous sucrose gradient essentially as described by Hohmann et al.
(1985). Acid and alkaline phosphatase activities were measured as described previously (Loomis, 1969
; Loomis and Kuspa, 1984
). For lysis in
the presence of Triton X-100, lysis buffer (10 mM MES, pH 6.1, 138 mM
KCl, 3 mM MgCl2, 2 mM EDTA, 1% Triton X-100) was added to a cell
pellet after disaggregation. The lysate was then ultracentrifuged as above.
Miscellaneous Methods
Standard molecular biology methods were as described by Sambrook et al.
(1989). RT-PCR was performed using a Tth DNA polymerase kit (Boehringer Mannheim GmbH) according to the instructions supplied by the
manufacturer. Cell size and resistance to osmotic shock were quantitated
as previously described (Rivero et al., 1996a
). To analyze agglutination
cells were adjusted to an OD600 of 0.9 (roughly equivalent to a density of
1 × 107 cells/ml) in 5 ml Soerensen phosphate buffer and were shaken at
160 rpm. To follow the time course of agglutination, changes in optical
density (600 nm wave length) were monitored using a LKB Ultrospec III
spectrophotometer (Pharmacia Biotech Sverige, Uppsala, Sweden).
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Results |
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Molecular Cloning of abpD
We have used a YAC-based approach to identify the
abpD locus (Fig. 1), which has been mapped to the Dictyostelium chromosome 4 and codes for interaptin, an actin-binding protein of the -actinin superfamily. The coding
region of the abpD gene spans 5.7 kb and is interrupted by
two introns. The first intron is inserted in the middle of the
serine codon AGT at amino acid 173 and has two features
that make it unusual for Dictyostelium introns: its length (417 bp) and the presence of GC-rich stretches. RT-PCR
was used to confirm the intron-exon boundaries of this intron. To this end a reverse primer was designed downstream of the first intron and was used for reverse transcription of total RNA from developed cells. The resulting
cDNA was used as a template for PCR using the same reverse primer and a forward primer derived from a sequence upstream of the first intron. The second intron is
placed at alanine codon GCA corresponding to amino
acid 1679, and is 82 bp long. cDNA clone C5 allowed confirmation of the intron-exon boundaries. The coding region
is flanked by noncoding sequences containing extensive
homopolymeric A+T rich stretches, as is characteristic of
Dictyostelium intergenic regions (Kimmel and Firtel,
1983
). The 5' intergenic region is 777 bp long and contains
the promoter. Several GC-rich stretches are interspersed
in this region. A polyadenylation signal was found 115 bp
after the stop codon. The abpD locus is flanked by one
ORF on either side. We did not find any homology in the
data bases for the ORF upstream of abpD. The ORF situated downstream shows homology to a variety of carboxylases.
The genomic organization of the abpD gene was studied
by Southern blot analysis (not shown). Genomic DNA was
cut with a variety of restriction enzymes and hybridized
under high and low stringency conditions with probes generated along the abpD gene (P1 to P4). Under high-stringency conditions all probes hybridized to one or two DNA
fragments, as expected from the location of restriction sites deduced from the gene sequence. Under low stringency conditions additional bands were apparent with
probe P1, which contains sequences for the ABD. This result was expected, since the high similarity among the
ABD of members of the -actinin superfamily was exploited to clone abpD. Additional faint bands were also
apparent under low stringency conditions with probes P2
to P4 in a pattern consistent with the presence of more
than one related gene. Screening of a
ZAP cDNA library
using probe P3 under low stringency conditions yielded
numerous clones corresponding to proteins all of them characterized by the presence of polyglutamine stretches.
Similar stretches are present throughout the central portion of interaptin and could be responsible for the pattern
observed in Southern blots under low stringency conditions. On the other hand, rescreening of the YAC library
under low stringency conditions with probe P2, which is
devoid of sequences for the ABD, did not reveal additional loci, indicating that abpD is present as a single locus.
Sequence and Structural Features of Interaptin
The abpD gene sequence predicts a protein composed of
1,737 amino acids, with a calculated molecular mass of
204,300 D (Fig. 2). Based on structural and functional
analyses interaptin exhibits three distinct domains linked
by serine-rich amino acid stretches (Fig. 3). An ABD of
the -actinin superfamily of ~250 amino acid residues is
situated in the NH2-terminal region. The sequence of this
domain showed the highest identity (40.4%) to the ABD of gelation factor (ABP-120). The identity to the ABD of
other members of the
-actinin superfamily like
-actinin,
cortexillin,
-spectrin, dystrophin, utrophin, ABP-280, plectin, and dystonin was 33-39%, and dropped to ~20% for
members of the fimbrin/plastin family (Fig. 3 A). Due to
low solubility of a bacterially expressed peptide containing
most of the ABD of interaptin, biochemical analyses of
the actin-binding properties of this domain could not be
performed. However, from the high degree of conservation it is likely that the ABD of interaptin is capable of
binding to filamentous actin.
Structural predictions provided by the COILS algorithm
(Lupas et al., 1991) define a central rod domain of 1,180 amino acids with a predominant coiled-coil
-helical structure (Fig. 3 B). This domain displays a particular amino acid
composition, with a high content of glutamine (22.5%),
leucine (14.3%), glutamic acid (12.7%), lysine (10.9%),
and asparagine (9.7%). Two stretches of 179 and 117 residues (underlined in Fig. 2) are composed of four incomplete
and nearly identical repeats of 57 and 28 residues, respectively, which probably arose by internal gene duplications. A putative tyrosine phosphorylation site is present at the
end of the rod domain (boxed in Fig. 2), in a region of
lower probability of coiled-coil structure. Search of sequence databases with the rod portion of interaptin revealed weak homology (19-24% identity; up to 49% similarity) to >50 entries corresponding to tail sequences of
myosins, indicating a structural relationship, since the tails of certain myosins also possess a coiled-coil structure.
The last ~120 amino acid residues of interaptin constitute a domain involved in membrane association. Evidence on the role of this domain in determining the subcellular localization of interaptin is presented below. This domain shows a particularly high content (41.5%) of hydrophobic and nonpolar amino acid residues. A stretch of 12 consecutive hydrophobic or nonpolar residues is double-underlined in Fig. 2. No sequence homology was found between this domain and known proteins in the databases.
Developmental regulation of the abpD Gene
One prominent feature of the Dictyostelium life cycle is
the transition from single cell amoebae to a multicellular
fruiting body consisting of at least two differentiated cell
types. This transition is induced by starvation of the cells
and involves coordinated transcription of certain genes
and differentiation and sorting out of cell populations.
This process is regulated by diffusible signals and strongly
depends on the integrity of the actin cytoskeleton. We
have used Northern blot analysis to study the expression
of the abpD gene during synchronized development on nitrocellulose filters. Hybridization with probes P1, P3, and P5 revealed a transcript of 5.8 kb that is expressed at very
low levels during growth phase and the early aggregation
period (Fig. 4 A). Levels of this transcript reached a maximum at 12-16 h, a stage when differentiation and sorting
out into prespore and prestalk cells take place and in fact,
this period of maximal expression is concomitant with expression of prespore- and prestalk-specific genes, particularly pspA (Fig. 10 C). Expression of the 5.8-kb transcript
decreased to basal levels during maturation of the fruiting
body. Hybridization with probes P3 and P5 revealed an additional 5.4-kb transcript that is expressed at very low
levels throughout the Dictyostelium developmental cycle.
Since this transcript was not detected by probe P1 (not
shown), it appears to arise by differential splicing or by the
use of a second promoter presumably located in the first
intron, which displays unusual features (see above). In
fact, experiments with a fusion of the first intron with a
-galactosidase reporter gene indicated that this intron can display promoter activity (unpublished). High amounts
of RNA and prolonged exposure times after hybridization
were necessary to detect both transcripts, indicating that
expression levels are low. When abpD transcript accumulation was analyzed in cells starved in suspension, an increase in the 5.8-kb transcript was observed after 12 h of
starvation. Levels continued to increase until at least 18 h
of starvation, paralleling the behavior of the pspA mRNA
(not shown).
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Total cell homogenates of cells developed under the
same conditions were analyzed using Western blotting
with mAb 260-60-10. This mAb, raised against a fragment
of the interaptin rod domain, recognized bands of 200 and
160 kD (Fig. 4 B). The 160-kD band appeared as a constitutive protein, and almost equal amounts were present
throughout the entire developmental cycle, including 24 h.
The 200-kD band, whose size is in agreement with the predicted molecular mass of interaptin, was present at very
low levels during the early stages of development. The
amount of the 200-kD protein increased during late aggregation, reached a maximum and was maintained during
culmination and maturation of the fruiting body. Western blot analysis with mAb 234-151-9, raised against the ABD,
recognized both the 160- and the 200-kD bands, and additionally two weak bands of ~95 and 120 kD, probably corresponding to -actinin and ABP-120, respectively (not
shown). Comparison of Fig. 4, A and B shows that the
stage at which mRNA and protein levels rise are coincident. At later stages mRNA levels decrease, whereas protein levels are maintained, indicating that interaptin remains in the cell as a stable protein (see also Fig. 6). The
relationship between the 200- and the 160-kD proteins and
of both to the 5.8 and 5.4 transcripts is still unclear. Both
proteins appear to be immunologically related and behave
identical in extraction and sucrose gradient experiments
(see below). Size and developmental pattern of the 200-kD
band suggest that this protein is the product of the 5.8-kb transcript of the abpD gene, and this was confirmed by the
generation of mutants in which abpD was knocked out
(see below) or overexpressed (not shown). The origin of
the 160-kD protein is not clear yet; although it could derive from the 5.4-kb transcript, its presence in the abpD
mutant, although at low levels, argues against the hypothesis of both proteins being encoded by the same gene.
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Transcription of the abpD Gene Is Regulated by cAMP
The timing of appearance of the abpD transcript strongly
suggested that expression of this gene might also be subject to control by cAMP. To test for cAMP dependence of
transcription, cells were allowed to develop on agar to the
stage of tight mounds. After stimulation of disaggregated
multicellular structures with 2 mM cAMP an increase in
the mRNA levels of the 5.8-kb abpD transcript was apparent, that paralleled the behavior of the pspA gene, a known cAMP-dependent late gene used as a control (Fig.
5 A). At the protein level an increase in the amount of the
200-kD protein recognized by mAb 260-60-10 was noted,
whereas the 160-kD protein remained unchanged (Fig. 5
B). When tested on cells starved in suspension and stimulated with 2 × 108 M cAMP pulses, the abpD transcript
appeared 3 h earlier as compared with nonstimulated cells,
and was maintained at high levels at least until 18 h. A
similar pattern of expression was noted for the pspA gene
(not shown).
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Many genes in Dictyostelium are regulated by cAMP,
and the promoter regions of several of them have been
studied in detail, allowing the identification of distinct GC-rich elements. The abpD promoter contains several regions with a high content of G + C. One of these regions is
highly similar to G-rich sequence elements (GSE) of two
cAMP-regulated genes: cprB, the gene coding for cysteine
proteinase 2, that is transcribed at highest levels from late
aggregation to culmination, exhibits cAMP inducible transcription and contains two GSEs arranged in tandem
(Pears and Williams, 1987), and dscA, the gene coding for
discoidin I
, that is transcriptionally repressed by cAMP
and possesses one GSE in the promoter region (Poole and
Firtel, 1984
; Fig. 5 C). Studies carried out with deletion
constructs coupled to reporter genes have provided evidence for a role of GSEs in developmental and hormonal
regulation of gene expression in Dictyostelium. Since there
are several GC-rich regions in the abpD promoter in addition to the one referred to here, detailed analysis of this
promoter is necessary to identify all control elements and
establish their function.
Interaptin Is Enriched in Prestalk Cells
The pattern of regulation of the abpD gene suggested that
accumulation of interaptin might be restricted to a particular cell type during differentiation and morphogenesis. To
analyze this we carried out immunofluorescence staining
with mAb 260-60-10 on whole mount preparations at different developmental stages. In slugs, intensely stained
cells appeared scattered at the rear, superimposed on a homogeneous weaker staining present along the whole multicellular structure (Fig. 6 A). This population of intensely stained cells, which resemble in distribution the anterior-like cells (ALC), was not apparent at previous developmental stages. Furthermore, slime trails left by migrating
slugs were spotted by these strongly stained cells. As development proceeded an enrichment of interaptin was also
noted at the tip of early culminants (Fig. 6 B, see also Fig.
6 A, left), and in late culminants and mature fruiting bodies most of the staining localized to the upper and lower
cup, a behavior coincident with the fate described for the ALC (Williams, 1997), with few interaptin-rich cells still
scattered in the prespore or spore region (Fig. 6, C-F). A
funnel shaped structure at the entrance of the stalk tube
was visible in late culminants (Fig. 6 C). Staining was absent from the stalk tube itself, which was only very weakly
lined by surrounding immunostaining (Fig. 6, D and F).
Beyond the sorus, the stalk and the basal disc were spotted
by few interaptin-rich cells. In AX2 cells transformed with
promoter::lacZ reporter gene constructs that contained 5' flanking sequences of the abpD gene (as depicted in Fig.
1)
-galactosidase staining was observed through the
whole developmental cycle, and a cell type-specific pattern was not apparent. Since
-galactosidase is characterized by its high stability, and basal abpD mRNA as well as
protein levels are observed in vegetative cells and at early
stages of development, staining at early stages masked a possible cell type-specific staining arising at later stages. Taken together, our data indicate that the abpD promoter
directs expression of interaptin at low levels in a constitutive manner, and at high levels in a developmentally and
cell type specific manner.
Subcellular Localization of Interaptin
The intracellular distribution of interaptin was studied by immunofluorescence experiments with mAb 260-60-10 on methanol fixed growth phase and developing cells (Fig. 7). Axenically grown vegetative cells display a conspicuous perinuclear staining accompanied by an intense Golgi-like staining. Additionally, a much weaker punctate staining is distributed all over the cytoplasm. For studies on developing cells slugs were mechanically dissociated before fixation. Developing cells are smaller than vegetative cells. Like these, they display a pattern of intense perinuclear and weaker cytoplasmic staining except for a population of cells (see also Fig. 6), in which a very intense staining is apparent also in the cytoplasm. Comparable results were obtained after fixation of the cells with picric acid/paraformaldehyde, although the pattern of staining was more punctate with this fixative. Since in Western blots of vegetative cells mAb 260-60-10 recognizes a more abundant 160-kD and a less abundant 200-kD protein, staining of vegetative cells should be due in part to the 160-kD protein.
|
The immunofluorescence pattern described above, confirmed by immunoelectron microscopy experiments (not shown), is suggestive of an association of interaptin with intracellular membranes, and is therefore unusual for an actin-associated protein. Inspection of the interaptin amino acid sequence suggested that the COOH-terminal portion of the molecule could be responsible for membrane association. To investigate the role of the COOH-terminal domain of interaptin, that we have termed MAD, we fused GFP to a COOH-terminal fragment of interaptin containing the MAD and a short portion of the rod domain. A vector allowing constitutive expression of this fusion protein was electroporated into AX2 cells and GFP-MAD-expressing cells were used in double labeling fluorescence studies. GFP-MAD-expressing cells display a pattern of prominent perinuclear and Golgi-like staining along with an intense cytoplasmic staining (Fig. 8 A'). This result allowed us to attribute the COOH-terminal domain of interaptin a role in membrane association. Although simultaneous immunofluorescence staining with mAb 260-60-10 (Fig. 8 A) showed an overall colocalization of both fluorescent signals (Fig. 8 A''), wild-type Dictyostelium cells, in contrast to GFP-MAD-expressing cells, are characterized by a comparatively more prominent perinuclear staining with mAb 260-60-10 (compare Fig. 8, A with B and also with Fig. 7). This difference might be due to competition of GFP-MAD with the endogenous 160- and 200-kD proteins for limited amounts of binding sites on the membranes.
|
Pericentrosomal localization of the mAb 260-60-10 antigen was confirmed using an -tubulin-GFP expressing
Dictyostelium mutant (Neujahr et al., 1998
). As shown in
Fig. 8 B', bundles of microtubules emanate from the centrosomal region of the cell. Unpolymerized
-tubulin-GFP
appears as a diffuse cytoplasmic staining. Immunostaining with mAb 260-60-10 (Fig. 8 B) is intense around the nucleus, as well as in the pericentrosomal region (Fig. 8 B'').
To further identify the membrane compartments with
which interaptin associates, GFP-MAD-expressing cells
were labeled with mAb 221-135-1 directed against a PDI
as an ER protein (Monnat et al., 1997
; Fig. 8 C), and with
mAb 221-35-2 directed against the A subunit of the V/H+-ATPase, a protein present on membranes of the contractile vacuole and the endo/lysosomal system of Dictyostelium
(Jenne et al., 1998
; Fig. 8 D). Fig. 8 C'' shows colocalization of GFP-MAD and the ER marker. The ER appears as
a tubular-vesicular meshwork distributed all over the cell
and is continuous with the nuclear envelope. By contrast,
the membrane compartments defined by GFP-MAD and
the V/H+-ATPase do not overlap (Fig. 8 D''). Therefore,
we conclude that interaptin associates with membranes of
specific intracellular compartments constituted by the nuclear envelope and the ER.
Interaptin Is Associated with Intracellular Membranes
The association of interaptin with intracellular membranes suggested by the immunofluorescence studies was further investigated in extraction experiments on developed cells (Fig. 9 A). After lysis by passage through Nuclepore filters, both the 160- and the 200-kD proteins were quantitatively recovered in the membrane pellet. Further treatment of this pellet with high salt (1 M KCl) or with alkali (0.1 M NaOH) did not result in extraction of significant amounts of any of the proteins, indicating a very strong membrane association. Approximately 50% of the actin cosedimenting with membranes was extracted with 1 M KCl, whereas no actin remained in the membrane pellet after treatment with alkali. When cells were lysed in the presence of Triton X-100 both the 160- and the 200-kD proteins were solubilized; under these conditions the 200-kD band was rapidly degraded to slightly smaller fragments (not shown).
|
To confirm the results obtained in the immunolocalization experiments presented above, we performed fractionation experiments of a membrane pellet (including nuclei)
of developed cells in a discontinuous sucrose gradient. The
resulting fractions were analyzed in Western blots using a
panel of mAbs directed against distinct cellular compartments (Fig. 9 B). Probing with mAb 260-60-10 indicated
that the 200-kD protein (almost completely degraded despite the use of protease inhibitors) as well as the 160-kD protein were enriched in the highest density fractions,
where nuclei were also present. A similar distribution pattern was noted for PDI, a protein of the ER, and for comitin, a Golgi- and vesicle-associated protein (Weiner et al.,
1993; not shown). Mitochondria, monitored by mAb100
against porin, a protein of the mitochondrial outer membrane (Troll et al., 1992
), peaked at fractions 8 and 9, and psA, a protein present in prespore vesicles (Gregg et al.,
1982
), is apparent in fractions 7-10. Furthermore, the distribution pattern observed with mAb 260-60-10 was also
distinct from the distribution of the enzyme markers alkaline phosphatase, a marker for plasma membrane and the
contractile vacuole, and acid phosphatase, a marker for lysosomes.
Generation of an abpD Mutant
To gain insight into the function of interaptin we have generated an abpD mutant by homologous recombination.
To this end we made a construct in which the blasticidin
(Bsr) resistance cassette was inserted in an ~4-kb genomic
fragment of the abpD gene, between the sequences coding
for the ABD and the linker to the rod domain (Fig. 10 A). Southern blot analysis was used to characterize the recombination event (Fig. 10 B). The results allowed us to conclude that a gene disruption event had occurred in the
abpD
mutant. The deduced genomic organization of the
disrupted gene is depicted in Fig. 10 A. Northern blot analysis confirmed the absence of transcripts derived from the
abpD gene in the mutant, whereas the Bsr resistance gene
was expressed (Fig. 10 C). In Western blot analysis with mAb
260-60-10 absence of the developmentally and cAMP-regulated 200-kD protein that we identify with interaptin was
noted. The 160-kD protein was present as a very faint
band, apparent only after long exposure of the autoradiogram (Fig. 10 D). Furthermore, in immunofluorescence
studies with mAb 260-60-10 on growth phase abpD
cells,
only a weak discontinuous perinuclear staining could be observed which should be due to the presence of the
160-kD protein. It appears that the 160-kD protein becomes instable in the absence of interaptin, suggesting a
functional relationship between both proteins.
abpD Cells Are Delayed in Their Developmental
Program and Display an Adhesion Defect
Since in wild-type cells basal levels of the 200-kD interaptin are present in vegetative cells, studies of the abpD
mutant were extended to growth phase cells. Growth of
abpD
cells both in axenic medium, in suspension with E.
coli and on a bacterial lawn was comparable to wild type.
No differences were appreciated in the size of mutant cells
when compared with AX2, and application of an osmotic
stress did not result in an altered viability of abpD
cells.
However, when the developmental pattern of mutant cells on a solid substratum (agar or nitrocellulose filters) was
examined, a 3-4-h delay was consistently observed. The
morphology of multicellular structures was unimpaired,
and mature normal-looking fruiting bodies were formed
(not shown). We next examined in detail the timing and
levels of expression of developmentally regulated genes that are specifically expressed during aggregation (contact
site A or csaA gene) and during cell type differentiation
(pspA as prespore-specific marker and ecmA and ecmB as
prestalk-specific markers). Apart from a delay in the onset
of development, no remarkable differences in the levels of
expression of these genes were observed with the exception of the ecmB gene, which showed lower levels of transcripts in the abpD
mutant than in control cells (Fig. 10 C).
When suspensions of abpD cells were shaken for more
than 12 h in Soerensen phosphate buffer, only very loose
aggregates were formed, and the pale yellowish staining
characteristic of AX2 cells due to pigment molecules accumulating in cell aggregates did not develop. Therefore, we
examined if lack of interaptin had an effect on the ability
of cells to aggregate. For this we monitored the time
course of agglutination of abpD
cells in shaking suspension by measuring the decrease in light scattering at 600 nm (Fig. 11 A). Under these conditions, aggregates are
formed that recapitulate many of the developmental
events that occur on a solid surface (Takeuchi et al., 1988
).
Although slightly delayed, mutant cells were able to agglutinate and build multicellular aggregates similar to the
wild-type strain, as revealed by examination under the microscope after 8 h of starvation (Fig. 11 B). In agreement
with this, Western blotting and immunofluorescence analyses did not reveal differences between both strains in the amount of contact site A glycoprotein, which is responsible for EDTA-stable cell-cell contacts in early aggregation (Noegel et al., 1986
; not shown). When shaking
was prolonged, aggregates of AX2 cells became larger,
whereas abpD
aggregates dissociated, as shown in the
pictures taken after 15 h. This suggests that an adhesion
mechanism that is switched on at later stages of development is affected by lack of interaptin.
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Discussion |
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A New Member of the -Actinin Superfamily
We have taken advantage of the wide distribution and the
high degree of conservation of the ABD that characterizes
the members of the -actinin superfamily of cytoskeletal
proteins to identify interaptin in D. discoideum. Based on
analyses of the amino acid sequence, three regions can be
distinguished in the interaptin molecule that are connected by serine-rich linking peptides: an NH2-terminal
ABD, a central coiled-coil rod domain and a COOH-terminal MAD. Like interaptin, all other members of the
-actinin superfamily are characterized by a modular organization (Matsudaira, 1991
). According to the disposition and structure of the different modules, all members
can be subgrouped into four classes. Members of the fimbrin/plastin class are characterized by a tandem arrangement of two ABD preceded by a pair of EF-hands that allows for calcium regulation. A second class of proteins
comprises
-actinin, spectrin, and dystrophin (as well as
utrophin, the autosomal homologue of dystrophin). They
are characterized by a rod domain consisting of triple
-helical repeat segments (Yan et al., 1993
) that allow antiparallel dimerization, and by the presence of EF-hands.
Members of a third class of proteins, constituted by the
gelation factor (ABP-120) and filamin (ABP-280) possess
a rod domain composed of segments of
-sheet structure
arranged in an immunoglobulin-like folding (Fucini et al.,
1997
).
Finally, a fourth class is constituted by proteins whose
rod domains have a coiled-coil conformation. In this conformation consecutive heptads of amino acid residues
adopt an -helical structure with a particular distribution
of charged and nonpolar residues, allowing interaction of
two or more of these coiled-coil regions, usually in a parallel arrangement (Lupas et al., 1991
). Besides interaptin reported here, two other proteins of this class, cortexillin I
and II, have been described in Dictyostelium (Faix et al.,
1996
). Cortexillins are able to form parallel dimers by virtue of the coiled-coil domain, which confers the molecule
the appearance of a small double headed myosin. Additionally, cortexillin I possesses a putative phosphatidylinositol (4,5) bisphosphate (PIP2) recognition site, probably responsible for the cortical distribution of this protein.
Plectin and dystonin have been described recently in
mammalian cells (Brown et al., 1995
; Liu et al., 1996
). Dystonin is the neural isoform of the previously described
bullous pemphigoid antigen 1 (BPAG-1), an isoform lacking an ABD. Plectin and dystonin have a similar domain
structure, with an NH2-terminal globular domain that contains the ABD, a central coiled-coil rod domain and a
COOH-terminal domain constituted by globular repeats related to the ones present in the desmosomal proteins
desmoplakin and envoplakin (Fuchs and Cleveland, 1998).
Both proteins have also the capacity to bind simultaneously to actin filaments and intermediate filaments, localize to hemidesmosomes and contribute to mechanical
resistance of the cell. Although data on the oligomerization of interaptin as well as plectin and dystonin are not
yet available, members of this class appear to constitute bifunctional links, directly connecting actin networks to
other intracellular structures like membrane compartments in the case of interaptin.
A Developmentally and cAMP-regulated Actin-binding Protein
The contribution of cytoskeletal proteins to the developmental program of Dictyostelium has been well established by mutant analysis. The cytoskeletal proteins
themselves are in general not strongly regulated during
development (Noegel and Luna, 1995). Interaptin constitutes a remarkable exception, its message being expressed
at highest levels at a decisive stage of the developmental cycle, namely when differentiation and sorting of prespore
and prestalk cell populations to multicellular structures
takes place, clearly suggesting a stage-specific role for this
protein. Protein levels are also developmentally regulated,
but whereas mRNA levels rapidly decrease before culmination, the protein is still present at high levels. A similar
phenomenon has been reported for ponticulin, an integral
membrane protein that links the plasma membrane to the underlying actin cortex in Dictyostelium. Ponticulin
transcript levels decrease dramatically after aggregation,
whereas the protein remains present, although at lower
levels (Hitt et al., 1994
).
Regulation by hormone-like signaling molecules, like
cAMP in the case of interaptin, is also an unusual feature
for a cytoskeletal protein. Cyclic AMP plays an essential
role during the Dictyostelium life cycle. In addition to its
function as chemoattractant, cAMP also regulates the expression of almost all classes of developmentally regulated
genes, either directly or through stimulation of DIF (differentiation inducing factor), another Dictyostelium morphogen (for a review see Verkerke-van Wijk and Schaap,
1997). Cyclic AMP pulses in the nanomolar concentration
stimulate expression of genes involved in aggregation, like
csaA (csA glycoprotein) and carA (a cAMP receptor subtype), while repressing growth phase genes. At a later
stage during aggregation, low concentration cAMP pulses
induce expression of so-called early and intermediate genes like cprB (cysteine proteinase 2), pde (phosphodiesterase), rasD (a small G protein), and lagC (an adhesion
molecule, see below). After aggregates have formed, micromolar concentrations of cAMP accumulate, leading to
expression of prespore specific genes, like pspA, as well as
non-cell-specific genes. Cyclic AMP also stimulates synthesis of DIF, which in turn induces expression of the
prestalk genes ecmA and ecmB.
The abpD gene appears to display a bimodal pattern of
expression that probably relies on promoter features that
remain to be elucidated. On the one hand, the abpD gene
is expressed at low levels in a constitutive manner. This is
based on immunofluorescence studies on whole mounts, in
which perinuclear staining with mAb 260-60-10 was apparent at all developmental stages, and on results obtained
with promoter::lacZ reporter gene fusions. Superimposed to this basal pattern of expression, on the other hand, the
abpD gene is expressed at high levels in a strictly regulated
manner, as demonstrated in Northern and Western blot
analyses, as well as in immunofluorescence studies on
whole mounts. In this case interaptin is enriched in a distinct population of cells first detectable at the slug stage.
According to the distribution and behavior of these cells,
we identify them with so-called ALC, a sub-type of
prestalk cells first described as neutral red-staining cells
scattered in the prespore region of the slug (Sternfeld and
David, 1981). The interaptin-rich cells, initially present at
the rear of the slug, appear to migrate to the front and to the base as culmination proceeds. At the front they form
part of the upper cup, and do not seem to enter the stalk
tube and differentiate into stalk cells. At the base they are
lifted up the stalk adherent to the spore mass, and constitute the lower cup, a structure of very variable size. Few
interaptin-rich cells appear to be left behind at the basal
disc and attached to the stalk during culmination. In general, ALC constitute a heterogeneous cell population, and
the gene expression patterns of these and other prestalk
subtypes have been extensively studied (Williams 1997
).
The fate of the interaptin-rich cells described here remains to be established in terms of expression of well known
prestalk markers like ecmO, ecmA, and ecmB, that are expressed in distinct subpopulations of ALC. In any case, the
abpD gene constitutes a marker of potential use for future
studies of morphogenetic events in Dictyostelium.
Interaptin as a Link between Membrane Compartments and the Actin Cytoskeleton
We present immunological and biochemical evidence that
interaptin is strongly associated with intracellular membrane compartments. The existence of interactions of
membranes in general with actin networks and cables is
well established, and they have been shown to participate
in a variety of cell functions. Associations of the plasma
membrane to the underlying cytoskeleton contribute to
the cell shape by providing mechanical strength, and at
sites of interaction with the extracellular matrix they participate in signal transduction pathways (Hitt and Luna,
1994; Cowin and Burke, 1996
). In mammalian cells examples of actin-binding proteins involved in anchoring the
plasma membrane to the cortical cytoskeleton are provided by spectrin and dystrophin (see Introduction). In Dictyostelium a number of actin-binding proteins like ponticulin (Hitt et al., 1994
), hisactophilins (Scheel et al.,
1989
), and talin (Kreitmeier et al., 1995
) could fulfill this
function. For cortexillins the exact nature and the significance of the cortical distribution apparent in immunofluorescence studies is presently unknown (Faix et al., 1996
).
Finally, myosin Is can bind to the plasma membrane
through a polybasic COOH-terminal domain, where they
are proposed to contribute to the overall cortical tension of the cell (Uyeda and Titus, 1997
).
On the other hand, association of intracellular membrane compartments with the actin cytoskeleton has been
implicated in intracellular traffic as well as in endo- and
exocytotic processes. Here actin provides the tracks for attaching vesicles and directing their transport within the
cell (Kuznetsov et al., 1992). Apart from interaptin, only
one example of a vesicle-associated actin-binding protein
is known in Dictyostelium. The 24-kD protein comitin is located on Golgi and vesicle membranes (Weiner et al.,
1993
), and it has been proposed that comitin links membrane vesicles to the actin cytoskeleton via mannose residues (Jung et al., 1996
). This lectin-like activity would enable comitin to bind specifically intracellular membranes
carrying glycoconjugates on their cytoplasmic faces. Actin-based motors are involved in organelle movement, and
several classes of unconventional myosins, particularly of classes I, V, and VI, have been implicated in vesicular trafficking (reviewed in Mermall et al., 1998
). More recently a
nonmuscle myosin II has been described to associate with
vesicles budding off the trans-Golgi network in mammalian cell lines, but its role is still uncertain (Ikonen et al.,
1997
; Müsch et al., 1997
). In Dictyostelium, myosin II
heavy chain null mutants are characterized by defective intracellular particle movement (Wessels and Soll, 1990
).
On the contrary, data available on Dictyostelium myosin Is
indicate that they do not play a role in the intracellular trafficking of vesicles. Their function seems to be restricted to the phases of the endo- and exocytotic processes that take place at the cell cortex (Uyeda and Titus,
1997
).
Two cytoskeletal proteins of the -actinin superfamily
have been reported to associate with intracellular membranes in higher eukaryotes, where they might play distinct roles. A homologue of erythrocyte
-spectrin colocalizes with membranes of the Golgi complex in a variety of
cell types, from which it reversibly dissociates during mitosis and upon treatment with brefeldin A, a drug that perturbs Golgi structure (Beck et al., 1994
). This spectrin isoform supposedly contributes to the structural integrity of
the Golgi complex in a manner similar to its action at the
erythrocyte plasma membrane, and in fact, an ankyrin isoform, a protein that links spectrin to the cytoplasmic domains of specific integral membrane proteins, has also
been identified in the Golgi complex (Beck et al., 1997
).
ABP-280, on the other hand, binds to and is required for
efficient sorting of furin, a transmembrane endoprotease
implicated in proteolytic maturation of many proproteins
within the trans-Golgi network/endosomal system, and is
required for correct localization of late endosomes and lysosomes (Liu et al., 1997
). For interaptin we have shown
that the COOH-terminal domain is responsible for interaction with intracellular membranes. We have also shown
that interaptin binds to membranes of distinct compartments, mainly the ER. Since interaptin lacks a motor domain, as is the case for proteins like myosin, kinesin, and
dyenin that are involved in vesicle trafficking, this protein
could play a structural role and contribute to the integrity
and correct localization of vesicles along the secretory
pathway. Alternatively, interaptin could contribute to the
correct folding and/or targeting of membrane proteins by affecting their clustering with either themselves or with
other membrane proteins, as has been shown for the Golgi
spectrin-ankyrin skeleton (Devarajan et al., 1997
) and for
PDZ domain proteins (Craven and Bredt, 1998
).
Experiments on agglutination of abpD cells suggest a
defect in a specific cell adhesion system. At least four adhesion systems are responsible for ensuring multicellularity in Dictyostelium and three cell adhesion molecules
have been identified so far. DdCAD-1 (gp24), a calcium-binding glycoprotein with some similarity to E-cadherin,
mediates EDTA/EGTA-sensitive adhesion and accumulates during the first four hours of development (Wong
et al., 1996
). The contact site A glycoprotein (gp80) mediates the EDTA-resistant adhesion during the aggregation
stage (Noegel et al., 1986
). This adhesion system is not
substantially impaired in the abpD
mutant. Finally, gp150
is responsible for EDTA-resistant contacts during the
postaggregation stage, and there is evidence indicating that this protein is encoded by the lagC gene (Dynes et al.,
1994
). Two features make this glycoprotein a potential
candidate to explain the phenotype of abpD
cells. First,
like abpD, the lagC gene is cAMP-regulated. And second,
gp150 is expressed at moderate levels during cell aggregation, and rapidly accumulates in the late postaggregation
stage, where it seems to constitute the last and only adhesion molecule expressed, since lagC
cells arrest at the
loose mound stage (Dynes et al., 1994
). However, abpD
cells are able to develop to the fruiting body stage. Therefore, it is possible that a functionally altered but still active
adhesion molecule is synthesized in our mutant. Glycosylation is one of the most common forms of posttranslational protein modification and takes place in the ER and
in the Golgi apparatus. One possible explanation for the
mutant phenotype would be that lack of interaptin brings
about an altered trafficking of vesicles from the ER to the
Golgi apparatus, disturbing maturation of glycoproteins.
In summary, we describe a new actin-binding protein of
the -actinin superfamily. Like all members of this superfamily, interaptin has a modular organization and is structurally related to cortexillins, plectin and dystonin through
its central coiled-coil rod domain. Additionally, interaptin
possesses a novel domain for association with membranes
of the ER. This specific feature, combined with the fact
that interaptin is expressed mainly at a late stage in the
Dictyostelium developmental cycle are suggestive of a specific role in membrane trafficking at this stage.
![]() |
Footnotes |
---|
Received for publication 8 April 1998 and in revised form 22 June 1998.
Address all correspondence to Angelika A. Noegel, Institut für Biochemie I, Medizinische Fakultät, Universität zu Köln, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany. Tel.: 49 221 478 6980. Fax: 49 221 478 6979. E-mail: noegel{at}uni-koeln.deWe thank L. Graciotti and J. Köhler for assistance during cloning and Dr.
M. Schleicher for discussion and Dr. W. Loomis for providing a ZAP library. We are grateful to Dr. G. Gerisch for mAbs against Dictyostelium
porin, to R. Neujahr for
-tubulin GFP-expressing cells, to M. Westphal
for supplying the red-shifted GFP mutant gene, and to Dr. M. Maniak for
mAbs against Dictyostelium PDI and V/H+-ATPase A subunit. We thank
R. Albrecht, J.-M. Schwartz and J. Murphy for help in the use of the confocal microscope and image processing. We are especially grateful to Dr.
M. Stewart for guidance on structural features of interaptin.
This work was supported by the Deutsche Forschungsgemeinschaft No. 113/5-5, European Union grant CHRX-CT93-0250, Project grant no. 40 from the Zentrum für Molekulare Medizin Köln and National Institutes of Health Grant GM52359.
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Abbreviations used in this paper |
---|
ABD, actin-binding domain; ALC, anterior-like cells; BPAG-1, pemphigoid antigen 1; Bsr, blasticidin; DAPI, 4',6-diamidino-2-phenylindole; GSE, G-rich sequence elements; MAD, membrane-associated domain; PDI, protein disulfide isomerase; PIP2, phosphatidylinositol (4,5) bisphosphate; PVDF, polyvinylidene difluoride; YAC, yeast artificial chromosomes.
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