Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

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., 1996b).

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.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

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.

View larger version (59K): [in this window] [in a new window]   Fig. 3.   Structural features of interaptin. (A) Alignment of the ABD of interaptin with the ABD of other members of the -actinin superfamily described in Dictyostelium. For fimbrin only the first ABD has been considered. The alignment was generated using the Clustal W program. Dashes indicate gaps introduced in the sequence for optimal alignment. Residues that are identical between interaptin and any of the other members of the -actinin superfamily are boxed. Arrows indicate the sequences used to design the degenerate primers for cloning the abpD gene. GeneBank accession numbers: interaptin, AF057019; -actinin, Y00689; ABP-120, X15430; cortexillin 1, L49527; cortexillin 2, L46371; fimbrin, L36202. (B). Probability of forming -helical coiled-coil structures. Matrix MTIDK of the COILS version 2.1 algorithm (Lupas et al., 1991) was used with a window size of 28 and weighting. High values are reached through the central portion of the protein, defining a rod domain. The proposed structure of interaptin is shown below. The protein consists of three functional domains connected by serine rich stretches: an NH2-terminal ABD (Met-1 to Leu-255), a central rod domain (Lys-417 to Gln-1597) and a COOH-terminal MAD (Asn-1624 to Thr-1737).

View larger version (5K): [in this window] [in a new window]   Fig. 1.   Strategy of cloning and genomic organization of the abpD gene. Five overlapping genomic clones (G1 to G5) encompassing 8.6 kb were isolated and sequenced in both directions. Additionally, a cDNA clone (C5) was obtained after screening a ZAP cDNA library. The coding sequence is interrupted by two introns (shaded) of 417 and 82 base pairs. The abpD locus has been mapped to chromosome 4 of the Dictyostelium genome. DNA fragments used as probes for Northern and Southern blot analyses are depicted as P1 to P5. DNA fragments used for construction of lacZ fusions are depicted as L1 and L2. R, EcoRI restriction sites.

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.

View larger version (58K): [in this window] [in a new window]   Fig. 2.   Deduced amino acid sequence of interaptin. The sequence predicts a protein of 204.3 kD. Internal repeats in the rod domain are underlined. A putative tyrosine phosphorylation site is boxed. A stretch of nonpolar amino acids at the COOH terminus is double underlined. Interaptin has received the GeneBank accession number AF057019.

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., 1996b) 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.

View larger version (28K): [in this window] [in a new window]   View larger version (36K): [in this window] [in a new window]   Fig. 10.   Generation of an abpD mutant by homologous recombination. (A) A construct was made in which the blasticidin resistance cassette (Bsr) was inserted in a genomic clone between the ABD and a portion of the rod domain. The arrows at the bottom indicate the position of the oligonucleotide primers used for the PCR screening of mutants. (B) Southern blot analyses demonstrate that a gene disruption event has occurred. Genomic DNA was digested with EcoRV and blots probed with the 32P-labeled DNA fragments indicated below. With probe 1 insertion of the Bsr cassette causes the shift of a 0.75-kb band to a 2.2-kb band in the mutant. With probe 2 an additional band of ~6 kb is apparent in the mutant which contains sequences of the pUC118 transformation vector, as shown after hybridization with a pUC probe. (C and D) Development of abpD mutant. AX2 and mutant cells were allowed to develop on nitrocellulose filters. At the indicated time points samples were taken for RNA extraction and Western blot analysis. Northern blots containing 30 µg RNA per lane (C) were probed with probe P1 to demonstrate that no RNA message is present in the mutant, whereas the blasticidin resistance gene (bsr) is expressed. Similar result was obtained with probe P4. Blots were also hybridized with probes for developmentally regulated genes. csaA codes for the cell adhesion molecule contact site A, responsible for cell-cell contacts in early aggregates (Noegel et al., 1986). pspA encodes the prespore-specific cell surface protein psA (Early et al., 1988). ecmA and ecmB encode the prestalk-specific extracellular matrix proteins ST340 and ST310, respectively (Jermyn et al., 1987). Myosin is a control for comparable loading. The abpD mutant shows a delay of ~3 h in the developmental pattern as compared with wild type. In Western blot analysis (D) the 200-kD band is absent, and the 160-kD band appears as an extremely faint band in the abpD mutant. The blot was subsequently probed with -actinin-specific mAb 47-62-17 (Schleicher et al., 1988) to show comparable loading.

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).

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

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).

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Developmental regulation of the abpD gene. Dictyostelium amebae were starved on nitrocellulose filters. At the indicated time points samples were taken for RNA extraction or preparation of total cell homogenates. (A) RNA blots containing 30 µg of RNA per lane were probed with probe P3 (see Fig. 1). Expression is highest at the mound and preculminant stage; very low levels are present at other stages of the developmental period. (B) Total cell homogenates of 4 × 105 cells were resolved in 6% polyacrylamide gels and blotted onto PVDF filters. Blots were incubated with mAb 260-60-10. This mAb recognizes two bands, a 160-kD constitutive band and a 200-kD developmentally regulated band.

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.

View larger version (68K): [in this window] [in a new window]   Fig. 6.   Investigation of cell type-specific expression of abpD. Cells were allowed to develop on agar, and multicellular structures were transferred to coverslips, fixed with methanol at room temperature, and then incubated with mAb 260-60-10. Optical sections were taken with a confocal laser scanning microscope. (A) Interaptin-rich cells appear scattered in the rear of a slug, resembling anterior-like cells. On the left, intensely stained tip of an early culminant. (B) Early culminant. Staining is enriched at the tip and in cells scattered along the culminant. (C and D) Two confocal sections, 16 µm apart, through a late culminant. Interaptin-rich cells accumulate at the upper and lower cups. The conus is apparent in C, and the stalk tube appears weakly lined by surrounding fluorescence in D. (E) Maximum projection image of a late culminant. 16 confocal sections 8 µm apart were taken. Images were superimposed and for each pixel the one with the highest intensity of all sections is represented. (F) Sorus of a mature fruiting body. Staining is most intense at the upper and lower cup and in few scattered cells in the spore region. Interaptin positive cells appear to line the stalk tube at the upper cup. Bar: (A-E) 100 µm; (F) 60 µm.

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).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Expression of the abpD gene is regulated by cAMP. Cells were allowed to develop on agar to the stage of tight mounds, disaggregated and incubated for 2 h in suspension in the presence of 5 mM cAMP. Cells were then collected for RNA extraction and Western blot analysis. (A) A Northern blot containing 30 µg RNA per lane was hybridized with probe P3. For control pspA, a known cAMP-stimulated gene, was used. (B) Total cell homogenates of 4 × 105 cells were resolved in 6% polyacrylamide gels and blotted onto PVDF. Blots were incubated with mAb 260-60-10. Only the 200-kD band is stimulated by cAMP. (C) Comparison of G-rich sequence elements upstream of cAMP-regulated genes. Numbers indicate the position relative to the ATG start codon. cprB, the gene coding for cysteine proteinase 2, is cAMP inducible and contains two of these elements (Pears and Williams, 1987); dscA, the gene coding for discoidin 1A, is repressed by cAMP (Poole and Firtel, 1984).

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.

View larger version (97K): [in this window] [in a new window]   Fig. 7.   Immunofluorescence labeling with interaptin-specific mAb 260-60-10 of vegetative (t0) and developed (t12) cells. Developed cells were disaggregated before fixation with methanol. Perinuclear and Golgi-like staining are apparent in vegetative cells, along with a weak punctate staining throughout the cytoplasm. DAPI, a fluorescent dye that binds to DNA, was used to confirm the perinuclear localization. The same pattern is observed in developed cells, but the cytoplasmic staining is more intense in a subpopulation of cells. Bar, 10 µm.

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.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Subcellular localization of interaptin. Confocal sections through double labeled vegetative cells. (A) Immunostaining with mAb 260-60-10 of cells expressing a MAD-GFP fusion (A'). The merge (A'') indicates that the COOH-terminal domain is responsible for the subcellular localization of interaptin. (B) Immunostaining with mAb 260-60-10 of cells expressing a GFP--tubulin fusion (B'). The merge (B'') shows an enrichment of immunostaining in the centrosomal region. (C) Immunostaining with mAb 221-135-1 (an ER marker; Monnat et al., 1997) of cells expressing a MAD-GFP fusion (C'). (D) Immunostaining with mAb 221-35-2 (a marker of membranes of the contractile vacuole and the endo/lysosomal system; Jenne et al., 1998) of cells expressing a MAD-GFP fusion (D'). Merges C'' and D'' indicate that interaptin localizes to vesicles of the ER compartment rather than to vesicles of the contractile vacuole or the endo/lysosomal system. Bar, 5 µm.

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).

View larger version (36K): [in this window] [in a new window]   Fig. 9.   Extraction and subcellular fractionation experiments on developed cells. Multicellular structures equivalent to 3 × 108 cells were washed, disaggregated, lysed and processed as described in Materials and Methods. Proteins were resolved in 6 or 12% polyacrylamide gels, blotted onto PVDF and incubated with the mAbs against the proteins indicated. (A) Salt and alkali extraction of a membrane fraction. Lane 1, total cell homogenate; lane 2, total cell lysate. Lanes 3 and 4, cytosolic and membrane (including nuclei) fraction, respectively, after 120,000 g centrifugation of a total cell lysate. Lanes 5 and 6, supernatant and pellet, respectively, after extraction of the membrane fraction of lane 4 with 1 M KCl. Lanes 7 and 8, supernatant and pellet, respectively, after extraction of the membrane fraction of lane 4 with 0.1 M NaOH. Blots were incubated with mAb 260-60-10 for interaptin and act-1 (Simpson et al., 1984) for actin. Both the 200- and the 160-kD proteins are recovered almost quantitatively in the membrane pellet after lysis and extraction with salt or alkali. The 200-kD band was partially degraded. (B) Sucrose gradient fractionation of a membrane (including nuclei) pellet. Samples were centrifuged to equilibrium on 30-50% (wt/vol) sucrose gradients atop 84% (wt/vol) cushions (fraction 11). After centrifugation 1 ml fractions were collected from the top and analyzed. 5-10% of each fraction was analyzed in Western blots using mAbs 260-60-10 for interaptin, 221-135-1 for PDI (an ER marker; Monnat et al., 1997), mAb100 for porin (a mitochondrial protein; Troll et al., 1992) and MUD-1 for psA (a protein of prespore vesicles; Gregg et al., 1982). The enzymes assayed were alkaline phosphatase (a marker for plasma membrane and the contractile vacuole; dotted line) and acid phosphatase (a marker for lysosomes; solid line). A fraction of the acid phosphatase has been released during the lysis step and appears at the top of the gradient. The 200-kD (almost completely degraded) and the 160-kD protein were enriched in the highest density fractions, in a pattern similar to PDI and distinct from all other markers tested.

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.

View larger version (36K): [in this window] [in a new window]   Fig. 11.   Alterations in the pattern of agglutination of the abpD mutant. Axenically grown AX2 and abpD cells were washed and starved in Soerensen's phosphate buffer at a density of 1 × 107 cells/ml with shaking at 160 rpm. At the time points indicated optical density (600 nm wave length) was measured in a spectrophotometer (A). Each point represents the average ± SD of three independent experiments. The pattern observed in the mutant after 9 h of starvation indicates a dissociation of the aggregates previously formed as shown in the photographs (B), and occurs at the time when the abpD gene begins to be expressed. Differences between AX2 and abpD cells were significant (P < 0.001, Student's t test) from 3 h on. Bar, 100 µm.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

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 st