Correspondence to: Annette Müller-Taubenberger, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany., amueller{at}biochem.mpg.de (E-mail), 49-89-8578-2322/2339 (phone), 49-89-8578-3885 (fax)
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Abstract |
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The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes.
We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions.
Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.
Key Words: actin, Dictyostelium discoideum, endocytosis, motility, WD-repeat
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Introduction |
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THE actin cytoskeleton is involved in cell locomotion, cytokinesis, cell-cell and cell-substratum interactions, the organization of the cytosol, vesicle and organelle transport, and the establishment and maintenance of cellular morphology. The organization of F-actin in different cytoskeletal structures is controlled by accessory proteins, some of which facilitate the assembly of actin filaments into a three-dimensional meshwork, whereas others regulate filament turnover or remodel the actin cytoskeleton in response to external signals.
Cytoskeletal fractions from Dictyostelium cells contain myosin II, actin, and actin-binding proteins such as -actinin, ABP120, fimbrin, cortexillin, and coronin (
Here we describe a new 64-kD protein from Dictyostelium that coprecipitates with actin-myosin complexes. Like coronin, this protein is a WD40-repeat protein. Therefore, its sequence has been indexed as WD-repeat protein 2 (Wdp2) in the EMBL/GenBank/DDBJ databases (accession number
U36936). We refer now to this protein as Dictyostelium Aip1 (DAip1), because it became evident that Wdp2 is the Dictyostelium homologue of the actin-interacting protein 1 (Aip1p) from Saccharomyces cerevisiae. The yeast protein has been discovered in a two-hybrid screen for proteins that interact with actin (
DAip1 is strongly accumulated in dynamic regions of the cell cortex, in particular crowns and leading edges, as shown by monitoring the localization of a green fluorescent protein (GFP)1-tagged DAip1 fusion protein. Mutants lacking DAip1 are severely impaired in endocytosis and motility, and have a moderate cytokinesis defect. Our results provide in vivo evidence that proteins of this type play a role in the control of complex processes that are based on the turnover of actin.
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Materials and Methods |
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Growth and Development of Dictyostelium Cells
Dictyostelium discoideum cells of the wild-type strain AX2, and DAip1-null and HG1569 coronin-null cells were cultivated in nutrient medium (
cDNA Cloning and Sequence Analysis
A partial clone of DAip1 was isolated from a gt11 cDNA library of D. discoideum strain AX3 (Clontech Inc.). The sequence was completed in both directions using a PCR-based strategy using DAip1 and
gt11 sequence-specific primers and the cDNA library as template. DNA was sequenced on an Applied Biosystems sequencer ABI PrismTM 377 (Toplab). Sequences were analyzed using the UWGCG (
Protein Purification, mAbs, and Immunoblotting
The contracted actin-myosin complex was prepared from AX2 cells starved for 14 h essentially as described previously (
A cDNA fragment encompassing the entire coding region of DAip1 beginning with Ser-2 was amplified by PCR using primers designed to obtain a BamHI site at the 5' end and a HindIII site at the 3' end. The product was cloned into the expression vector pQE-30 (Qiagen Inc.), and the His-tagged protein was expressed in Escherichia coli M15. The recombinant protein was purified on a Ni2+-NTA-agarose column (Qiagen) to homogeneity using denaturing conditions (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0).
Antibodies were obtained by immunizing BALB/c mice with recombinant DAip1 using either aluminum hydroxide or Freund's adjuvant. Spleen cells were fused with PAIB3Ag8 myeloma cells. mAbs 245-308-1, 246-466-6, 246-258-1, and 246-153-2 that specifically recognized DAip1 were used in this study. For detection of coronin, mAb 176-3-6 (
Gene Replacement in Dictyostelium
For construction of the DAip1 targeting vector, 5' and 3' fragments of the DAip1 sequence were generated by PCR using the primers 5'-GTGAAGCTTGAATTCAACACCAGCAACTACTCGTG-3' and 5'-GTGAAGCTTACCATCATAAACAAAGGCTTTC-3' for the 5' fragment, and 5'-GTGTCTAGAAGCCCAACAACATACTGGTGG-3' and 5'-GTGGAATTCACCTTCATTACCTGCAGAG-3' for the 3' fragment. The 3' fragment was cleaved by XbaI and EcoRI and the 5' fragment by HindIII, and both fragments were cloned subsequently into the plasmid pBsr2 (
Southern blotting was performed as described (
Expression of DAip1-GFP and GFP-Actin Constructs, and Overexpression of DAip1
Mutants expressing a DAip1-GFP fusion protein were produced by transformation of wild-type and DAip1-null cells with a vector conferring resistance to G418 essentially as described (
For constructing the expression vector for GFP-(N)-DAip1, a PCR fragment was generated using the primers 5'-GTGAATTCAAAATGTCTGTAACTTTAAAAAATATT-3' and 5'-GTGGAATTCTTAATTTGATACATACCAAATTTTAATAG-3', and a Dictyostelium cDNA library in gt11 (Clontech) as template. The fragment was cleaved with EcoRI, and cloned into the EcoRI site of pDEX gfp (
For expression of GFP-actin, DAip1-null cells were transformed with the same vector that has been used previously for the expression and analysis of GFP-actin in wild-type cells (
Immunofluorescence Microscopy
AX2 wild-type or mutant cells were allowed to settle onto glass coverslips for 30 min, fixed with picric acid/formaldehyde for 20 min, and postfixed with 70% ethanol as described (
In Vivo Microscopy
For observing the morphology of dividing cells, a double-view microscope was used, which combines phase-contrast and RICM imaging (
Motility and Endocytosis Assays
Quantitative analysis of cell motility was performed according to the method of
Phagocytosis assays using TRITC-labeled, heat-killed yeast in shaken suspension were carried out essentially as described by
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Results |
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DAip1 Is a Member of the WD-Repeat Family
During a cDNA screen for a PAK/STE20-related kinase from D. discoideum (GenBank accession number
U51923), we isolated a partial clone encoding a protein that showed homology to the previously identified WD-repeatcontaining cytoskeletal protein coronin. Sequence analysis of the complete cDNA revealed a polypeptide of 597 amino acid residues with a calculated molecular mass of 64 kD (Figure 1 A), and an isoelectric point of 7.4. The gene is present in a single copy per haploid genome, and is constitutively expressed.
Comparison of the complete sequence to the protein databases revealed a significant degree of homology to Aip1p from S. cerevisiae, and during our studies it became evident that both proteins are also functionally related. Therefore, we refer to the new protein as Dictyostelium Aip1 (DAip1). An alignment of the DAip1 amino acid sequence with yeast Aip1p is shown in Figure 1 A. The two sequences show 33% identity over their entire length. Aip1 homologues were also found in C. elegans and P. polycephalum (Figure 1 A), and recently in Arabidopsis thaliana (GenBank accession number
O23240), Xenopus laevis (
The DAip1 sequence contains nine WD40-repeat motifs extending from residues 56 to 594 (Figure 1 B). When compared with the consensus ( helix is positioned between residues 85 and 100.
DAip1 Is Enriched in Cell Surface Projections
Antibodies that specifically recognized DAip1 in total cell lysates were used to show that DAip1 is a component of precipitated actin-myosin complexes (Figure 2 A). Association of DAip1 with the actin system is supported by its intracellular localization. Immunofluorescence labeling of Dictyostelium cells in the growth phase showed an enrichment of DAip1 in the cell cortex. Crown-shaped extensions of the dorsal cell surface were most prominently labeled (Figure 2B and Figure C). These funnel-shaped protrusions are sites where nutrients from liquid medium are taken up by macropinocytosis (
Generation of DAip1-Null Cells
To study the function of DAip1 in vivo, we eliminated DAip1 by gene replacement in the Dictyostelium wild-type strain AX2 using a blasticidin resistance cassette as marker for selection (Figure 3 A). Mutants were identified by a shift in size of an EcoRI fragment of the DAip1 gene by 1.4 kb corresponding to the size of the blasticidin resistance gene cassette (Figure 3 B). DAip1 was no longer detectable on Western blots of mutant cell lysates, whereas it was clearly recognized in wild-type and coronin-null cells (Figure 3 C).
Examination of DAip1-null cells by phase-contrast microscopy revealed that the mutant cells were larger than wild-type cells and tended to become multinucleate. Growth of DAip1-null cells on bacteria and in liquid medium was reduced. DAip1 proved not to be essential for multicellular development. On agar plates, DAip1-null cells formed normal fruiting bodies. In the following, we present a detailed analysis of the defects of the DAip1-null mutant in growth and cytokinesis, which suggests that the defects are a direct consequence of changes in the actin cytoskeleton dynamics caused by the lack of DAip1.
DAip1-Null Cells Show Altered Cytokinesis
Staining of DAip1-null cells with 4,6-diamidino-2-phenylindole (DAPI) revealed the conspicuous presence of multinucleate cells (Figure 4 A). A quantitative analysis confirmed the shift in the DAip1-null cell population toward a larger number of nuclei per cell (Figure 4 B). Furthermore, changes of the cell shape that occur during cytokinesis of DAip1-null cells show distinct alterations when compared with cytokinesis of wild-type cells (Figure 4 C;
DAip1-Null Cells Are Impaired in Macropinocytosis, Phagocytosis, and Motility
Loss of DAip1 led to a reduced growth rate of mutant cells both in liquid culture and on bacteria. In liquid medium, the generation time of DAip1-null cells was prolonged to 12 h as compared with 8 h determined for the wild-type. A large portion, if not the entire uptake of soluble nutrients in Dictyostelium, occurs through macropinocytosis (
Next, we wanted to assay a pathway where the activity of the F-actin cytoskeleton can be affected by an experimental stimulus. In Dictyostelium, phagocytosis is induced by adhesion of a particle to the cell surface as opposed to macropinocytosis, which is a constitutive process. Therefore, we determined the rate of phagocytosis of yeast particles in wild-type and in DAip1-null cells. The rate of particle uptake in DAip1-null cells was reduced to 26% of the wild-type rate (Figure 6 A). This strong deficiency in phagocytosis of yeast particles was paralleled by slow growth on bacterial lawns (Figure 6 B).
To monitor the process of phagocytosis in vivo, we used GFP-tagged actin expressed in wild-type and DAip1-null cells. In wild-type cells, the formation of a phagocytic cup, from inception until the complete engulfment of a yeast particle, took between 20 and 40 s (Figure 6 C). In the DAip1-null cells, the duration of this process was prolonged to 70120 s (Figure 6 D).
Since the radius of a colony is a cumulative function of both growth rate and of cell motility, we also compared the motility of wild-type and DAip1-null cells by a quantitative assay. The speed of locomotion of DAip1-null cells was reduced to 46% of the wild-type values (Table 1).
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DAip1 Overexpression Suggests an Involvement of DAip1 in the Regulation of Actin Depolymerization
Since disruption of the DAip1 gene did not allow us to draw a definitive conclusion about its mode of action in vivo, we expressed DAip1 under control of an actin-15 promoter in DAip1-null cells. DAip1 accumulated to a level ~20-fold higher than the endogenous DAip1 in the wild-type (data not shown). A quantitative analysis of the number of nuclei per cell in the DAip1-overexpressing cells indicated that the cytokinesis defect was partially rescued (data not shown). The motility of the DAip1-overexpressing cells was reversed to wild-type values (Table 1).
Whereas the rate of pinocytosis in DAip1-overexpressing cells was almost identical to wild-type cells (Figure 7 A), they phagocytosed yeast particles at a rate that was ~50% higher than the rate determined for wild-type (Figure 7 B). Overexpression of cytoskeletal proteins can sometimes be simulated by the addition of drugs that mimic the effect of actin-binding proteins. Cytochalasin A, known to block polymerization at the barbed end of actin filaments, decreases the rate of particle uptake and fluid-phase internalization in Dictyostelium (
DAip1 and Cofilin Localize to Cortical Protrusions
Recent in vitro data suggested a functional interaction of DAip1 with cofilin (
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Dynamics of GFP-tagged DAip1 in Living Cells
To study the distribution of DAip1 in vivo, gene fusions were constructed comprising the full-length coding sequences of DAip1 and GFP. GFP was either fused to the NH2 terminus or to the COOH terminus of DAip1. The constructs were named GFP-(N)-DAip1 and DAip1-(C)-GFP, respectively. In DAip1-null cells, no functional rescue was achieved by expression of DAip1-(C)-GFP. Nevertheless, the intracellular distribution of the DAip1-(C)-GFP hybrid protein, both in wild-type and in DAip1-null cells, was similar to the antibody labeling of crowns and lamellipodia in wild-type cells (Figure 2).
The expression of GFP-(N)-DAip1 in DAip1-null cells partially rescued the defects of DAip1-null cells. Their phagocytosis rate recovered to 80% of the wild-type rate, and the distribution of the number of nuclei was similar to that of wild-type cells (data not shown).
DAip1-null cells expressing the GFP-(N)-DAip1 construct showed a distinct labeling of cell surface protrusions. Using confocal microscopy, we recorded the dynamic redistribution of the GFP-(N)-DAip1 fusion protein in cells during cytokinesis, pinocytosis, directed movement, and phagocytosis (Figure 9). A common feature of all these activities is the formation of specialized cortical structures rich in filamentous actin (
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By local stimulation with the chemoattractant cAMP through a micropipette, the polarity of aggregation-competent Dictyostelium cells can be changed at will (
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Discussion |
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DAip1 Is a Constituent of the Actin Cytoskeleton
D. discoideum Aip1 (DAip1) belongs to the WD-repeat family of proteins (Figure 1;
DAip1 is homologous to Aip1p from S. cerevisiae, an actin-interacting protein that has been identified using a two-hybrid screen (
DAip1 is found in purified actin-myosin complexes and colocalizes with regions of the cell cortex known to be enriched in filamentous actin: phagocytic cups, crowns, leading edges of cells migrating toward a source of chemoattractant, and poles of dividing cells (Figure 2, Figure 9, and Figure 10). The elimination of DAip1 by gene replacement results in defects both in macropinocytosis and in phagocytosis (Figure 5 and Figure 6), the main pathways responsible for fluid-phase and particle uptake in Dictyostelium. In both cases, the cell protrudes F-actinrich extensions from its surface in order to capture and engulf the endocytic cargo (
DAip1 May Contribute to the Regulation of Actin Depolymerization
Overexpression of DAip1 in a DAip1-null mutant did not only rescue phagocytosis, but markedly increased particle uptake over wild-type rates, whereas fluid-phase uptake was barely affected. The effects of Lat-A observed in wild-type cells also distinguish phagocytosis, which is stimulated by the drug (Figure 7 B), from macropinocytosis and cell migration, which both are inhibited by the same concentration of Lat-A (Figure 7 A;
One possibility is that different stimuli make different sources of actin monomers available for polymerization. During phagocytosis, spreading of a lamella around a particle depends on a steady supply of monomeric actin. Depolymerization of actin filaments may be stimulated as a response to the continuous signal induced by adhesion of a particle, and could provide monomers for local actin assembly at the phagocytic cup. In distinction to phagocytic cups, surface ruffles for macropinocytosis and pseudopodia during directed migration are not necessarily supported by a solid substratum (
A second possible explanation is based on the fact that a phagocytic cup needs to follow precisely the shape of the particle during protrusion. If existing actin filaments do not depolymerize fast enough during this process, the structure becomes too rigid to allow close interaction between the membrane and the particle. To form a macropinosome in the absence of spatial guiding cues, the protruding surface ruffles must be rigid and persist until they fuse at their distal edges. The same argument applies to cell migration. Polarization of the cytoskeleton during protrusion of a leading edge needs to be maintained for a certain time, in order to achieve persistent directional migration.
Consistent with these ideas, DAip1-null cells are most severely affected in phagocytosis, which is reduced to 26% of the wild-type rate. The major reason for this reduction in the mutant appears to be a threefold slower protrusion of the phagocytic cup (Figure 6). Fluid-phase uptake is only reduced to 56% of the wild-type rate. The DAip1-null cells migrate with 46% of the wild-type's velocity, suggesting that the requirements on cytoskeletal dynamics are similar for macropinocytosis and cell motility.
Cofilin as a Putative Effector Protein of Aip1
There is evidence in vitro that Aip1 exerts its effect on actin depolymerization through the stimulation of cofilin. Cofilin is a member of the ADF family known to depolymerize actin filaments at their pointed ends (
Support for an interaction of Aip1 and cofilin is provided by the observation that GFP-tagged cofilin (
Although the results discussed above suggest that Aip1 acts through cofilin to enhance actin depolymerization (
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Acknowledgements |
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We are grateful to Dr. Günther Gerisch for his continuous support and stimulating discussions at all stages of the work. We thank Drs. Hiroyuki Aizawa and Ichiro Yahara for their generous gift of anti-cofilin antiserum, and John Murphy and Jean-Marc Schwartz for their help with image processing and for three-dimensional image reconstructions.
The work was supported by a grant of the Deutsche Forschungsgemeinschaft (MU 1415) to A. Müller-Taubenberger.
Submitted: December 24, 1998; Revised: June 11, 1999; Accepted: June 14, 1999.
1.used in this paper: GFP, green fluorescent protein; Lat-A, latrunculin-A
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