CAP in Dictyostelium

3195

Journal of Cell Science 112, 3195-3203 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0525

Assessing the role of the ASP56/CAP homologue of Dictyostelium

discoideum and the requirements for subcellular localization Angelika A. Noegel1, Francisco Rivero1, Richard Albrecht2, Klaus-Peter Janssen4, Jana Köhler2, Carole A. Parent3 and Michael Schleicher4,* 1Institut für Biochemie I, Medizinische Einrichtungen der Universität zu Köln, Joseph-Stelzmann-Str. 2Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany 3Department of Biological Chemistry, Johns Hopkins University, School of Medicine, North Wolfe St,

52, 50931 Köln, Germany

Baltimore, Maryland 21205, USA 4Institut für Zellbiologie der Ludwig-Maximilians-Universität München, Schillerstrasse 42, 80336 München, Germany *Author for correspondence (e-mail: [email protected])

Accepted 2 July; published on WWW 22 September 1999

SUMMARY The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5bisphosphate (PIP2) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP2-regulation of the sequestering activity. Here we have constructed GFPfusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP

were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.

INTRODUCTION

(Gerst et al., 1991). The latter domain was subsequently shown to sequester actin (Freeman et al., 1995; Gottwald et al., 1996). This activity is, however, no longer PIP2-regulated and it appears that both domains have to interact to allow for PIP2sensitivity. Proline rich sequences often function as SH3 domain binding sites which are thought to mediate formation of specific protein complexes, and the proline rich region 2 (P2) of yeast CAP was identified as an SH3 domain binding site and shown to interact with the SH3 domain of yeast actin binding protein Abp1p (Freeman et al., 1996). Both Abp1p and CAP are localized in cortical actin patches in yeast and the subcellular localization of CAP seems to be mediated by the SH3 domain of Abp1p, since CAP/Srv2p carrying a mutated SH3 domain binding site did no longer show localization in cortical patches (Lila and Drubin, 1997). Recent work identified a complex of proteins consisting of End4p1, a talin related protein, Rvs167p, an amphiphysin homologue, Abp1p and CAP/Srv2p, that is involved in endocytosis (Wesp et al., 1997). Dictyostelium CAP homologue has a single proline rich

CAP/Srv2p was first described in yeast with roles in Ras mediated responses of adenylyl cyclase and cell morphology (Fedor-Chaiken et al., 1990; Field et al., 1990). ASP56, the mammalian homologue of CAP, was isolated from pig platelets on the basis of its actin sequestering activity (Gieselmann and Mann, 1992). We have isolated and characterized the Dicytostelium homologue as an actin sequestering protein and shown that this activity is regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Gottwald et al., 1996). An actin monomer binding activity has also been demonstrated for the yeast protein (Freeman et al., 1995). CAP and CAP-homologues have two domains, an N- and a C-terminal domain separated by one (D. discoideum) or two (yeast, human) proline rich stretches, and distinct functions have been attributed to the domains as well as to the proline rich region. The N-domain of Saccharomyces cerevisiae CAP is responsible for mediating the Ras sensitivity of adenylyl cyclase, the C-domain for regulating cellular morphology

Key words: Actin binding protein, GFP-fusion, Endocytosis, Cytokinesis

3196 A. A. Noegel and others region which can be aligned with the proline rich region P1 of the yeast protein. The protein is present throughout the cytoplasm and shows enrichment at the plasma membrane, especially at the rear and front ends. Furthermore it undergoes rapid rearrangements in moving cells (Gottwald et al., 1996). We have analyzed the localization of N- and C-domain fusions of CAP homologue with green fluorescent protein (GFP) in living cells, and found that the N-domain of CAP is important for the localization of the protein. The proline rich region seems to be dispensable. To further study the in vivo role of CAP, we generated a mutant expressing strongly reduced protein levels. The most prominent changes as compared to the parent strain were altered cell morphology and cell growth associated with a cytokinesis and endocytosis defect. MATERIALS AND METHODS Cell culture D. discoideum strain AX2 and transformants of this strain expressing various fusion proteins of CAP with GFP were cultivated in nutrient medium as described by Watts and Ashworth (1970) in suspension culture at 21°C. For development cells were washed in 17 mM Soerensen phosphate buffer, pH 6.0, resuspended at a density of 1×107 cells /ml in the buffer and starved for 6 hours (Malchow et al., 1972). Generation of mutant cells A 1.4 kb XbaI/HindIII fragment carrying the blasticidin resistance cassette was retrieved from plasmid pUCBsr∆Bam kindly provided by Dr H. Adachi (Adachi et al., 1994), blunt-ended using Klenow enzyme and cloned into the similarly blunt-ended BglII site of the full length CAP cDNA in pUC19 (Gottwald et al., 1996). The BglII site roughly separates the two domains of CAP. The gene targeting vector was transformed into AX2 cells. Transformants were analyzed in colony blots using mAb 223-445-1 directed against the C-terminal half of CAP (Gottwald et al., 1996) and as secondary antibody 125Isheep anti-mouse antibody. Cloning and expression of CAP-GFP fusion proteins For expression of specific domains fused to GFP we used the polymerase chain reaction to amplify the corresponding gene sequences. The N-terminal domain of D. discoideum CAP without (amino acid residues 1 to 215) and with the proline rich region (amino acid residues 1 to 254) and residues 1 to 93 were amplified by PCR using a full length cDNA as template and cloned in frame into the BamHI-site of the pDdA15gfp vector kindly provided by Dr R. Kay giving rise to pN-CAP-Pro-GFP, pN-CAP-GFP and pN102-CAP-GFP. For the C-terminal domain, plasmids pC-CAP-GFP and pPro-C-CAPGFP coding for amino acid residues 216 to 464 and 255 to 464 were generated. Full length CAP was cloned into pDEX-T65S-GFP (Westphal et al., 1997). In both vectors CAP-proteins are N-terminally fused to GFP and transcription of fusion proteins is under control of the actin 15 promoter allowing expression during growth and development (Cohen et al., 1986). The sequence of the PCR products was verified by DNA sequence analysis. For all GFP-fusions the wildtype GFP gene was used with the exception of full length CAP-GFP where a GFP gene was used that encoded red-shifted GFP carrying the T65S mutation. Plasmids were introduced into D. discoideum AX2 cells by electroporation (Rivero et al., 1996), selection of transformants was with G418. Transformants were analyzed for the presence of fusion proteins by western blot using domain specific monoclonal antibodies (Gottwald et al., 1996) and by fluorescence microscopy. Mutant analysis For analysis of development, cells were either starved in suspension,

on Millipore filters (type HA) or phosphate agar plates (Rivero et al., 1996). Samples for RNA or protein analysis were taken at the indicated time points. Determination of growth rates under various conditions, of cell size, phagocytosis and motility were done according to the method of Rivero et al. (1996) and fluid phase endocytosis according to the method of Aubry et al. (1993). For quantitative phago- and endocytosis assays, equal cell volumes were used. In general, experiments were performed three to five times, usually the results of one typical experiment are shown. For rescue experiments a full length CAP cDNA was cloned into expression vector pDEXRH (Faix et al., 1992) and expressed under control of the actin 15 promoter. Adenylyl cyclase assays were performed as previously described on cells starved for 5 hours and repeatedly stimulated with 50 nM cAMP (Parent and Devreotes, 1995). Image analysis Living cells expressing GFP fusions with domains of CAP were examined using a Zeiss LSM-410 laser scanning microscope (Zeiss, Oberkochen, FRG) or a Leica TCS-SP laser scanning microscope (Leica Lasertechnik GmbH, Heidelberg, FRG). Miscellaneous methods DNA manipulations were done according to the methods of Sambrook et al. (1989). SDS-PAGE was performed according to the method of Laemmli (1970) and immunoblotting according to the method of Towbin et al. (1979). Bacterial expression and purification of recombinant CAP, immunofluorescence methods and antibodies are described by Gottwald et al. (1996), nuclei were stained with 4,6diamidino-2-phenylindole (DAPI). Monoclonal antibody 223-317 originated from the previously described generation of monoclonal antibodies against a C-terminal bacterially expressed CAP polypeptide (Gottwald et al., 1996).

RESULTS Targeting of CAP to membranes is mediated by its N-domain In Dicytostelium CAP is enriched in peripheral regions, the cytoplasm shows a week overall staining (Gottwald et al., 1996). For yeast CAP a localization in the cortical actin cytoskeleton was reported and the protein’s proline rich region P2 was shown to function as an SH3 domain binding site and to be required for targeting CAP to its proper cellular location in the cortical actin cytoskeleton (Freeman et al., 1996; Lila and Drubin, 1997). D. discoideum CAP harbors a single proline rich stretch which, based upon homology, might also function as an SH3 domain binding site (Gottwald et al., 1996). We have therefore constructed expression plasmids carrying PCR-generated gene sequences that coded for fusions of CAP and the N- and the C-terminal domain with GFP, and either contained or lacked the proline rich region (CAP-GFP, N-CAPPro-GFP, N-CAP-GFP, Pro-C-CAP-GFP, C-CAP-GFP; Fig. 1). This allowed us to analyze the distribution of the fusion proteins in living cells. By immunoblot analysis we found that all fusion proteins were stably produced and not subject to proteolysis, furthermore the overall amount of GFP-fusion proteins paralleled that of the one of the endogenous protein (data not shown), although at the single cell level we observed considerable differences between cells, a phenomenon not uncommon with GFP fusion proteins (Westphal et al., 1997). C-CAP-GFP and Pro-C-CAP-GFP fusions were present throughout the cytosol without being enriched at leading or

ASP56/CAP in Dictyostelium 3197 Cyclase Associated Protein: CAP-N 1

P

CAP-C

216 255

464 aa

CAP

GFP constructs: GFP

N-CAP-GFP N-CAP-Pro-GFP N102 -CAP-GFP Pro-C-CAP-GFP C-CAP-GFP CAP-GFP

posterior edges unlike full length CAP (Fig. 2A,B). In moving cells the distribution did not change. N-CAP fusions behaved differently. GFP-tagged N-CAP and N-CAP-Pro fusion proteins were present throughout the cytoplasm and showed an accumulation at anterior and posterior ends (Fig. 2C). This distribution is nearly identical to the one we observed for full length CAP-GFP fusion protein (Fig. 2A) as well as to that of CAP described previously (Gottwald et al., 1996). We also investigated N- and C-CAP-GFP expressing cells in more detail and determined the fluorescence intensities throughout the cells by analyzing more than 150 cells each. This showed a significant enrichment of N-CAP-GFP at cell borders as compared to C-CAP-GFP (Fig. 2F). To identify the regions responsible for this localization, we generated by PCR N102CAP-GFP, a fusion protein consisting of the first 93 amino acid residues of CAP. N102-CAP encompasses a region of CAP that codes for a conserved amino acid stretch with the potential to form an amphipathic helix and in yeast CAP it interacts with adenylyl cyclase (Nishida et al., 1998). This fusion protein still retained the property to accumulate at anterior and posterior ends of cells (Fig. 2D) and resembled in its distribution similar the one of endogenous CAP. When N102-CAP-GFP expressing cells were fixed and analyzed in double immunofluorescence studies with CAP specific monoclonal antibodies the GFP signal of the fusion protein and the one derived from the antibody staining overlaped in front regions (Fig. 2E). Isolation of a CAP mutant For isolation of a CAP-deficient mutant we constructed a vector which was designed in such a way as to allow a gene replacement event (Fig. 3). Transformants were screened using monoclonal antibodies specific for the C-domain of CAP. After several attempts that included use of different vector constructs we finally isolated a CAP deficient mutant cell line designated CAP bsr expressing strongly reduced amounts of CAP. When we compared dilutions of AX2 homogenates in western blots to homogenates derived from CAP bsr cells we found that the remaining protein amounted to less than 5% of the wild-type level (Fig. 4A). The protein still present in CAP-bsr cells was recognized by all monoclonal antibodies available as well as

Fig. 1. Schematic representation of GFP fusion proteins of CAP and its domains.

polyclonal antibodies specific for CAP (data not shown). The monoclonal antibodies had been generated against recombinant N- and C-domain polypeptides ensuring that they recognize different epitopes (Gottwald et al., 1996), polyclonal antibodies were generated against a C-terminal polypeptide (Gottwald, 1995). Furthermore, by two-dimensional gel analysis the mutant protein proved indistinguishable from wild-type CAP, exhibiting an experimentally determined pI between 6.9 and 7.2. BglII digests of mutant DNA showed that the vector had inserted with several copies into the endogenous gene (Fig. 4B). The hybridization pattern was consistent with a homologous recombination that had occurred in the Cterminal part of the gene leaving the coding region of the gene intact but separating it from its original 3′-non-coding sequences (see Fig. 3). Due to the recombination event the latter were replaced by vector sequences. The proposed insertion was further confirmed by RNA analysis. Instead of the 1.5 kb CAP message, two mRNAs, a stronger and a weaker hybridising one, of approximately 5 kb were detected with a CAP specific cDNA probe (Fig. 4C). Consistent with the assumption, that homologous recombination resulted in replacement of the original 3′-end by sequences of the transformation vector, these RNAs hybridized with pUC19 sequences. They did not hybridize with a blasticidin resistence cassette specific probe which suggested that the regulatory sequences of the bsr cassette functioned as terminator for the read through RNAs. The overall amount of these RNAs was greatly reduced, this being most likely responsible for the observed reduced protein level. Reduction of CAP level affects cell morphology and is associated with a cytokinesis defect Altered CAP levels led to changes both at the single cell level and during multicellular development. The most dramatic alterations were noted when single cells were analyzed. During microscopic inspection of mutant cells grown in suspension we observed a rather heterogeneous population of cells with regard to cell size (Fig. 5A). In a quantitative analysis the diameters varied from 10 µm to 30 µm (Fig. 5B) with a mean diameter of 17±5.2 µm. For AX2 we observed a mean diameter of

3198 A. A. Noegel and others

Fig. 2. Distribution of GFP fusions of CAP and its domains in AX2 transformants visualized by confocal microscopy. (A) In cells expressing CAP-GFP the fusion protein is present in the cytosol and most prominently close to the membrane and at fronts. (B) C-domain fusions of CAP with GFP. The protein is present throughout the cell and does not show specific enrichment. (C) shows cells expressing N-terminal fusions of CAP with GFP. The protein is present throughout the cell and shows particular enrichment at front regions. (D) A similar distribution is observed for N102-CAP-GFP. Representative pictures are shown for each fusion protein in living cells (A-D). (E) Double immunofluorescence studies. Cells expressing N102CAP-GFP were fixed with methanol and labeled with CAPspecific mAb 223-317 followed by Cy3 labeled secondary antibody. Left panel, detection of the GFP fusion protein, middle panel, detection of CAP with the monoclonal antibody, right panel, overlay. Bar, 10 µm. (F) The relative fluorescence intensity of C-CAP-GFP and N-CAP-GFP expressing cells throughout the cells was determined for more than 150 cells each as described by Hanakam et al. (1996).

12.0±2.8 µm. Similar data were obtained by analyzing cell size in a Coulter Counter. The enlarged mutant cells were multinucleated with as many as 10 nuclei per cell pointing to a cytokinesis defect (Fig. 5A). For CAP bsr cells grown in suspension in axenic medium we found that 11.2% of all cells had more than five nuclei as compared to 0.75% for AX2 wild type (Table 1). Multinuclearity was seen in cells grown in axenic medium in suspension as well as on plastic surfaces.

Altered CAP levels result in growth and endocytosis defect AX2 wild-type cells grow in axenic medium to cell densities of more than 1×107 cells per ml before reaching stationary phase. By contrast, CAP bsr mutant cells consistently reached saturation at lower densities (7×106 cells/ml for CAP bsr as compared to 1.2×107 cells/ml for wild type in the experiment shown, Fig. 6A). Growth defects can be due to cytokinesis

ASP56/CAP in Dictyostelium 3199 construct for gene disruption: Bgl ATG

TAA

CAP cDNA

blunt

100 bp

bsr resistance cassette

disruption event: gene disruption vector pUC

bsr

cDNA

cDNA

pUC

D. discoideum genome endogenous CAP gene

Fig. 3. Disruption of the CAP gene. Schematic representation of the vector generated to disrupt the CAP gene and the proposed recombination event in the CAP bsr mutant. A 1.4 kb fragment encoding the DNA for the blasticidin resistance gene (bsr) driven by the actin 15 promoter was cloned into the blunt ended BglII-site of full length CAP cDNA in pUC19. This plasmid was inserted by homologous recombination into the C-terminal half of the CAP gene 3′ of the endogenous BglII-site thereby separating the gene from its 3′-noncoding sequences.

recombinational event pUC + insert / tandem insertion

complete CAP gene

expected bands in Southern blot:

vector / tandem insertion 1 kb

defects, however, often they also reflect pinocytosis defects as has been shown for Dictyostelium mutants lacking phosphatidylinositide 3-kinases (Buczynski et al, 1997; Zhou et al., 1998), we assayed pinocytosis using fluorescently labeled dextran. To take into account the difference in cell size of wild-type and mutant, the results obtained were normalized to cell mass. We found that uptake of fluid was reduced to 70% of wild type. Doubling time was increased from 12 hours for wild type to 15 hours for CAP bsr. By contrast, growth on bacteria in shaking suspension was not affected and doubling times of between 2.5 and 3 hours for mutant and parent strain were observed. This assay shows the ability of cells to phagocytose. Similarly, growth on agar plates in association with bacteria was comparable to wild type as was uptake of fluorescently labeled yeast cells by quantitative analysis. The morphological defect as well as the growth and cytokinesis defect were reverted to wild type once mutant cells were transfected with a plasmid allowing expression of CAP under the control of the A15 promoter and terminator.

Motility, chemotaxis and development are impaired in the CAP bsr mutant The observed redistribution of CAP to newly formed fronts of D. discoideum during motile events prompted us to analyze the motile behaviour of the mutant. Motility was reduced both in the presence and absence of a cAMP gradient, and orientation towards the cAMP source was impaired (Table 2). The onset of development was delayed by more than four hours both in shaking suspension and on solid substratum as analysed with stage specific probes (shown for starvation in suspension, Fig. 7). Then development proceded normally, and on solid substratum it ended with formation of fruiting bodies. Yeast CAP is an adenylyl cyclase associated protein and is required by Ras to stimulate adenylyl cyclase activity (Field et al., 1990). In D. discoideum cAMP is an important signaling molecule involved in coordinated cell movement and cell type differentiation. It binds to a cAMP receptor (cAR), and signaling by the βγ-subunit of the G protein is required for Table 2. Motility of wild-type and mutant cells Speed (µm/minute)

Table 1. Cytokinesis defect in CAP bsr cells Strain

Strain Nuclei/cell 1 2 3 4 5 >5

AX2 (%)

CAP bsr (%)

40 43 11 5 1 0.75

19 26 13 10 6 11

Wild-type AX2 and mutant CAP bsr cells were fixed and nuclei stained with DAPI and counted.

AX2 CAP bsr

Buffer 8.57±1.36 3.83±0.77*

Gradient

Orientation (cosθ) Buffer

11.76±1.42 0.010+0.078 5.45±0.94* −0.051+0.076

Gradient

N

0.176±0.006 3 0.040+0.032* 4

*P