Disruption of Endocytosis with the Dynamin Mutant shibirets1 Suppresses Seizures in Drosophila

One challenge in modern medicine is to control epilepsies that do not respond to currently available medications. Since seizures consist of coordinated and high-frequency neural activity, our goal was to disrupt neurotransmission with a synaptic transmission mutant and evaluate its ability to suppress seizures. We found that the mutant shibire, encoding dynamin, suppresses seizure-like activity in multiple seizure–sensitive Drosophila genotypes, one of which resembles human intractable epilepsy in several aspects. Because of the requirement of dynamin in endocytosis, increased temperature in the shi^ts1 mutant causes impairment of synaptic vesicle recycling and is associated with suppression of the seizure-like activity. Additionally, we identified the giant fiber neuron as critical in the seizure circuit and sufficient to suppress seizures. Overall, our results implicate mutant dynamin as an effective seizure suppressor, suggesting that targeting or limiting the availability of synaptic vesicles could be an effective and general method of controlling epilepsy disorders.     

Morphology and dynamics of the endocytic pathway in Dictyostelium discoideum

Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes. It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis, and phagocytosis that are not performed by other model organisms or are difficult to study. D. discoideum is becoming a popular system to study molecular mechanisms of endocytosis, but the morphological characterization of the organelles along this pathway and the comparison with equivalent and/or different organelles in animal cells and yeasts were lagging. Herein, we used a combination of evanescent wave microscopy and electron microscopy of rapidly frozen samples to visualize primary endocytic vesicles, vesicular-tubular structures of the early and late endo-lysosomal system, such as multivesicular bodies, and the specialized secretory lysosomes. In addition, we present biochemical and morphological evidence for the existence of a micropinocytic pathway, which contributes to the uptake of membrane along side macropinocytosis, which is the major fluid phase uptake process. This complex endosomal compartment underwent continuous cycles of tubulation/vesiculation as well as homo- and heterotypic fusions, in a way reminiscent of mechanisms and structures documented in leukocytes. Finally, egestion of fluid phase from the secretory lysosomes was directly observed.     

Overexpression of a Novel Rho Family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects Phagocytosis in Dictyostelium discoideum

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin–rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif–containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.     

radE, a new radiation-sensitive locus in Dictyostelium discoideum

Dictyostelium discoideum strain M28, which has been used widely in genetic studies, was found to carry a radiation-sensitive mutation. This allele, termed rad-100, was recessive in heterozygous diploids and mapped in linkage group III. Complementation analysis and survival studies on strains carrying rad-100 suggested that this allele defines a new radiation-sensitive locus in D. discoideum, and this locus has been designated radE. radE strains were moderately sensitive to ultraviolet light (D10 90 J m-2) and slightly sensitive to 137Cs gamma rays D10 255 krad). radE strains also exhibited increased sensitivity to killing by N-methyl-N'-nitro-N-nitrosoguanidine but not by other alkylating agents such as ethyl methanesulphonate or methyl methanesulphonate. The frequency of spontaneous methanol-resistant (acrA) mutants was approximately the same in cultures of radE and radE+ strains. However, when amoebae of these strains were irradiated with ultraviolet light, the frequency of induced mutants was significantly lower in cultures of the radE strain. Furthermore, when amoebae of wild-type strain NC4 were plated in the presence of caffeine after ultraviolet-irradiation, the survival curves were very similar to the curves obtained for amoebae of radE strains in the presence or in the absence of caffeine. These results suggest that the radE100 mutation and caffeine interfere with an error-prone DNA repair pathway in D. discoideum.     

Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology

Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.In vertebrates, ionotropic glutamate receptors (iGluRs in animals) are ligand-gated cation channels that mediate the majority of the excitatory neurotransmission in the central nervous system (Dingledine et al., 1999). In the model plant Arabidopsis (Arabidopsis thaliana), 20 genes encoding homologs of animal iGluRs have been identified (Lam et al., 1998). According to phylogenetic analyses, the Arabidopsis GLUTAMATE RECEPTOR (AtGLR) homologs can be subdivided into three separate subgroups (Chiu et al., 1999, 2002). Some evidence for the channel-forming ability by plant ionotropic glutamate receptors (iGLRs) has been obtained only recently, and only for AtGLR3.4 and AtGLR1.4 expressed in heterologous systems (Vincill et al., 2012; Tapken et al., 2013). Studies with transgenic plants suggested roles of members of the plant GLR family in Ca²⁺ fluxes (AtGLR2; Kim et al., 2001), coordination of mitotic activity in the root apical meristem (Li et al., 2006), regulation of abscisic acid biosynthesis and water balance (AtGLR1.1; Kang and Turano, 2003; Kang et al., 2004), carbon/nitrogen sensing (AtGLR1.1; Kang and Turano, 2003), resistance against fungal infection (Kang et al., 2006), leaf-to-leaf wound signaling (Mousavi et al., 2013), and lateral root initiation (Vincill et al., 2013). Application of antagonists and agonists of animal iGluRs revealed that plant GLRs might be involved in the regulation of root growth and branching (Walch-Liu et al., 2006), in light signal transduction (Lam et al., 1998), and in the response to aluminum (Sivaguru et al., 2003). In various plant cell types, the agonists Glu- and Gly-induced plasma membrane depolarization and a rise in intracellular Ca²⁺ concentration that were inhibited by blockers of nonselective cation channels (NSCCs) and by antagonists of animal iGluRs (Dennison and Spalding, 2000; Dubos et al., 2003; Meyerhoff et al., 2005; Krol et al., 2007; Kwaaitaal et al., 2011; Michard et al., 2011). Furthermore, Glu-activated cation currents in patch-clamped root protoplasts were inhibited by NSCC blockers such as La³⁺ and Gd³⁺ (Demidchik et al., 2004). Therefore, it was proposed that plant iGLRs can form Ca²⁺-permeable NSCCs, are inhibited by animal iGluR antagonists, and might contribute to the shaping of plant Ca²⁺ signaling (McAinsh and Pittman, 2009). Studies using AtGLR3.3 mutant plants showed that intracellular Ca²⁺ rise and membrane depolarization induced by Glu in Arabidopsis hypocotyls and root cells are correlated with the presence of AtGLR3.3 (Qi et al., 2006; Stephens et al., 2008).However, most plant iGLRs, when expressed in heterologous systems, do not give rise to any current (e.g. in Xenopus spp. oocytes) or are toxic to host cells (e.g. in mammalian cells; Davenport, 2002). Recently, to examine whether AtGLR homologs possess functional ion channel domains, Tapken and Hollmann (2008) transplanted the pore loop together with two adjacent intracellular loops of 17 AtGLR subunits into two rat iGluR subunits and tested the resulting chimeric receptors for ion channel activity in the heterologous expression system Xenopus spp. oocyte. They showed that AtGLR1.1 and AtGLR1.4 have functional ion pore domains. The AtGLR1.1 pores are permeable to Na⁺, K⁺, and Ca²⁺ and are blocked by the nonspecific cation channel blocker La³⁺ (Tapken and Hollmann, 2008). Recent work has demonstrated that the expression of full-length AtGLR1.4 in oocytes gives rise to an amino acid-activated, nonselective, calcium-permeable channel that was found to be inhibited by the animal iGluR modulators 6,7-dinitroquinoxaline-2,3-dione and 6-cyano-7-nitroquinoxaline-2,3-dione (Tapken et al., 2013).The study of these channels has so far been restricted to those members that are located in the plasma membrane and were proved to be functional in the expression systems used. Instead, various localization prediction tools suggest that some of the plant GLRs might have chloroplast and mitochondrial targeting. In general, determining the subcellular localization of a protein is an important step toward understanding its function. We recently reported the localization of GLR3.4 to the inner chloroplast membrane (Teardo et al., 2011), which was also shown to harbor a 6,7-dinitroquinoxaline-2,3-dione-sensitive, calcium-permeable channel activity (Teardo et al., 2010). No other studies have addressed the eventual subcellular localization of other putative Glu receptors.In this work, we show that an isoform of GLR3.5 is efficiently targeted to the mitochondria. Functional expression of the channel in this organelle is indicated by the fact that its absence in knockout plants leads to a dramatically altered ultrastructure of mitochondria that impacts the plant physiology, ultimately leading to an anticipated senescence.     

Genetics of phototaxis in a model eukaryote,Dictyostelium discoideum

The life cycle of Dictyostelium discoideum offers a unique opportunity to study signal transduction in eukaryotic cells at both the unicellular and multicellular levels of organization. Adding to the already extensive knowledge of the unicellular stages, classical and molecular genetics have begun to unravel transduction of signals controlling morphogenesis and behaviour (phototaxis and thermotaxis) in the multicellular ‘slug’ stage of the life cycle. Distributed over all seven genetic linkage groups are probably about 20, but possibly as many as 55, genes of importance for slug behaviour. The encoded proteins appear from pharmacological studies and mutant phenotypes to govern transduction pathways involving the intracellular second messengers cyclic AMP, cyclic GMP, IP₃ and Ca²⁺. Pathways from the photo- and thermoreceptors converge first with each other and thence, at the level of the second messengers, with those from extracellular tip activation (cyclic AMP) and inhibition (Slug Turning Factor and/or ammonia and/or adenosine) signals that control slug movement and morphogenesis.     

Dipeptidyl-aminopeptidases and aminopeptidases in Dictyostelium discoideum

Extracts prepared from culminating cells of Dictyostelium discoideum have been found to contain dipeptidyl-aminopeptidases I (EC 3.4.14.1), II (EC 3.4.14.2), III (EC 3.4.14.4), arginine aminopeptidase (EC 3.4.11.6) and valine aminopeptidase. Dipeptidyl-aminopeptidase III was the most active of the dipeptidyl-aminopeptidases; its molecular weight was 158,000, with a pH optimum of 10.2 and gave a single peak of activity on gel-filtration or when fractionated by chromatofocusing. The specific activities of dipeptidyl-aminopeptidases I and III increased during development being highest during the culmination stage before decreasing during sorocarp formation; dipeptidyl-aminopeptidase II and arginine aminopeptidase decreased progressively throughout development. The presence of these dipeptidyl-aminopeptidases suggests the possibility that processing of peptides may be necessary during the development of Dictyostelium.     

Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.     

Expression of a spore-specific gene in Dictyostelium discoideum

The expression of a previously cloned Dictyostelium discoideum spore-specific gene (Julien et al., EMBO J. 1, 1089-1093 (1982)) was investigated in wild type and mutant strains. In vitro translation of this spore-specific mRNA gave a protein of a molecular weight consistent with the mRNA size. Expressed at a low level during vegetative growth development and in stalk cells, the accumulation of this mRNA reached high values only in spore cells.     

Evidence for a membrane-bound pyrophosphatase in Dictyostelium discoideum

Plasma membrane enriched fractions of Dictyostelium discoideum contain a Des-insensitive ATPase activity that can be fractionated by DEAE-Sephacel into a major vanadate-sensitive activity and a minor vanadate-insensitive activity. The vanadate-insensitive activity hydrolyzed pyrophosphate considerably more rapidly than ATP or any other substrate tested, and the enzyme was therefore designated a pyrophosphatase. The enzyme had no activity on AMP or p-nitrophenyl phosphate. The pyrophosphatase activity was maximal at alkaline pH values and stimulated by Mg2+ but not by Ca2+, properties of the enzyme that are very similar to those of the previously characterized pyrophosphatases of the plant tonoplast membrane. The pyrophosphatase activity of total membrane extracts changed very little during Dictyostelium differentiation.     

Evidence for a Sex Hormone in Dictyostelium discoideum

A dialyzable sex hormone is produced by one strain of Dictyostelium discoideum (NC-4) that induces macrocyst formation (sexual stage) in its opposite mating type (V-12).     

Trishanku, a novel regulator of cell-type stability and morphogenesis in Dictyostelium discoideum

We have identified a novel gene, trishanku (triA), by random insertional mutagenesis of Dictyostelium discoideum. TriA is a Broad complex Tramtrack bric-a-brac domain-containing protein that is expressed strongly during the late G2 phase of cell cycle and in presumptive spore (prespore (psp)) cells. Disrupting triA destabilizes cell fate and reduces aggregate size; the fruiting body has a thick stalk, a lowered spore: stalk ratio, a sub-terminal spore mass and small, rounded spores. These changes revert when the wild-type triA gene is re-expressed under a constitutive or a psp-specific promoter. By using short- and long-lived reporter proteins, we show that in triA(-) slugs the prestalk (pst)/psp proportion is normal, but that there is inappropriate transdifferentiation between the two cell types. During culmination, regardless of their current fate, all cells with a history of pst gene expression contribute to the stalk, which could account for the altered cell-type proportion in the mutant.     

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn(2+)-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn(2+) and Mg(2+) activated PdeE and PdeD maximally at 10 mm, whereas Mn(2+) activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 microm. PdeE is an allosteric enzyme, which is approximately 4-fold activated by cAMP, with half-maximal activation occurring at about 10 microm and an apparent K(m) of approximately 1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.     

Essential Role of the Dynamin Pleckstrin Homology Domain in Receptor-Mediated Endocytosis

Pleckstrin homology (PH) domains are found in numerous membrane-associated proteins and have been implicated in the mediation of protein-protein and protein-phospholipid interactions. Dynamin, a GTPase required for clathrin-dependent endocytosis, contains a PH domain which binds to phosphoinositides and participates in the interaction between dynamin and the βγ subunits of heterotrimeric G proteins. The PH domain is essential for expression of phosphoinositide-stimulated GTPase activity of dynamin in vitro, but its involvement in the endocytic process is unknown. We expressed a series of dynamin PH domain mutants in cultured cells and determined their effect on transferrin uptake by those cells. Endocytosis is blocked in cells expressing a PH domain deletion mutant and a point mutant that fails to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. In contrast, expression of a point mutant with unimpaired PI(4,5)P₂ interaction has no effect on transferrin uptake. These results demonstrate the significance of the PH domain for dynamin function and suggest that its role may be to mediate interactions between dynamin and phosphoinositides.     

Cell-cell adhesion and morphogenesis in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire EDTA-resistant cell-cell adhesion at the aggregation stage. The EDTA-resistant cell binding activity is associated with a cell surface glycoprotein of Mr 80,000 (gp80), which mediates cell-cell binding via homophilic interaction. Analysis of the structure of gp80 deduced from cDNA sequence reveals the presence of three internally homologous segments in the NH2-terminal domain, which also contains regions with homology to the neural cell adhesion molecule. Secondary structure predictions show an abundance of beta-structures and very few alpha-helices. This is confirmed by circular dichroism measurements. It is likely that the homologous segments are organized into globular structures, extended from the cell surface by a Pro-rich stalk domain. The cell binding activity of gp80 resides within the first globular repeat of the NH2-terminal domain and has been mapped to a 51 amino acid region between Val123 and Leu173. Synthetic oligopeptides corresponding to sequences within this region have been prepared and assayed for their ability to bind to cell surface gp80. Results lead to identification of the homophilic binding site to an octapeptide sequence within this region. Synthetic peptides containing this octapeptide sequence and univalent antibodies directed against this site block the formation of organized cell streams during aggregation. Although cell aggregates are eventually formed, most fail to undergo further development to give rise to slugs and fruiting bodies, indicating that cell-cell adhesion involving gp80 is an important step in normal morphogenesis.     

Characterization of a novel calmodulin from Dictyostelium discoideum

We have purified calmodulin from the eukaryotic microorganism Dictyostelium discoideum (Clarke, M., Bazari, W. L., and Kayman, S. C. (1980) J. Bacteriol. 141, 397-400) and have compared it to calmodulin purified from bovine brain. The two proteins behaved almost identically during fractionation on ion exchange and gel filtration columns and on isoelectric focusing gels. Dictyostelium calmodulin had one-third the specific activity of brain calmodulin in the Ca2+-dependent activation of brain cyclic nucleotide phosphodiesterase; this activation was inhibited for both proteins by 25 microM trifluoperazine. Dictyostelium calmodulin also activated erythrocyte (Ca2+ + Mg2+)-ATPase and interacted with the inhibitory subunit of skeletal muscle troponin. Competition radioimmune assays showed that Dictyostelium calmodulin could compete with brain calmodulin for antibodies to brain calmodulin. These similarities indicate a close relationship between Dictyostelium and brain calmodulin and suggest that the functional capabilities of the protein have been conserved even among evolutionarily distant species. However, substantial differences in primary structure were detected by amino acid analyses and peptide mapping. Most interesting is the lack of trimethyllysine in Dictyostelium calmodulin. This unusual amino acid, which is commonly found in calmodulins, is therefore not essential for interaction between calmodulin and the calmodulin-regulated proteins tested here.     

Characterization of a vacuolar proton ATPase in Dictyostelium discoideum

Of the total ATPase activity in homogenates of the ameba, Dictyostelium discoideum, approximately one-third was inhibited at pH 7 by 25 microM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Upon isopycnic sucrose density gradient centrifugation, the bulk of the NBD-CI-sensitive ATPase activity was recovered in a major membrane fraction with a broad peak at 1.16 g/ml, well-resolved from markers for plasma membranes, mitochondria, lysosomes and contractile vacuoles. The gradient peak had a specific activity of 0.5 mumol/min per mg protein. The activity was half-inhibited by 1 microM silicotungstate, 2 microM diisothiocyanatostilbene disulfonate (DIDS), 2.5 microM dicyclohexylcarbodiimide (DCCD), 4 microM NBD-CI and 20 microM N-ethylmaleimide (NEM) but was resistant to conventional inhibitors of mitochondrial and plasma membrane ATPase. That this ATPase activity constituted a proton pump was shown by the MgATP-dependent uptake and quenching of Acridine orange fluorescence by partially purified vacuoles. The Acridine orange uptake was specifically blocked by the aforementioned inhibitors. The generation of proton electrochemical gradients was suggested by the stimulation of enzyme activity by protonophores (fatty acids) and cation exchangers (nigericin). Uncoupling stimulated the ATPase activity as much as 20-fold, revealing an unusually high impermeability of the membranes to protons. ATPase activity was also stimulated by halide ions, apparently through a parallel conductance pathway. Under a variety of sensitive test conditions, the reverse enzyme reaction (i.e., incorporation of 32Pi into ATP) was not detected. We conclude that this major H+-ATPase serves to acidify the abundant prelysosomal vacuoles found in D. discoideum (Padh et al. (1989) J. Cell Biol. 108, 865-874). The finding of a vacuolar H+-ATPase in a protist suggests the ubiquity of this enzyme among the eukaryotic kingdoms.     

Pleiotropic roles of a ribosomal protein in Dictyostelium discoideum

The cell cycle phase at starvation influences post-starvation differentiation and morphogenesis in Dictyostelium discoideum. We found that when expressed in Saccharomyces cerevisiae, a D. discoideum cDNA that encodes the ribosomal protein S4 (DdS4) rescues mutations in the cell cycle genes cdc24, cdc42 and bem1. The products of these genes affect morphogenesis in yeast via a coordinated moulding of the cytoskeleton during bud site selection. D. discoideum cells that over- or under-expressed DdS4 did not show detectable changes in protein synthesis but displayed similar developmental aberrations whose intensity was graded with the extent of over- or under-expression. This suggested that DdS4 might influence morphogenesis via a stoichiometric effect--specifically, by taking part in a multimeric complex similar to the one involving Cdc24p, Cdc42p and Bem1p in yeast. In support of the hypothesis, the S. cerevisiae proteins Cdc24p, Cdc42p and Bem1p as well as their D. discoideum cognates could be co-precipitated with antibodies to DdS4. Computational analysis and mutational studies explained these findings: a C-terminal domain of DdS4 is the functional equivalent of an SH3 domain in the yeast scaffold protein Bem1p that is central to constructing the bud site selection complex. Thus in addition to being part of the ribosome, DdS4 has a second function, also as part of a multi-protein complex. We speculate that the existence of the second role can act as a safeguard against perturbations to ribosome function caused by spontaneous variations in DdS4 levels.     

CyrA, a matricellular protein that modulates cell motility in Dictyostelium discoideum

CyrA, an extracellular matrix (slime sheath), calmodulin (CaM)-binding protein in Dictyostelium discoideum, possesses four tandem EGF-like repeats in its C-terminus and is proteolytically cleaved during asexual development. A previous study reported the expression and localization of CyrA cleavage products CyrA-C45 and CyrA-C40. In this study, an N-terminal antibody was produced that detected the full-length 63kDa protein (CyrA-C63). Western blot analyses showed that the intracellular expression of CyrA-C63 peaked between 12 and 16h of development, consistent with the time that cells are developing into a motile, multicellular slug. CyrA immunolocalization and CyrA-GFP showed that the protein localized to the endoplasmic reticulum, particularly its perinuclear component. CyrA-C63 secretion began shortly after the onset of starvation peaking between 8 and 16h of development. A pharmacological analysis showed that CyrA-C63 secretion was dependent on intracellular Ca(2+) release and active CaM, PI3K, and PLA2. CyrA-C63 bound to CaM both intra- and extracellularly and both proteins were detected in the slime sheath deposited by migrating slugs. In keeping with its purported function, CyrA-GFP over-expression enhanced cAMP-mediated chemotaxis and CyrA-C45 was detected in vinculin B (VinB)-GFP immunoprecipitates, thus providing a link between the increase in chemotaxis and a specific cytoskeletal component. Finally, DdEGFL1-FITC was detected on the membranes of cells capped with concanavalin A suggesting that a receptor exists for this peptide sequence. Together with previous studies, the data presented here suggests that CyrA is a bona fide matricellular protein in D. discoideum.