Involvement of Sib proteins in the regulation of cellular adhesion in Dictyostelium discoideum

Molecular mechanisms ensuring cellular adhesion have been studied in detail in Dictyostelium amoebae, but little is known about the regulation of cellular adhesion in these cells. Here, we show that cellular adhesion is regulated in Dictyostelium, notably by the concentration of a cellular secreted factor accumulating in the medium. This constitutes a quorum-sensing mechanism allowing coordinated regulation of cellular adhesion in a Dictyostelium population. In order to understand the mechanism underlying this regulation, we analyzed the expression of recently identified Dictyostelium adhesion molecules (Sib proteins) that present features also found in mammalian integrins. sibA and sibC are both expressed in vegetative Dictyostelium cells, but the expression of sibC is repressed strongly in conditions where cellular adhesion decreases. Analysis of sibA and sibC mutant cells further suggests that variations in the expression levels of sibC account largely for changes in cellular adhesion in response to environmental cues.     

LARGE expression augments the glycosylation of glycoproteins in addition to α-dystroglycan conferring laminin binding

Mutations in genes encoding glycosyltransferases (and presumed glycosyltransferases) that affect glycosylation and extracellular matrix binding activity of α-dystroglycan (α-DG) cause congenital muscular dystrophies (CMDs) with central nervous system manifestations. Among the identified genes, LARGE is of particular interest because its overexpression rescues glycosylation defects of α-DG in mutations of not only LARGE but also other CMD-causing genes and restores laminin binding activity of α-DG. It is not known whether LARGE protein glycosylates other proteins in addition to α-DG. In this study, we overexpressed LARGE in DG-deficient cells and analyzed glycosylated proteins by Western blot analysis. Surprisingly, overexpression of LARGE in α-DG-deficient cells led to glycosylation dependent IIH6C4 and VIA4-1 immunoreactivity, despite the prevailing view that these antibodies only recognize glycosylated α-DG. Furthermore, the hyperglycosylated proteins in LARGE-overexpressing cells demonstrated the functional capacity to bind the extracellular matrix molecule laminin and promote laminin assembly at the cell surface, an effect that was blocked by IIH6C4 antibodies. These results indicate that overexpression of LARGE catalyzes the glycosylation of at least one other glycoprotein in addition to α-DG, and that this glycosylation(s) promotes laminin binding activity.     

Differential glycosylation of α-dystroglycan and proteins other than α-dystroglycan by like-glycosyltransferase

Genetic defects in like-glycosyltransferase (LARGE) cause congenital muscular dystrophy with central nervous system manifestations. The underlying molecular pathomechanism is the hypoglycosylation of α-dystroglycan (α-DG), which is evidenced by diminished immunoreactivity to IIH6C4 and VIA4-1, antibodies that recognize carbohydrate epitopes. Previous studies indicate that LARGE participates in the formation of a phosphoryl glycan branch on O-linked mannose or it modifies complex N- and mucin O-glycans. In this study, we overexpressed LARGE in neural stem cells deficient in protein O-mannosyltransferase 2 (POMT2), an enzyme required for O-mannosyl glycosylation. The results showed that overexpressing LARGE did not lead to hyperglycosylation of α-DG in POMT2 knockout (KO) cells but did generate IIH6C4 and VIA4-1 immunoreactivity and laminin-binding activity. Additionally, overexpressing LARGE in cells deficient in both POMT2 and α-DG generated laminin-binding IIH6C4 immunoreactivity. These results indicate that LARGE expression resulted in the glycosylation of proteins other than α-DG in the absence of O-mannosyl glycosylation. The IIH6C4 immunoreactivity generated in double-KO cells was largely removed by treatment either with peptide N-glycosidase F or with cold aqueous hydrofluoric acid, suggesting that LARGE expression caused phosphoryl glycosylation of N-glycans. However, the glycosylation of α-DG by LARGE is dependent on POMT2, indicating that LARGE expression only modifies O-linked mannosyl glycans of α-DG. Thus, LARGE expression mediates the phosphoryl glycosylation of not only O-mannosyl glycans including those on α-DG but also N-glycans on proteins other than α-DG.     

SadA,a novel adhesion receptor in Dictyostelium

Little is known about cell-substrate adhesion and how motile and adhesive forces work together in moving cells. The ability to rapidly screen a large number of insertional mutants prompted us to perform a genetic screen in Dictyostelium to isolate adhesion-deficient mutants. The resulting substrate adhesion-deficient (sad) mutants grew in plastic dishes without attaching to the substrate. The cells were often larger than their wild-type parents and displayed a rough surface with many apparent blebs. One of these mutants, sadA-, completely lacked substrate adhesion in growth medium. The sadA- mutant also showed slightly impaired cytokinesis, an aberrant F-actin organization, and a phagocytosis defect. Deletion of the sadA gene by homologous recombination recreated the original mutant phenotype. Expression of sadA-GFP in sadA-null cells restored the wild-type phenotype. In sadA-GFP-rescued mutant cells, sadA-GFP localized to the cell surface, appropriate for an adhesion molecule. SadA contains nine putative transmembrane domains and three conserved EGF-like repeats in a predicted extracellular domain. The EGF repeats are similar to corresponding regions in proteins known to be involved in adhesion, such as tenascins and integrins. Our data combined suggest that sadA is the first substrate adhesion receptor to be identified in Dictyostelium.     

Control of cellular physiology by TM9 proteins in yeast and Dictyostelium

TM9 proteins constitute a well defined family, characterized by the presence of a large variable extracellular domain and nine putative transmembrane domains. This family is highly conserved throughout evolution and comprises three members in Dictyostelium discoideum and Saccharomyces cerevisiae and four in humans and mice. In Dictyostelium, previous analysis demonstrated that TM9 proteins are implicated in cellular adhesion. In this study, we generated TM9 mutants in S. cerevisiae and analyzed their phenotype with particular attention to cellular adhesion. S. cerevisiae strains lacking any one of the three TM9 proteins were severely suppressed for adhesive growth and filamentous growth under conditions of nitrogen starvation. In these mutants, expression of the FLO11-lacZ reporter gene was strongly reduced, whereas expression of FRE(Ty1)-lacZ was not, suggesting that TM9 proteins are implicated at a late stage of nutrient-controlled signaling pathways. We also reexamined the phenotype of Dictyostelium TM9 mutant cells, focusing on nutrient-controlled cellular functions. Although the initiation of multicellular development and autophagy was unaltered in Dictyostelium TM9 mutants, nutrient-controlled secretion of lysosomal enzymes was dysregulated in these cells. These results suggest that in both yeast and amoebae, TM9 proteins participate in the control of specific cellular functions in response to changing nutrient conditions.     

Inhibition of cell-cell adhesion and morphogenesis of Dictyostelium by carnitine.

Carnitine (gamma-trimethylammonium beta-hydroxy-butyric acid) possesses the novel property of preventing cell aggregation elicited by clusterin or by fibrinogen (I.B. Fritz and K. Burdzy, J. Cell. Physiol., 140:18-28 [1989]). In investigations reported here, we show that carnitine also affects cell-cell adhesion in Dictyostelium discoideum, a cellular slime mold whose cells interact in specific and complex manners during discrete stages of development. Two types of cell adhesion systems sequentially appear on the surface of developing Dictyostelium cells, involving the surface glycoprotein gp24 which mediates EDTA-sensitive binding sites, and the surface glycoprotein gp80 which mediates the EDTA-resistant binding sites. Addition of increasing concentrations of D(+)-carnitine and L(-)-carnitine resulted in a progressive inhibition of both the EDTA-sensitive binding sites and the EDTA-resistant binding sites of Dictyostelium cells at different stages of development. In contrast, comparable or higher concentrations of choline, acetyl-beta-methylcholine, or deoxycarnitine had no detectable effects on cell aggregation. Concentrations of carnitine required for 50% inhibition of EDTA-resistant adhesion sites were found to be dependent upon levels of gp80 expressed by Dictyostelium, with greatest inhibition by carnitine of reassociation of cells containing the lowest levels of gp80. Removal of carnitine from cells by washing resulted in the rapid restoration of the ability of Dictyostelium to form aggregates and to resume normal development. We discuss possible mechanisms by which carnitine inhibits the aggregation of cells.     

Direct implication of surface mannosyl residues in cell adhesion of Dictyostelium discoideum

In the cell adhesion of aggregation-competent Dictyostelium cells, the requirement for the carbohydrate moiety of the glycoprotein appeared to be indirect in that it acts to protect the protein moiety from proteolytic degradation; however, the effect was limited to the tunicamycin (TM)-sensitive carbohydrate moiety (Hirano, T., et al. (1983) J. Biochem. 93, 1249-1257). In the present study, we showed that the EDTA-stable adhesion of aggregation-competent Dictyostelium cells was abolished by the treatment of intact cells with jack bean alpha-mannosidase, whereas neuraminidase, beta-galactosidase, beta-N-acetylhexosaminidase, or alpha-L-fucosidase had no effect. The EDTA-stable cohesiveness of TM-treated cells in the presence of leupeptin (TM/LP cells) was also abolished by the treatment of the cells with alpha-mannosidase. The effect of alpha-mannosidase was not prevented in the presence of LP. The N-glycoside-deficient contact site A (an adhesion-mediating glycoprotein) was obtained from TM/LP cells and was shown to have a molecular weight of 70,000. This protein (p 70) was shown to still have carbohydrates as detected by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and subsequent staining of the gel with periodic acid-silver stain. Moreover, p 70 reacted with anti-gp 68, which has a specificity against alpha-mannosyl residues of carbohydrate chains. However, p 70 treated with alpha-mannosidase showed decreased reactivity with anti-gp 68. The monovalent antibody fragment of anti-contact site A or anti-p 70 inhibited EDTA-stable cell adhesion of both control and TM/LP cells. These results indicated that TM-resistant mannosyl residues of contact site A are directly involved in EDTA-stable adhesion of aggregation-competent cells. This is the first report of the direct involvement of the carbohydrate moiety in cell adhesion of aggregation-competent Dictyostelium cells. A schematic model is presented of the role of the carbohydrate moiety in EDTA-stable cell adhesion, including the direct effect of carbohydrates.     

Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.     

Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca2+]i) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca2+]i. We observed small, brief, Ca2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd3+) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells.     

Overlapping functions of the two talin homologues in Dictyostelium

Talin is a cytoskeletal protein involved in constructing and regulating focal adhesions in animal cells. The cellular slime mold Dictyostelium discoideum has two talin homologues, talA and talB, and earlier studies have characterized the single knockout mutants. talA(-) cells show reduced adhesion to the substrates and slightly impaired cytokinesis leading to a high proportion of multinucleated cells in the vegetative stage, while the development is normal. In contrast, talB(-) cells are characterized by reduced motility in the developmental stage, and they are arrested at the tight-mound stage. Here, we created and analyzed a double mutant with a disruption of both talA and talB. Defects in adhesion to the substrates, cytokinesis, and development were more severe in cells with a disruption of both talA and talB. The talA(-) talB(-) cells failed to attach to the substrates in the vegetative stage, exhibited a higher proportion of multinucleated cells than talA(-) cells, and showed more-reduced motility during the development and an earlier developmental arrest than talB(-) cells at the loose-mound stage. Moreover, overexpression of either talA or talB compensated for the loss of the other talin, respectively. The analysis of talA(-) talB(-) cells also revealed that talin was required for the formation of paxillin-rich adhesion sites and that there was another adhesion mechanism which is independent of talin in the developmental stage. This is the first study demonstrating overlapping functions of two talin homologues, and our data further indicate the importance of talin.     

A novel consensus motif in fibronectin mediates dipeptidyl peptidase IV adhesion and metastasis

Lung endothelial dipeptidyl peptidase IV (DPPIV/CD26) is a vascular address for cancer cells decorated with cell-surface polymeric fibronectin (poly-FN). Here, we identified the DPPIV-binding sites in FN and examined the effect of binding site peptides on DPPIV/poly-FN adhesion and metastasis. Using proteolytic fragments and maltose-binding protein fusion proteins that together span full-length FN, we found DPPIV-binding sites in type III repeats 13, 14, and 15 (FNIII13, -14, and -15, respectively). DPPIV binding was mediated by the consensus motif T(I/L)TGLX(P/R)G(T/V)X and was confirmed by swapping this motif in FNIII13, -14, and -15 with the corresponding region in FNIII12, which did not bind DPPIV. DPPIV binding was lost in swapped FNIII13, -14, and -15 and gained in swapped FNIII12 (FNIII12(14)). Peptides containing the DPPIV-binding domain of FNIII14 blocked DPPIV/poly-FN adhesion and impeded pulmonary metastasis. This study adds to the classes of cell-surface adhesion receptors for FN and will help in the further characterization of the functional implications of the DPPIV/poly-FN adhesion in metastasis and possibly in cell-mediated immunity involving DPPIV-expressing lymphocytes.     

Impaired sexual behavior in male mice deficient for the beta1-3 N-acetylglucosaminyltransferase-I gene

The beta1-3 N-acetylglucosaminyltransferase-1 (B3gnt1) gene encodes a poly-N-acetyllactosamine synthase which can initiate and extend poly-N-acetyllactosamine chains [Gal(beta1-4)GlcNAc (beta1-3)(n)]. Previous investigations with heterozygous and homozygous null mice for this gene have revealed the importance of poly-N-acetyllactosamine chains for the formation of olfactory axon connections with the olfactory bulb and the migration of gonadotropin releasing hormone neurons to the hypothalamus. The possible long-term effects of these developmental defects, however, has not yet been studied. Here we have examined a reproductive phenotype observed in B3gnt1-null mice. Whereas the B3gnt1 null females were fertile, the B3gnt1 null males were not able to sire litters at the expected rate when mated to either wildtype or B3gnt1-null females. We assessed male sexual behavior as well as male reproduction parameters such as testes size, spermatogenesis, sperm number, morphology, and the development of early embryos in order to identify the source of a reduced rate of reproduction. Our findings show that the B3gnt1 null male reproductive organs were functional and could not account for the lower rate at which they produced offspring with wildtype conspecifics. Hence, we propose that the phenotype observed resulted from an impaired sexual response to female mating partners.     

Screening of genes involved in cell migration in Dictyostelium

A single cell of wild-type Dictyostelium discoideum forms a visible colony on a plastic dish in several days, but due to enhanced cell migration, amiB-null mutant cells scatter over a large area and do not form noticeable colonies. Here, with an aim to identify genes involved in cell migration, we isolated suppresser mutants of amiB-null mutants that restore the ability to form colonies. From REMI (restriction enzyme-mediated integration)-mutagenized pool of double-mutants, we identified 18 responsible genes from them. These genes can be categorized into several biological processes. One cell line, Sab16 (Suppressor of amiB) was chosen for further analysis, which had a disrupted phospholipase D pldB gene. To confirm the role of pldB gene in cell migration, we knocked out the pldB gene and over-expressed gfp-pldB in wild-type cells. GFP-PLDB localized to plasma membrane and on vesicles, and in migrating cells, at the protruding regions of pseudopodia. Migration speed of vegetative pldB-null cells was reduced to 73% of that of the wild-type. These results suggest that PLDB plays an important role in migration in Dictyostelium cells, and that our screening system is useful for the identification of genes involved in cell migration.     

A precise group size in Dictyostelium is generated by a cell-counting factor modulating cell-cell adhesion

A remarkable aspect of Dictyostelium development is that cells form evenly sized groups of approximately 2 x 10(4) cells. A secreted 450 kDa protein complex called counting factor (CF) regulates the number of cells per group. We find that CF regulates group size by repressing cell-cell adhesion. In both experiments and computer simulations, high levels of CF (and thus low adhesion) result in aggregation streams breaking up into small groups, while no CF (and thus high adhesion) results in no stream breakup and large groups. These results suggest that in Dictyostelium and possibly other systems a secreted factor regulating cell-cell adhesion can regulate the size of a group of cells.     

Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced actin polymerization

We have investigated the role of the Arp2/3 complex in Dictyostelium cell chemotaxis towards cyclic-AMP and in the actin polymerization that is triggered by this chemoattractant. We confirm that the Arp2/3 complex is recruited to the cell perimeter, or into a pseudopod, after cyclic-AMP stimulation and that this is coincident with actin polymerization. This recruitment is inhibited when actin polymerization is blocked using latrunculin suggesting that the complex binds to pre-existing actin filaments, rather than to a membrane associated signaling complex. We show genetically that an intact Arp2/3 complex is essential in Dictyostelium and have produced partially active mutants in two of its subunits. In these mutants both phases of actin polymerization in response to cyclic-AMP are greatly reduced. One mutant projects pseudopodia more slowly than wild type and has impaired chemotaxis, together with slower movement. The second mutant chemotaxes poorly due to an adhesion defect, suggesting that the Arp2/3 complex plays a crucial part in adhering cells to the substratum as they move. We conclude that the Arp2/3 complex largely mediates the actin polymerization response to chemotactic stimulation and contributes to cell motility, pseudopod extension and adhesion in Dictyostelium chemotaxis.