The position of the microtubule-organizing center relative to the nucleus is independent of the direction of cell migration in Dictyostelium discoideum

Dictyostelium amoebae can migrate in several different modes. We tested for correlations of the direction of cell locomotion with the relative positions of the nucleus and microtubule-organizing center (MTOC). Five cases were analyzed on electron micrographs with a microcomputer. Each mode of movement showed characteristic locations of the MTOC relative to the nucleus; however, they differed in the various cases. In randomly migrating interphase amoebae, the number of cells with the MTOC located behind the nucleus was twice as great as those with the MTOC located ahead of the nucleus. During chemotactic migration toward folic acid, cells with the MTOC behind the nucleus were more numerous, with a concomitant reduction of anterior MTOCs. When amoebae aggregated on agar plates, a posterior location of the MTOC was most strikingly favored, whereas in cells aggregating under submerged conditions, the MTOC was indifferently anterior or posterior to the nucleus. (It may be significant that EDTA-resistant cell-cell adhesion was fully expressed in the former cells, but weaker in the latter.) Finally, in the case of chemotactically migrating cells from dissociated pseudoplasmodia, which adhere by means of other molecules, the MTOC was consistently ahead of the nucleus. Thus the MTOC shows no necessary preferential position anterior or posterior to the nucleus; its position, rather, correlates with the type of migration and perhaps with the nature of cell-cell adhesion.     

Mechanism of centrosome positioning during the wound response in BSC-1 cells

Locomoting cells are characterized by a pronounced external and internal anterior-posterior polarity. One of the events associated with cell polarization at the onset of locomotion is a shift of the centrosome, or MTOC, ahead of the nucleus. This position is believed to be of strategic importance for directional cell movement and cell polarity. We have used BSC-1 cells at the edge of an in vitro wound to clarify the causal relationship between MTOC position and the initiation of cell polarization. We find that pronounced cell polarization (the extension of a lamellipod) can take place in the absence of MTOC repositioning or microtubules. Conversely, MTOCs will reposition even after lamellar extension and cell polarization have occurred. Repositioning requires microtubules that extend to the cell periphery and is independent of selective detyrosination of microtubules extending towards the cell front. Significantly, MTOCs maintain, or at least attempt to maintain, a position at the cell's centroid. This is most clearly demonstrated in wounded monolayers of enucleated cells where the MTOC closely follows the centroid position. We suggest that the primary response to the would is the biased extension of a lamellipod, which can occur in the absence of microtubules and MTOC repositioning. Lamellipod extension leads to a shift of the cell's centroid towards the wound. The MTOC, in an attempt to maintain a position near the cell center, will follow. This will automatically put the MTOC ahead of the nucleus in the vast majority of cells. The nucleus as a reference for MTOC position may not be as meaningful as previously thought.     

Wild-Type Strains ofDictyostelium discoideumCan Be Transformed Using a Novel Selection Cassette Driven by the Promoter of the Ribosomal V18 Gene

Almost all methods for transformation of the social amebaDictyostelium discoideumrely on axenic growth, that is, growth in a synthetic medium, for at least part of the procedure. Axenic growth requires several mutations. Here we describe a procedure that can be used to transform wild-type strains which are able to grow only on the natural food source, bacteria. The method relies on a new selection cassette driven by the V18 promoter, a promoter that we show is substantially more active during growth on bacteria than the actin-6 promoter, which is widely used for axenic transformation. The procedure gives transformation frequencies of about 10⁻⁵with both strains Ax2 (capable of axenic growth) and NC4 (capable of growth only on bacteria). Using this vector, we have obtained NC4 strains carrying several β-galactosidase reporter cassettes. Our vector can also be used in axenic transformations.     

Mota1552, a Mutation of Dictyostelium Discoideum Having Pleiotropic Effects on Motility and Discoidin I Regulation

The Dictyostelium discoideum mutant MC2 exhibits temperature-sensitive growth, temperature-sensitive motility, and temperature induction of discoidin I synthesis. These three phenotypes of MC2 were not separated in the genetic experiments reported here. They were therefore assigned to the mutation motA1552, which was mapped to linkage group II by segregation analysis and by analysis of mitotic recombinant diploids. In one motA1552 strain, loss of motility preceded accumulation of discoidin I by 3 hr, indicating that discoidin I is not involved in generation of the motility defect. Expression of motA1552 phenotypes varied both among strains carrying the mutation, and among different clones of a particular strain. MC2 and its derivatives displayed elevated levels of recombination between whiA and acrA on linkage group II, and yielded highly unstable mutations at the acrA locus. Accumulation of large amounts of discoidin I during axenic growth of strain AX3 was found to depend on the presence of a second linkage group II mutation, daxA1551. This mutation was already present in the strain mutagenized to isolate motA1552, complicating explication of motA1552 action.     

Characterization of temperature-sensitive strains of Neospora caninum in mice

Temperature-sensitive (ts) strains of the Neospora caninum tachyzoites were selected by chemical mutagenesis and selection for growth at 32 C. Three ts strains and the parental, N. caninum wild-type strain, NC-1, were examined in the present study for their ability to cause disease in inbred BALB/c mice, outbred ICR mice, and chemically immunosuppressed ICR mice. In BALB/c mice, all 3 strains failed to induce clinical disease, whereas infection with the NC-1 strain caused central nervous system disease and death in some mice. No disease was observed in ICR mice inoculated with the 3 ts strains or the NC-1 strain. All immunosuppressed ICR mice inoculated with the NC-1 strain died, whereas no immunosuppressed mice inoculated with the NCts-4 strain and only 1 of 5 mice inoculated with the NCts-8 and NCts-12 strains died. The NCts-4 and NCts-12 strains reverted to a wild-type phenotype when grown at 37 C. Vaccination of BALB/c mice with live, but not frozen NCts-8 strain tachyzoites induced significant (P < 0.05) protection following NC-1 strain challenge.     

Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture

1. The DNA, RNA, protein and carbohydrate contents of myxamoebae of Dictyostelium discoideum strain Ax-2 were measured after growth on bacteria or in various axenic media. 2. Myxamoebae grown in the different axenic media have similar DNA, RNA and protein contents, but there are marked differences in the contents of glycogen and free sugars. The DNA and protein contents of myxamoebae grown on bacteria are different from those in myxamoebae grown axenically. 3. Approximately half the DNA found in myxamoebae grown on bacteria is of bacterial rather than of slime-mould origin. 4. The specific activities of some enzymes (including UDP-glucose pyrophosphorylase) are higher in myxamoebae grown axenically than in myxamoebae grown on bacteria. Nevertheless the characteristic increase in the specific activity of UDP-glucose pyrophosphorylase occurring during differentiation of cells of the wild-type strain NC-4 is also found in cells grown axenically. 5. The rate of amino acid oxidation during axenic growth of the myxamoebae is decreased when the cells are supplied with glucose.     

Relationship between axenic growth of Dictyostelium discoideum strains and their track morphology on substrates coated with gold particles

Amoebae of Dictyostelium discoideum produce tracks with two distinct morphologies on gold-coated coverslips. The wild-type strain and other strains that feed only by phagocytosis produced indistinct, fuzzy tracks, whereas mutants capable of axenic growth produced clear, sharp tracks. The sharp track morphology was found to be a recessive phenotype that segregates with axenicity and probably requires a previously unidentified axenic mutation. Axenic and nonaxenic strains also differed in their ability to pinocytose. When the two types of cells were shifted from bacterial growth plates to nutrient media, within 24 h the axenic strain established a rapid rate of pinocytosis, approximately 100-fold higher than the low rate detectable for the nonaxenic strain. However, track formation did not appear to be directly related to endocytosis. Electron microscopic examination of cells during track formation showed that both axenic and nonaxenic strains accumulated gold particles on their surfaces, but neither strain internalized the gold to any significant degree. Observation of living cells revealed that axenic strains collected all particles that they contacted, whereas wild-type strains left many particles undisturbed. The size of the gold particle clusters discarded by the cells also contributed to track morphology.     

Distribution of microtubule organizing centers in migrating sheets of endothelial cells

This study was designed to investigate the relationship between the position of the microtubule organizing center (MTOC) and the direction of migration of a sheet of endothelial cells (EC). Using immunofluorescence and phase microscopy the MTOC's of migrating EC were visualized as the cells moved into an in vitro experimental wound produced by mechanical denudation of part of a confluent monolayer culture. Although the MTOC's in nonmigrating EC were randomly positioned in relation to the nucleus, in migrating cells the position of the MTOC's changed so that 80% of the cells had the MTOC positioned in front of the nucleus toward the direction of movement of the endothelial sheet. This repositioning of the MTOC occurred within the first 4 h after wounding and was associated with the beginning of migration of EC's into the wounded area as seen by time-lapse cinemicrophotography. These studies focus attention on the MTOC as a cytoskeletal structure that may play a role in determining the direction of cell movement.     

Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA^Val(GUU) and a tRNA^Val(GUA) gene. The method used involved a combination of classitics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA^Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F,), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VIII (A, H). Six tRNA^Val(GUA) genes from the NC4 isolate were mapped; one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).     

The Use of Restriction Fragment Length Polymorphisms and DNA Duplications to Study the Organization of the Actin Multigene Family in DICTYOSTELIUM DISCOIDEUM

The techniques of restriction fragment length polymorphism analysis and examination of gene copy number in duplication-bearing Dictyostelium discoideum strains have been used to map four actin genes of the wild-type strain NC4 to specific linkage groups. In part, this was accomplished by identification of restriction fragments corresponding to particular cloned actin genes using gene-specific probes from unique sequence 5' and 3' untranslated regions. Cloned gene Actin 8 (designation act-8) maps to linkage group I; Actins 12 (act-12) and M6 (actM6) to linkage group II. An uncloned gene (act-100) also maps to linkage group II in the same region as actM6, as defined by a chromosomal duplication. From analysis of other wild isolates of D. discoideum, it was determined that in these isolates at least two actin genes map to linkage group I and at least four map to linkage group II. These results demonstrate the utility of molecular techniques in genetic analysis of Dictyostelium, particularly for developmentally regulated genes that have been cloned but that have no identified mutant phenotypes.     

Nuclear Behavior in Artificially Induced Multi-Nuclear Cells of Dictyostelium discoideum

The culture medium of the strain CK-8 of the cellular slime mold Polysphondylium pallidum contains a cell-fusion induction factor. Cells of the two opposite mating type strains NC-4 and HM1 of Dictyostelium discoideum were treated to induce cell fusion with the diluted fraction of CK-8 cultures, F2, which contains the factor and consequently numerous multinuclear cells were produced. NC-4 and HM1 usually fuse in the sexual cycle and form large multinuclear cells, called giant cells, which develop into macrocysts. These cells are very similar in morphology to the multinuclear cells produced following F2 treatment, however, the latter cells did not develop into macrocysts. In the sexually formed multinuclear cells, only two haploid nuclei fused to form a diploid nucleus and all others degenerate as previously reported. However, in the artificially produced multinuclear cells, no nuclear-fusion and degeneration took place. They stayed as heterokaryons and seem to lyse within 20 h incubation.     

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.     

Dictyostelium MTOC: Structure and linkage to the nucleus

Summary The microtubule organizing center (MTOC) was isolated fromDictyostelium discoideum to investigate the fine structure of the components as the first step in clarifying its molecular organization and function. The isolation protocol was designed to preserve microtubules bound to the MTOC by using indirect immunofluorescence employing anti--tubulin. After cell lysis with Triton X-100, the MTOCs were isolated in association with the nucleus by centrifugation in a microtubule-stabilizing buffer. The MTOC was found to be bound to the nucleus via an electron-dense fibrous structure, and this linkage could not be destroyed by KI, KCl, or sonication. We named this complex composed of microtubules, MTOC, and the anchor the MTOC-complex. Negative staining of the isolated MTOC-complex revealed that distinct vesicles decorated with 11-nm tacks were associated with microtubules radiating from the MTOC. Fine filaments, 4–5 nm wide, were also present close to the MTOC, aligned parallel to the microtubules. The three-dimensional profile of the central core of the MTOC, examined by transmission electron microscopy of serial thin sections of the isolated MTOC fraction supplemented by a microcomputer analysis, was concluded to be a matchbox-like cuboid (180 × 210 × 370 nm) of 15 layers.We propose that theDictyostelium MTOC is the structural domain of a more complicated unit composed of 1. MTOC, 2. microtubules, and 3. a firm fibrous linkage connecting the MTOC to the nucleus, with the MTOC core being a multilayered cuboid, associated with nodules and surrounded by amorphous electron-dense material including peculiar vesicles with 11 nm-tacks. The possible functions of these domains are discussed.     

Investigation of the het genes that control heterokaryon incompatibility between members of heterokaryon-compatibility (h-c) groups A and G1 of Aspergillus nidulans

A chromosome assay method was used to determine the heterokaryon compatibility relationships between strains belonging to heterokaryon-compatibility (h-c) groups A and G1 of Aspergillus nidulans. A hybrid strain (RD15) was isolated following protoplast fusion of strains 65-5 (h-cA) and 7-141 (h-cG1). The morphology of RD15 was severely abnormal compared to diploid strains of A. nidulans produced from heterokaryon-compatible haploid parents. Inocula of RD15 were induced to haploidize on medium containing Benlate and a parasexual progeny sample of 291 haploid segregants was obtained. The progeny strains were genotyped for standard markers. Allelic ratios and pairwise marker segregations were determined. Pairs of progeny strains that carried different alleles for the standard markers on each linkage group in turn were tested for compatibility. Strain pairs that possessed different alleles for the markers on linkage groups II, III, V, VI and VII were incompatible indicating the presence of heterokaryon-incompatible (het) genes on these linkage groups. Backcrosses to an h-cGl strain showed that two het genes were located on linkage group III and confirmed a total of six het gene differences between the h-cA and h-cGl strains.     

Genomic organization of the yeast Yarrowia lipolytica

We produced electrophoretic karyotypes of the reference strain E150 and of seven other isolates from different geographical origins to study the genomic organization of the dimorphic yeast Yarrowia lipolytica. These karyotypes differed in the number and size of the chromosomal bands. The karyotype of the reference stain E150 consisted of five bands of between 2.6 and 4.9 Mb in size. This strain contained at least five rDNA clusters, from 190 to 620 kb in size, which were scattered over most of the chromosomes. The assignment of 43 markers, including rRNA genes and three centromeres, to the E150 bands defined five linkage groups. Hybridization to the karyotypes of other isolates with pools of markers of each linkage group showed that linkage groups I, II, IV and V were conserved in the strains tested whereas group III was not and was split between at least two chromosomes in most strains. Use of a meganuclease I-SceI site targeted to one locus of E150 linkage group III showed that two chromosomes actually comigrated in band III of this strain. Our results are compatible with six chromosomes defining the haploid complement of strains of Y. lipolytica and that, despite an unprecedented chromosome length polymorphism, the overall structure of the genome is conserved in different isolates. Received: 27 March 1997; in revised form: 8 July 1997 / Accepted: 9 July 1997     

Localization of the ribosomal genes in Caenorhabditis elegans chromosomes by in situ hybridization using biotin-labeled probes

The site of the ribosomal gene cluster on embryonic metaphase chromosomes of Caenorhabditis elegans has been mapped by in situ hybridization using probe DNAs that have been nick-translated to incorporate biotin-labeled UTP. The hybridized probe DNA was detected by a double-layer fluorescent antibody technique. Since chromosomes from wild-type C. elegans embryos are indistinguishable, in situ hybridization was carried out with chromosomes from C. elegans strains carrying cytologically distinct translocation or duplication chromosomes in order to identify the right end of linkage group I as the site of the ribosomal genes. Chromosomes carrying a lethal mutation, let-209 I displayed smaller hybridization signals than wild-type, suggesting that these chromosomes carried a partial deficiency of the ribosomal gene cluster. A duplication of the ribosomal genes, eDp20(I;II) rescued let-209 homozygotes. Chromosomes carrying the alterations in the ribosomal genes were combined with mnT12(IV;X) to facilitate the mapping of genes in C. elegans by in situ hybridization. Linkage groups I and II are then labeled by the distinctive hybridization signals from the ribosomal probes, linkage groups IV and X are together distinguishable morphologically and linkage group V is labeled by hybridization to a 5S gene probe.     

Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum

Background

Duplications of stretches of the genome are an important source of individual genetic variation, but their unrecognized presence in laboratory organisms would be a confounding variable for genetic analysis.

Results

We report here that duplications of 15 kb or more are common in the genome of the social amoeba Dictyostelium discoideum. Most stocks of the axenic 'workhorse' strains Ax2 and Ax3/4 obtained from different laboratories can be expected to carry different duplications. The auxotrophic strains DH1 and JH10 also bear previously unreported duplications. Strain Ax3/4 is known to carry a large duplication on chromosome 2 and this structure shows evidence of continuing instability; we find a further variable duplication on chromosome 5. These duplications are lacking in Ax2, which has instead a small duplication on chromosome 1. Stocks of the type isolate NC4 are similarly variable, though we have identified some approximating the assumed ancestral genotype. More recent wild-type isolates are almost without large duplications, but we can identify small deletions or regions of high divergence, possibly reflecting responses to local selective pressures. Duplications are scattered through most of the genome, and can be stable enough to reconstruct genealogies spanning decades of the history of the NC4 lineage. The expression level of many duplicated genes is increased with dosage, but for others it appears that some form of dosage compensation occurs.

Conclusion

The genetic variation described here must underlie some of the phenotypic variation observed between strains from different laboratories. We suggest courses of action to alleviate the problem.     

Transformation of type polysaccharide antigen synthesis and hemolysin synthesis in streptococci

Transformation of the ability to synthesize type polysaccharide antigen and beta-hemolysin has been obtained in group F streptococci. Colonies possessing cells transformed to antigen synthesis were detected on the agar surface with fluorescein-labeled anti-type serum. This selection method, in contrast to those with antibiotics, allowed both transformed and nontransformed cells to grow, resulting in sectored colonies. These colonies could be subcultured to further establish the synthesis of antigen. Group F, group A, and group-like z deoxyribonucleic acid (DNA) labeled with type II antigen and hemolysin, and streptomycin resistance transferred each marker to a group F strain lacking a type antigen. DNA from group F and z3 strains labeled with type III antigen, and streptomycin resistance transferred both markers to group F and z3 strains lacking type antigen. A second F strain without type antigen was not transformed with any of these markers. A group H strain was transformed to streptomycin resistance only by the same types of DNA. Transformation to type II antigen synthesis always resulted in the formation of beta-hemolysin. All strains isolated from natural sources contained both markers. A mutant, obtained by nitrosoguanidine treatment of an FII(sr) strain, did not synthesize either the hemolysin or the antigen. This mutant still possessed the group antigen and streptomycin resistance. A close linkage of type II antigen and beta-hemolysin is indicated. The fluorescent-antibody staining of cells containing both group and type antigens showed a more intense ultraviolet adsorption for type than group antigen. A surface location (microcapsular) for the type antigen appeared likely. These results are of interest for studies on antigen biosynthesis, genetics, and classification of the streptococci.     

Genetics of aggregation pattern mutations in the cellular slime mould Dictyostelium discoideum.

A class of aggregation pattern mutants called 'streamers' have been isolated from Dictyostelium discoideum and analysed genetically. The streamer phenotype is the formation of very large streams of centripetally moving amoebae which are collected from abnormally large territories during the aggregation phase of this organism. Such mutants do not show the pleiotropic developmental defects seen with most other classes of aggregation mutants and after the abnormal aggregation phase they develop into normally differentiated stalk cells and spores. Twenty-four haploid streamers were isolated and assigned to seven complementation groups, stmA to stmG, after selecting diploids formed between pairs of the mutants. The complementation loci were assigned to the following linkage groups using parasexual genetic techniques: stmA and stmF, linkage group VII; stmB, stmD and stmG, linkage group II; stmC and stmE, linkage group III. Use was made of a new temperature sensitive for growth marker, tsgK21, which was assigned to linkage group VII. The total number of complementation groups giving the streamer phenotype is estimated from statistical calculation, based on the frequency of allelism, to be between seven and nine.     

Filopodelike projections induced with dimethyl sulfoxide and their relevance to cellular polarity in Dictyostelium

When 5% dimethyl sulfoxide (DMSO) was applied to Dictyostelium cells, the cells rounded up in shape and cytoplasmic streaming ceased. The cells resumed both cytoplasmic streaming and locomotion in 20 min. SDS PAGE of isolated plasma membrane fractions showed that actin and myosin apparently became dissociated from the plasma membrane by the action of DMSO. Scanning electron microscopy revealed that many filopodelike projections formed on the surface of cells treated with 5% DMSO for 5 min. Interestingly, the projections were formed on a restricted portion of the cell surface. The phagokinetic track technique of Albrecht- Buehler (1977, Cell, 11: 395-404) showed that the projection region corresponded to the anterior part of a migrating cell. The possible relationship between the DMSO-induced projection region on the cell surface and intracellular organization of cell organelles was investigated using serial thin sections. The DMSO-induced projections contained arrays of microfilaments; and the microtubule organizing center (MTOC), nucleus, and vesicular structure were usually located in this order from the anterior end of the cell. The indirect immunofluorescent study using monoclonal anti-alpha-tubulin antibody was performed with a new fixation technique, which greatly improved the phase as well as immunofluorescent microscopy. It was verified that the intracellular positioning of the MTOC and nucleus had significant correlation with the cell polarity. The results show that DMSO is a powerful tool with which to manipulate the cellular microfilaments and to make visible the differentiation in the cortex layer, which apparently is relevant to the intracellular positioning of cell organelles and cell polarity.