Relationships between cell-cell interactions,cAMP, and gene expression in a developmental mutant ofDictyostelium discoideum

Previous work has led us to propose that close cell-cell associations duringD. discoideum development serve as a signal to deactivate expression of discoidin I mRNA, and that intracellular cAMP serves as a mediator of this regulatory pathway. This model is based in part on the failure of a morphogenetic mutant, EB-21, to deactivate discoidin I expression under conditions where these cells fail to acquire cell-cell cohesiveness and hence remain as single cells, unlike the wild type strain which forms multicellular aggregates. Here we show that the failure of EB-21 to express specific cohesiveness depends on developmental conditions, and that under conditions where close cell-cell associations are allowed to form, discoidin I mRNA expression is deactivated normally. Furthermore, in both wild type and EB-21 there is a close correlation between formation close cell-cell associations and elevation of intracellular cAMP under different developmental conditions. Additional analyses of the biological behavior of EB-21 indicate that it acquires a normal cAMP chemotactic signal-response system, and that the morphogenetic defect cannot be corrected by co-development with wild type cells. The results are discussed in terms of possible relationships between cell-cell interactions, cAMP metabolism, and developmental gene expression in this organism.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).     

Expression of starvation-induced genes in zygotes ofDictyostelium discoideum

Two cDNA fragments induced in developing zygotes ofDictyostelium discoideum were isolated by mRNA differential display. the relevant genes were also found to be expressed during asexual development, suggesting that sexual and asexual development share common molecular mechanisms inD. discoideum.     

Measurement of the motive force of the migrating slug ofDictyostelium discoideum by a centrifuge method

Summary The motive force of the migrating slug of the cellular slime mouldDictyostelium discoideum was measured by the use of centrifugal force. Changes in shape of the slugs due to the use of centrifugal force were prevented by letting them migrate in an agar capillary. The motive force thus obtained was proportional to the slug volume, the value per unit volume being 5.8×10⁶ dyne/cm³ (58 N/cm³). This is in good agreement with the value measured by the use of hydrostatic pressure.     

Formation and organization of the spore coat ofDictyostelium discoideum

Macromolecular components of the spore coat ofDictyostelium discoideum have been localized by gold-labeled affinity cytochemistry. The outer electron-dense layer is the residence of three prominent glycoproteins that express a fucose-dependent epitope, whereas the inner electron-dense layer includes SP85 and the galactose/N-acetylgalactosamine-containing polysaccharide (GPS). The cellulosic layers are interposed between them. The outer-layer glycoproteins and the GPS also can be found in the interspore fluid, which is usually lost during collection of the spores. Assembly of the spore coat, examined over time, showed that all components, except for the cellulose, are found in an internal secretory vesicle population. All components are found in each vesicle but are not uniformly intermixed within them. Cellulose does not appear until after the outer electron-dense layer of the spore coat has been organized following secretion. The GPS is excluded from the outer dense layer and largely from the cellulosic layer, being more concentrated in the inner layer. SP85 remains localized in the inner dense layer near the cell surface with a circumferentially focal distribution. The distinct distributions of these macromolecular species in the mature spore coat are foreshadowed by their mosaic distribution in the prespore vesicles from which they originate.     

Analytical studies on migrating movement of the pseudo-plasmodium ofDictyostelium discoideum

Summary Migrating movement of a pseudoplasmodium (slug) of the cellular slime mouldDictyostelium discoideum was analyzed using a time-lapse video tape recorder. Since slugs usually migrated with repeated interruptions of advance, migrating velocities were measured only within a period of forward movement. On the basis of some known facts and assumptions, a dynamical model for slug movement was formulated, which consists of motive force generated by slug cells against their intrinsic resistance and resistance of slime sheath at the tip. The migrating velocity of a slug depended neither on its width nor its volume, but solely on its length. Under any experimental conditions tested, a linear relationship always held between reciprocals of the two variables. The results were in good agreement with predictions of the model. Quantitative analyses of experimental results by the use of the model lead to the conclusions that a decrease in velocity at a low temperature is due to an increase in resistance of slime sheath at the tip, but that a decrease in velocity during prolonged migration is due to a decrease in motive force of constituent cells. An anterior isolate dissected from a slug migrated at a velocity greater than that of an intact slug of the same length. This was interpreted by the model to be due to the fact that the anterior cells have greater motive forces and intrinsic resistances than the posterior cells. The heterogeneous distributions of the two variables in the cell mass is discussed in reference to the mechanism of sorting out of cells.     

The characterization and secretion pattern of the lysosomal trehalases ofDictyostelium discoideum

The enzyme trehalase II ofDictyostelium discoideum is efficiently secreted into the matrix of sori along with seven known lysosomal enzymes. The vegetative form of the enzyme, trehalase I, is particulate but the enzyme is secreted prior to cell aggregation or when cells are starved in phosphate buffer under standard secretion conditions. The secreted enzyme possesses properties common to lysosomal enzymes. Polyclonal and monoclonal antibodies raised against purified lysosomalN-acetylglucosaminidase precipitate the enzyme. The enzyme is released efficiently and about 62% of the initial cellular enzyme becomes extracellular. The secretion of trehalase is slightly sensitive to cycloheximide and completely blocked by sodium azide. Secretion is enhanced in the presence of disaccharides such as sucrose, lactose, and trehalose. Electrophoretograms of intracellular and secreted enzyme reveal no major processing of the enzyme during secretion. The pI of the trehalases has been estimated to be less than 2.5.     

Activation ofDictyostelium discoideum spores with guanidine and methyl derivatives of urea

A majority ofDictyostelium discoideum spores were activated with guanidine hydrochloride and tetramethylurea treatments. Dimethylurea could be utilized over a wide range of concentrations to activate spores. The minimal concentration was 2 M dimethylurea employed for 45–60 min, and the maximal concentration was 5 M dimethylurea employed for 20–30 min. Moderate overstimulation with dimethylurea resulted in an increase in the postactivation lag time, while severe overstimulation caused lysis and death of the spores. Partial spore deplasmolysis was a requirement for activation with dimethylurea at 23,5°C; deplasmolysis and activation did not occur at 0°C. The time required to produce an LD₅₀ was twice the time required for optimal activation when spores were treated with high concentrations of urea derivatives. A correlation was found for the hydrophobicity of the urea family of compounds and the molar concentration required for maximal activation with a 30-min treatment (2 M tetramethylurea, 5 M dimethylurea, and 8 M urea).     

Cytochemical localization of alkaline phosphatase in the cell membrane ofDictyostelium discoideum amebae

Histochemistry and Cell Biology - We demonstrated that alkaline phosphatase was localized on the cell membrane ofDictyostelium discoideum amebae and on isolated plasma membranes. The enzyme...     

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance

A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wild- type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models. ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.     

Responses of Dictyostelium discoideum to Multiple Environmental Stimuli

The movement responses of the cellular slime mold Dictyostelium discoideum to multiple stimuli were investigated. The responses were found to differ depending on the developmental stage of the organism. A novel response, positive gravitaxis, was found in Dictyostelium slugs but not in amoebae. In the presence of a simultaneous light stimulus, gravitaxis is effective only at low fluence rates. Slugs showed positive thermotaxis in a thermal gradient (0.2 °C cm^?1) and ignored the simultaneous light stimulus at low fluence rates (< 10^?3 W m^?2), while at higher fluence rates they moved toward the light source. With a combination of a thermal gradient and gravity Dictyostelium slugs clearly oriented thermotactically ignoring the gravistimulus.     

Cellular responsiveness to cyclic AMP in a phosphodiesterase-defective mutant of Dictyostelium discoideum

Responsiveness of Dictyostelium discoideum amoebae to cAMP, a chemotactic mediator, was investigated in a strain defective in cAMP-phosphodiesterase production. Cells were subjected to a high cAMP signal (10(-6) M) in the presence or absence of exogenous phosphodiesterase, and the changes of intracellular cAMP and cGMP concentrations and of adenylate cyclase activity were measured. In the presence of cAMP hydrolysis, both adenylate and guanylate cyclases are transiently activated. In the absence of hydrolysis, the high and constant extracellular cAMP concentration is sufficient to elicit a re-activation of adenylate cyclase a few minutes after the first transient response. In contrast, levels of cGMP remain basal for at least 20 min after termination of the initial response to the cAMP addition.     

The effect of tunicamycin on cell-surface changes associated with development and differentiation ofDictyostelium discoideum

Developmental changes in surface properties ofDictyostelium discoideum cells have been detected by partitioning. Changes occurring between 0 and 6 h development were not affected by tunicamycin but subsequent changes associated with formation of presumptive spore cells were inhibited. It would seem that only the latter changes are dependent on glyco-protein synthesis.     

Quantitative analysis of the spatial distribution of ultrastructural differentiation markers during development ofDictyostelium discoideum

Summary The appearance and spatial distrubution of ultrastructural markers ofDictyostelium discoideum differentiation were quantitatively analysed. Our results combined with data from the literature on the functions of cells at various stages of development lead to the following conclusions. When food is no longer available all amoebae initially develop an autophagic apparatus in order to sustain metabolism. After slugs have been formed, autophagy is suppressed in the prespore cells. During aggregation a number of cells gradually form prespore characteristics. These cells arise at random but later they become located in the basal part of the tip-forming aggregate. From the early slug stage onwards, cells of the posterior two third region gradually enter into the prespore pathway. During prolonged slug migration the optimal acquirement of prespore characteristics is blocked. Cells of the anterior region show no active differentiation but they maintain the morphology and most of the functions of aggregating cells. At the rear-guard of the slug and later on in the basal region of the maturing fruiting body, a second anteriorlike region appears. Actual stalk cell differentiation takes place only at the apex and at the base of the developing fruiting body.     

Alteration of cellular ionic constituents by external ionic conditions,and its significance in the development ofDictyostelium discoideum

Effects of external ionic conditions ofD. discoideum cells were examined in relation to intracellular ionic concentrations, the activity of pyruvate kinase and the amount of ATP. Main components of metal cations in heat extracts of vegetative cells were K⁺, Na⁺, Mg²⁺ and Ca²⁺ whose concentrations in a cell were about 35.0, 3.6, 10.6 and 2.3 mM, respectively. External Na⁺ at the concentration more than 50 mM inhibited the formation of cell aggregates in the presence of 10^?4M Ca²⁺. Such an inhibitory effect of Na⁺ was completely nullified by the addition of more than 10 mM K⁺. External Na⁺ caused a rapid decrease in intracellular K⁺, but an increase in intracellular Na⁺. Furthermore, it was found that the cells containing a high concentration of Na⁺ can develop normally in the presence of exogenous 10 mM K⁺, where intracellular K⁺ was maintaned at about 30 mM, irrespective of a high concentration of intracellular Na⁺ (about 30 mM). These suggest that the Na⁺-inhibition of the development is caused by a decrease in intracellular K⁺, but not by an increase in intracellular Na⁺. Pyruvate kinase extracted from the organism required K⁺ for its activation. The vegetative cells incubated in 50 mM Na⁺ contained only about 10 mM K⁺ which is insufficient for the enzyme activation. However, the amount of ATP in the cells containing less K⁺ was similar to that in those with much K⁺. These results are discussed in relation to the activity of glycolysis.     

The spore coat of a fucosylation mutant in Dictyostelium discoideum

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.     

Altered prestarvation response in a nystatin resistant Dictyostelium discoideum mutant

Abstract Wild-type Dictyostelium amoebae secrete an autocrine, prestarvation factor (PSF) that allows them to measure the amount of food bacteria compared to their cell density. When the ratio of PSF to bacteria reaches a threshold, the cells are signaled to prepare for eventual starvation. This prestarvation response (PSR) usually starts three to four generations before the end of exponential growth, leading to the accumulation of several aggregation specific genes during growth. We characterize a nystatin-resistant mutant, HK19, that expresses the PSR genes three generations earlier than wild type but has an otherwise wild-type PSR. Although HK19 has a full PSR during growth, HK19 continues to grow at the wild-type rate and reaches normal cell densities. Because HK19 temporally separates the PSR from starvation, it became possible to test whether starvation is required for development. Since HK19 growing at low density can be induced to clump with either cAMP or folate, it appears that the PSR and an external signal are sufficient for entry into development. These data suggest that the PSR is a complex genetic pathway that induces genes involved in the exit from growth and the entry into development.     

Altered prestarvation response in a nystatin resistant Dictyostelium discoideum mutant

Wild-type Dictyostelium amoebae secrete an autocrine, prestarvation factor (PSF) that allows them to measure the amount of food bacteria compared to their cell density. When the ratio of PSF to bacteria reaches a threshold, the cells are signaled to prepare for eventual starvation. This prestarvation response (PSR) usually starts three to four generations before the end of exponential growth, leading to the accumulation of several aggregation specific genes during growth. We characterize a nystatin-resistant mutant, HK19, that expresses the PSR genes three generations earlier than wild type but has an otherwise wild-type PSR. Although HK19 has a full PSR during growth, HK19 continues to grow at the wild-type rate and reaches normal cell densities. Because HK19 temporally separates the PSR from starvation, it became possible to test whether starvation is required for development. Since HK19 growing at low density can be induced to clump with either cAMP or folate, it appears that the PSR and an external signal are sufficient for entry into development. These data suggest that the PSR is a complex genetic pathway that induces genes involved in the exit from growth and the entry into development.     

Responses to novelty in staggerer mutant mice

Responses to novelty in normal C57BL/6 and staggerer mutant mice were recorded. The normal mice confronted a novel object in their familiar environment showed avoidance and burying responses while the staggerer mutant mice contacted it. When given the opportunity to move around freely in simultaneously presented novel and familiar environments, the mutant mice more quickly entered the novel areas than normal animals. these data reveal a significant decrease in the neophobic components of the neotic behaviour in the staggerer mice. However, since the mutant mice did not show a locomotor deficit, the impairment of neophobia seems not to be due to the gait abnormalities of these animals. The results support the view that the cerebellum may contribute to the organization of complex behaviours.     

A mathematical model for cell sorting,migration and shape in the slug stage ofDictyostelium discoideum

A mathematical model for cell sorting and migration in the slug stage of cellular slime moldsDictyostelium discoideum is proposed. Assuming that a slug is a “mixed fluid” of prespore and prestalk cells, a set of equations which describe the dynamics of cell distribution, internal pressure and velocity of hte slug are derived from the balance formula of individual cell movement. These equations are analyzed to obtain the spatial patterns of the two types of cells at dynamical equilibrium and the relationship between the migration velocity and the slug size. The body shape of the elongated slug at the migrating stage is also investigated, taking account of the law of surface tension. The stable shapes of slugs with different volumes are explicity obtaained and the existence of critical size of a slug is suggested.