Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

Calcium levels during cell cycle correlate with cell fate of Dictyostelium discoideum

Levels of intracellular calcium, (Ca(2+))(i), from different stages of cell cycle of Dictyostelium discoideum were monitored using the fluorescent Ca(2+)-sensitive dye, Indo 1. Combinations of Ca(2+)-ionophore (A23187) and Ca(2+)-chelator (EGTA) resulted in the inhibition of progression of cell cycle. This delay was caused due to block in G(2)/M-->S phase transition of the cell cycle. Rescue of the cell cycle progression was made with 0.5 m m of exogenous Ca(2+). High (Ca(2+))(i)levels overlapped with the S-phase, of the cell cycle.Results indicate that a high (Ca(2+))(i)level during S-phase is not required for cell cycle progression but for cell-type choice mechanism at the onset of starvation, and these cells tend to follow the prestalk pathway.     

On the effects of cycloheximide on cell motility and polarisation in Dictyostelium discoideum

Background  

Cycloheximide is a protein synthesis inhibitor that acts specifically on the 60S subunit of eukaryotic ribosomes. It has previously been shown that a short incubation of Dictyostelium discoideum amoebae in cycloheximide eliminates fluid phase endocytosis.     

Actin on disease--studying the pathobiology of cell motility using Dictyostelium discoideum

The actin cytoskeleton in eukaryotic cells provides cell structure and organisation, and allows cells to generate forces against membranes. As such it is a central component of a variety of cellular structures involved in cell motility, cytokinesis and vesicle trafficking. In multicellular organisms these processes contribute towards embryonic development and effective functioning of cells of all types, most obviously rapidly moving cells like lymphocytes. Actin also defines and maintains the architecture of complex structures such as neuronal synapses and stereocillia, and is required for basic housekeeping tasks within the cell. It is therefore not surprising that misregulation of the actin cytoskeleton can cause a variety of disease pathologies, including compromised immunity, neurodegeneration, and cancer spread. Dictyostelium discoideum has long been used as a tool for dissecting the mechanisms by which eukaryotic cells migrate and chemotax, and recently it has gained precedence as a model organism for studying the roles of conserved pathways in disease processes. Dictyostelium's unusual lifestyle, positioned between unicellular and multicellular organisms, combined with ease of handling and strong conservation of actin regulatory machinery with higher animals, make it ideally suited for studying actin-related diseases. Here we address how research in Dictyostelium has contributed to our understanding of immune deficiencies and neurological defects in humans, and briefly discuss its future prospects for furthering our understanding of neurodegenerative disorders.     

Reduced glutathione levels affect the culmination and cell fate decision in Dictyostelium discoideum

Glutaredoxins have been known to be glutathione-dependent oxidoreductases that participate in the redox regulation of various cellular processes. To understand the role of glutaredoxins in the development, we examined glutaredoxin 1 (Grx1) of Dictyostelium discoideum. Its mRNA was highly accumulated at the mound and the culmination stages. When Grx1-overexpressing cells were developed, their culmination was delayed, and the expression of marker genes for prespore and spore decreased. Interestingly, they had about 1.5-fold higher amount of reduced glutathione (GSH) compared with parental cells and their prolonged migration was repressed by the oxidant such as hydrogen peroxide. To confirm the effect of GSH on the culmination, glutathione reductase (Gsr) was overexpressed or underexpressed. Similar to Grx1-overexpressing cells, Gsr-overexpressing cells contained about 1.5-fold higher amount of GSH and exhibited the delayed culmination. In contrast, the knockdown mutant of Gsr had nearly 50% lower amount of GSH and showed accelerated culmination. Taken together, these data suggest that the culmination of Dictyostelium is controlled by GSH. In addition, the cells having higher GSH levels showed a prestalk tendency in the chimeric slugs with parental cells, indicating that the difference in the amount of GSH may affect the determination of cell fate.     

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.     

Prespore cell fate bias in G1 phase of the cell cycle in Dictyostelium discoideum

By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.     

The developmental regulation of single-cell motility in Dictyostelium discoideum

The velocity of single amebae in the absence of a chemotactic signal has been analyzed during growth, development, rapid recapitulation, and dedifferentiation in the cellular slime mold Dictyostelium discoideum. It is demonstrated that (1) the velocity of axenically grown cells in half that of bacterially grown cells, (2) the velocity of bacterially grown cells decreased to roughly the same low level as axenically grown cells approximately 5 hr after the removal of exogeneous bacteria, (3) the velocity remains low for a 7-hr period preceding the onset of aggregation in both axenically and bacterially grown cells, (4) the velocity increases transiently at the onset of aggregation for both axenically and bacterially grown cells, (5) the velocity decreases to a very low level after the formation of loose aggregates and remains at that level at least through the early culminate I stage, (6) the velocity is not stimulated in 13-hr developing cells (finger stage) by inducing rapid recapitulation, (7) the velocity decreases after the erasure event in cultures of 7-hr developing cells (ripple stage) stimulated to undergo dedifferentiation, but the inhibition of the erasure event by the addition of 10(-4) M cAMP does not block this decrease. These results demonstrate that the basal level of single-cell motility in growing cultures is significantly influenced by the nutrient composition of the supporting medium, and that the transient increase in single-cell motility at the onset of aggregation is under the rigid control of the initial developmental program. Both rapid recapitulation and the program of dedifferentiation appear to have no influence on the basal level of single-cell motility.     

Length distribution of F-actin in Dictyostelium discoideum

Inhibition of deoxyribonuclease I activity was used to assay the actin monomers and the pointed ends of actin filaments in lysates of Dictyostelium discoideum. The KD for the binding reaction was 0.2-0.3 nM. Total cellular actin was 93 microM in monomers (approximately 0.1 fmol/cell) of which roughly half was initially polymeric. Essentially all of the filamentous actin (F-actin) was readily pelleted in the microcentrifuge and was therefore presumed to be in the cytoskeleton. Free F-actin barbed ends, measured as pelletable [3H]cytochalasin B, numbered 1.8 x 10(5)/cell; nuclei for the polymerization of rabbit muscle globular (monomeric) actin numbered 2.0 x 10(5)/cell; and pointed ends, determined by their inhibition of deoxyribonuclease I, numbered 3.6 x 10(5)/cell. These values suggest that half the barbed ends might be occluded. On average, the filaments contained approximately 76 subunits and were therefore about 0.2 micron long. The distribution of their lengths was estimated from the time course of depolymerization following vast dilution. Three populations were defined. In one experiment, the smallest population contained 71% of the F-actin mass and 96% of the pointed ends; these filaments averaged 80 subunits or 0.22 microns in length. An intermediate population contained 14% of the F-actin mass and 3% of the filaments; these were roughly 460 subunits (1.3 microns) long. The largest population contained 15% of the F-actin mass in about 0.3% of the filaments; these were 13 microns in length, about the diameter of the cell. The numerous short filaments might populate a cortical mesh, while the long filaments might constitute endoplasmic bundles.     

Adaptation in the motility response to cAMP in Dictyostelium discoideum

When developing amebae of Dictyostelium discoideum are treated with constant concentrations of cAMP above 10(-8)M, the average rate of motility is depressed, with maximum inhibition at roughly 10(-6)M. It is demonstrated that shifting the concentration of cAMP from 0 M to concentrations ranging from 10(-8) to 10(-6)M in a perfusion chamber results in the immediate inhibition of motility. After shifting from 0 M to 10(-8) or 10(-7)M, the rate of cell motility remains low, then rebounds to a higher level, exhibiting a standard adaptation response. No adaptation is exhibited after a shift from 0 M to 10(-6)M, a concentration resulting in maximum inhibition. It is demonstrated that the level of inhibition and the extent of the adaptation period are dependent upon the concentration of cAMP after the shift, and that submaximal inhibition is additive. The characteristics of adaptation in this motility response are very similar to the characteristics of adaptation for the relay system and phosphorylation of the putative cAMP receptor.     

Subcellular distribution of ribosomal proteins in Dictyostelium discoideum

The distribution of ribosomal proteins in monosomes, polysomes, the postribosomal cytosol, and the nucleus was determined during steady-state growth in vegetative amoebae. A partitioning of previously reported cell-specific ribosomal proteins between monosomes and polysomes was observed. L18, one of the two unique proteins in amoeba ribosomes, was distributed equally among monosomes and polysomes. However S5, the other unique protein, was abundant in monosomes but barely visible in polysomes. Of the developmentally regulated proteins, D and S6 were detectable only in polysomes and S14 was more abundant in monosomes. The cytosol revealed no ribosomal proteins. On staining of the nuclear proteins with Coomassie blue, about 18, 7 from 40S subunit and 11 from 60S subunit, were identified as ribosomal proteins. By in vivo labeling of the proteins with [35S]methionine, 24 of the 34 small subunit proteins and 33 of the 42 large subunit proteins were localized in the nucleus. For the majority of the ribosomal proteins, the apparent relative stoichiometry was similar in nuclear preribosomal particles and in cytoplasmic ribosomes. However, in preribosomal particles the relative amount of four proteins (S11, S30, L7, and L10) was two- to four-fold higher and of eight proteins (S14, S15, S20, S34, L12, L27, L34, and L42) was two-to four-fold lower than that of cytoplasmic ribosomes.     

Constitutively active protein kinase A disrupts motility and chemotaxis in Dictyostelium discoideum

The deletion of the gene for the regulatory subunit of protein kinase A (PKA) results in constitutively active PKA in the pkaR mutant. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium discoideum, pkaR mutant cells were subjected to computer-assisted two- and three-dimensional motion analysis. pkaR mutant cells crawled at only half the speed of wild-type cells in buffer, chemotaxed in spatial gradients of cyclic AMP (cAMP) but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing toward an aggregation center in natural waves generated by wild-type cells that made up the majority of cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of the pkaR mutant cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR mutant cells shared with regA mutant cells are discussed by considering the pathway ERK2 —| RegA —| [cAMP] → PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateral-pseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave.     

Periodic cell aggregation in suspensions of Dictyostelium discoideum

Abstract. Periodic activities of Dictyostelium discoideum can be observed in cell suspension as two types of oscillations in the light-scattering properties, spike-shaped and sinusoidal. Responses of suspended cells to applied chemoattractants are also reflected by transient changes in light scattering. Alterations in the light-scattering properties are due to structural changes such as changes in cell shape and/or changes in the size of cell aggregates. Therefore, changes in the aggregation state during autonomous oscillations and during attractant-induced responses were investigated. In order to be able to withdraw multiple samples and larger sample volumes from optically monitored cell suspensions, a photometer comprising glass fiber optics immersable in a cell suspension was constructed. Samples were fixed with formaldehyde and photographed. The aggregation state of the samples was quantified by counting the number of particles (cells and cell aggregates) per volume. Folic acid elicited in suspensions of undifferentiated cells a transient decrease in the number of particles per volume as did cAMP in suspensions of preaggregation cells. Periodic changes in the number of particles per volume occurred synchronously with spike-shaped and sinusoidal oscillations. The relative amplitude of the oscillations in particle number was larger during sinusoids than during spikes. Photographs showed periodic changes in the aggregate size during sinusoidal oscillations. In each cycle, the cell-aggregation phase was followed by a phase of partial disaggregation. The recurring loosening of cell-cell contacts may be relevant for sorting out the different cell types. The potential role of contact site as synchronizer and as constituent of an oscillator is discussed.     

Correlates of developmental cell death in Dictyostelium discoideum

We have studied the correlates of cell death during stalk cell differentiation in Dictyostelium discoideum. Our main findings are four. (i) There is a gradual increase in the number of cells with exposed phosphatidyl serine residues, an indicator of membrane asymmetry loss and increased permeability. Only presumptive stalk cells show this change in membrane asymmetry. Cells also show an increase in cell membrane permeability under conditions of calcium-induced stalk cell differentiation in cell monolayers. (ii) There is a gradual fall in mitochondrial membrane potential during development, again restricted to the presumptive stalk cells. (iii) The fraction of cells showing caspase-3 activity increases as development proceeds and then declines in the terminally differentiated fruiting body. (iv) There is no internucleosomal cleavage of DNA, or DNA fragmentation, in D. discoideum nor is there any calcium- and magnesium-dependent endonucleolytic activity in nuclear extracts from various developmental stages. However, nuclear condensation and peripheralization does occur in stalk cells. Thus, cell death in D. discoideum shows some, but not all, features of apoptotic cell death as recognized in other multicellular systems. These findings argue against the emergence of a single mechanism of 'programmed cell death (PCD)' before multicellularity arose during evolution.     

The distribution of myosin II in Dictyostelium discoideum slug cells

While the role of myosin II in muscle contraction has been well characterized, less is known about the role of myosin II in non-muscle cells. Recent molecular genetic experiments on Dictyostelium discoideum show that myosin II is necessary for cytokinesis and multicellular development. Here we use immunofluorescence microscopy with monoclonal and polyclonal antimyosin antibodies to visualize myosin II in cells of the multicellular D. discoideum slug. A subpopulation of peripheral and anterior cells label brightly with antimyosin II antibodies, and many of these cells display a polarized intracellular distribution of myosin II. Other cells in the slug label less brightly and their cytoplasm displays a more homogeneous distribution of myosin II. These results provide insight into cell motility within a three-dimensional tissue and they are discussed in relation to the possible roles of myosin II in multicellular development.     

盘基网柄菌细胞分化及细胞比例的调控

本文综述了盘基网柄菌(Dictyosteliumdiscoideum)发育过程中调控细胞分化及细胞比例的一些信号分子,包括分化诱导因子(DIF-1、SDF-2)、糖原合成酶激酶(GSK-3)、环状亮氨酸拉链蛋白(rZIP)等,介绍了这些信号分子的功能及其作用机制。     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.