Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

Dictyostelium cells produce platelet-activating factor in response to cAMP.

Evidence is provided that Dictyostelium discoideum cells produce 1-O-alkyl-2-delta-acetyl-O-sn-glycero-3-phosphocholine (platelet-activating factor, PAF). D. discoideum PAF has been characterized as being identical with mammalian platelet-activating factor, based on its stimulation of rabbit platelet aggregation, its physicochemical properties and mass spectrum. The basal activity of PAF increases after starvation and during aggregation and declines at the slug stage. PAF is not detected in the extracellular space. Cell treatment with cAMP pulses stimulates a transient accumulation of PAF, probably via activation of a cAMP-dependent acetyltransferase, suggesting a possible involvement of PAF in cAMP-regulated processes in Dictyostelium.     

Analysis of the prestarvation response in growing cells of Dictyostelium discoideum

We have previously shown that growing cells of Dictyostelium discoideum (strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarke et al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth, Dictyostelium cells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF-regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response of Dictyostelium cells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic cultures.     

Input-output relations in computer-simulated nerve cells

Summary This communication examines, in digital computer simulated networks, the input-output relation established at synaptic level. It is restricted to excitatory junctions and analyzes the changes in post-synaptic discharge which occur when the number of pre-synaptic terminals increases while the EPSP size decreases, when the statistical structure or form (as measured by the interspike interval mean, standard deviation, histogram and by the autocorrelogram) of the spike train in each pre-synaptic fiber changes, and/or when the interdependence between pre-synaptic fibers varies from complete independence to strong dependence.1Independent Pre-synaptic Terminals. When the number of pre-synaptic terminals increases and the EPSP size decreases proportionally (while the input form remains constant), the post-synaptic interspike interval mean increases slightly, the standard deviation decreases markedly, the histogram becomes sharp and narrow and the autocorrelogram becomes periodic. When, on the other hand, the pre-synaptic form varies (while the number of terminals and the EPSP size remain constant), the effect upon the post-synaptio output depends upon the given number of terminals and EPSP size. If terminals are few and EPSP's large, the output varies with the pre-synaptic form. The post-synaptic coefficient of variation is linearly related to the pre-synaptic coefficient of variation, the slope decreasing as the number of inputs increases. If terminals are numerous and weak, the pre-synaptic form ceases to be influential and the post-synaptic cell generates the same output regardless of the detailed structure of the corresponding input. The output common to any combination of independent weak input forms is a very regular train of evenly spaced spikes. (This conclusion is valid unless pre-synaptic terminals fire at extremely low rates.) Such results are mathematically predictable in a simple and realistic model of membrane potential and threshold dynamics (see Appendix).As the EPSP size increases, all other variables being equal, the post-synaptic interval mean decreases monotonically. The decrease is smooth or in steps depending on whether the pre-synaptic form is Poisson or pacemaker, respectively. Post-synaptic spikes are effectively blocked by relatively small numbers of inhibitory terminals.2Dependent Pre-synaptic Terminals. When there is a physiological amount of interdependence between the presynaptic terminals that impinge upon the post-synaptic cell, the activity of the latter is a function of the statistical form of the input channels, even when the latter are numerous and weak. This happens when interdependence involves only a proportion of all terminals or only the terminals within separate and independent groups.In order to understand the transactions that take place in the nervous system, it is necessary to identify the presynaptic statistics that influence the corresponding post-synaptic discharge. When pre-synaptic terminals produce large PSP's their influence is dominant and exerted by way of the precise statistical form of the discharge. When terminals produce small PSP's their influence is contingent on their degree of interdependence. If they are uncorrelated, they act exclusively by way of their mean rates and provide a smooth adjustment of the post-synaptic membrane potential and firing rate. If terminals are correlated, they act by way of several statistical features and assume a dominant role that determines a precise relation between pre-synaptic timing and post-synaptic firing. The degree of inter-terminal correlation is thus a functionally significant variable.

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Zusammenfassung Mit Hilfe eines Digitalrechners wurden die Eingangs-und Ausgangsbeziehungen auf synaptischer Ebene untersucht und dargestellt. Diese Untersuchung erstreckt sich auf erregende Synapsen und analysiert die Veränderungen postsynaptischer Aktionspotentiale, die auftreten, 1. wenn die Anzahl der präsynaptischen axonischen Endigungen ansteigt, während andererseits die Amplitude des EPSP abnimmt; 2. wenn sich die statistische Struktur oder Form der Spike-Kette in jeder präsynaptischen Faser verändert; und/oder 3. wenn die Beziehungen zwischen den präsynaptischen Fasern von völliger Unabhängigkeit bis zu starker Abhängigkeit variiert werden.1Unabhängige präsynaptische Endigungen. Mit zunehmender Anzahl präsynaptischer Endigungen bei gleichzeitiger proportionaler Abnahme des EPSP (Input Form konstant) treten folgende Veränderungen auf: a) das durchschnittliche Intervall zwischen den postsynaptischen Spikes nimmt etwas zu; b) die mittlere statistische Abweichung (standard deviation) nimmt erheblich ab; c) die Form des Histogramms wird eng umschrieben; und d) das Autokorrelogramm nimmt periodischen Charakter an. Wenn andererseits die präsynaptische Form verändert wird (während die Anzahl der Endigungen sowie die Größe des EPSP konstant bleibt), hängt der am postsynaptischen Ausgang registrierte Effekt von der gegebenen Anzahl der Endigungen und von der Größe des EPSP ab. Ist die Anzahl der Endigungen gering und das EPSP groß, dann variiert der Ausgang mit der präsynaptischen Form. Der postsynaptische Variationskoeffizient steht dann in linearer Abhängigkeit vom präsynaptischen Variationskoeffizienten, wobei die Steigung der Geraden mit zunehmendem Eingang abnimmt. Sind die Endigungen zahlreich und die Größen der EPSPs gering, dann übt die präsynaptische Form keinen Einfluß mehr aus, und das von der postsynaptischen Zelle erzeugte Ausgangsprodukt wird unabhängig von der detaillierten Struktur des Eingangs. Für eine jegliche Kombination von unabhängigen und schwachen Eingangsformen stellt sich das Ausgangsprodukt in Form einer sehr regelmäßig gestalteten und durch gleichmäßige Abstände gekennzeichneten Spike-Kette dar (diese Folgerung gilt nur für die Fälle, in denen die präsynaptischen Endigungen sich nicht äußerst langsam entladen). Diese Resultate können mathematisch an Hand eines einfachen Membranmodells abgeleitet werden (s. Appendix).Wenn das EPSP in Größe ansteigt, alle anderen Variablen jedoch gleich bleiben, dann nimmt das postsynaptische Intervall fortwährend ab. Dieser Abfall ist entweder gleichmäßig (präsynaptische Form: Poisson) oder stufenweise (präsynaptische Form: pacemaker). Die postsynaptischen Aktionspotentiale werden durch eine vergleichsweise kleine Anzahl von hemmenden Endigungen wirkungsvoll blockiert.2Abhängige präsynaptische Endigungen. Wenn sich der Grad der Abhängigkeit zwischen den präsynaptischen Endigungen in physiologischen Grenzen bewegt, dann kann die Aktivität der postsynaptischen Zelle als eine Funktion der statistischen Form der Eingangskanäle angegeben werden, und das sogar, wenn die letzteren zahlreich und schwach sind. Dieser Fall tritt dann ein, wenn die Abhängigkeit zwischen den präsynaptischen Endigungen nur einen Teil aller Endigungen oder nur die Endigungen innerhalb getrennter und unabhängiger Gruppen betrifft.Um die im Nervensystem stattfindenden Übertragungen zu verstehen, ist es notwendig, diejenigen präsynaptischen Statistiken zu idendifizieren, die entsprechende postsynaptische Entladungen beeinflussen. Wenn präsynaptische Endigungen große PSPs hervorrufen, dann ist ihr Einfluß dominierend und wird entsprechend der präzisen statistischen Form ausgeübt. Wenn die Endigungen kleine PSPs hervorrufen, dann hängt ihr Einfluß weitgehend von dem Grad der Abhängigkeit voneinander ab. Wenn die präsynaptischen Endigungen unkorreliert sind, dann vermitteln ihre durchschnittlichen, präsynaptischen Entladungsgeschwindigkeiten eine gleichmäßige Regulierung des postsynaptischen Membranpotentials und der postsynaptischen Entladungsgeschwindigkeiten. Sind andererseits die präsynaptischen Endigungen korreliert, dann nehmen sie eine dominierende Funktion ein, und die Beziehungen zwischen präsynaptischer Regulierung und postsynaptischer Entladung können präzise definiert werden. Somit stellt sich der Grad der Abhängigkeit zwischen den präsynaptischen Endigungen als eine funktionell bedeutende Variable dar.

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Supported by a Research Career Program (J. P. S.) and by Grants from the USPHS (NB-02501, NB-05264, NB-07325), and by Air Force Project Rand. Computing assistance was obtained from the UCLA Health Science Computing Facility sponsored by NIH Grant FR-3, and from the Brain Research Institute Data Processing Laboratory.     

Heat shock response of Dictyostelium

In response to a shift from 22 to 30°C the relative rate of synthesis of a small number of proteins is dramatically increased in Dictyostelium discoideum. The cells neither grow nor develop at this temperature but die slowly with a half-life of 18 hr. The major protein synthesized in response to a heat shock to 30°C in either growing cells or developing cells has an apparent molecular weight of 70,000 (70K). An increase in the relative rate of synthesis of 70K can be seen as early as 20 min following heat shock. Synthesis of 70K remains high for 4 hr at 30°C and then decreases. Similar kinetics of 70K synthesis occur during recovery at 22°C following a 1-hr heat shock. RNA synthesis during the first half-hour of heat shock is essential for the high rate of 70K measured 2 hr later. By isoelectric focusing the 70K protein can be separated into two spots, one of which overlaps one of the major heat shock proteins of Drosophila melanogaster. The relative rate of synthesis of several other proteins (82K, 60K, 43K) increases less dramatically in Dictyostelium during heat shock at 30°C. A heat shock to 34°C results in rapid synthesis of these proteins but not of 70K. The relative rates of synthesis of most other proteins made at 22°C decreases, most notably that of actin. Synthesis of heat shock proteins at 30°C does not significantly affect viability at 30°C but dramatically prolongs the period of time the cells can survive at 34°C. Thus, 30°C appears to be a stasis condition for Dictyostelium which elicits a response essential for protection from lethal temperatures. The similarity of the heat shock response in Dictyostelium to that in Drosophila and vertebrate cells suggests that certain aspects of the response may be universal in eukaryotes.     

Fusion of cell ghosts with intact cells in Dictyostelium discoideum: Differential response of opposite mating-type cells

The sexual cycle of Dictyostelium discoideum is initiated by the fusion of cells that are of opposite mating types (e.g. NC4- and HM1-type cells). Cells grown in light on agar plates are not capable of sexual cell fusion, but become capable when cultured in the dark in a liquid medium. Cells in the incapable state are called fusion-incompetent cells, and cells in the latter state, fusion-competent cells. To gain some understanding of the mechanism of cell fusion, cell ghosts prepared by freeze-thawing intact cells were incubated with intact cells. The cell ghosts killed the intact cells by directly fusing with them, the extent of fusion depending on the particular strains employed and the fusion-competency of the intact cells and of the cells from which the cell ghosts had been prepared. A detailed examination revealed that fusion-competent NC4 cells were always more easily killed by cell ghosts than fusion-incompetent NC4 cells. It also became apparent that cell ghosts prepared from fusion-competent NC4 cells killed all cell types far more efficiently than did those prepared from fusion-incompetent NC4 cells. However, fusion-competent and fusion-incompetent HM1 cells were equally sensitive to cell ghosts, and cell ghosts prepared from fusion-competent HM1 cells had the same ability to kill as those prepared from fusion-incompetent HM1 cells. From these findings, it thus appears that opposite mating-type cells have distinct membrane properties related to sexual cell fusion.     

Rapid patterning in 2-D cultures of Dictyostelium cells and its relationship to zonal differentiation

Rapid patterning has been observed in confined 2-D cultures of Dictyostelium discoideum Ax-2 cells as an outer dark zone and a inner light zone. The width of outer zone was usually approximately100 microm, irrespective of the size of cell masses under atmospheric conditions. The width of the outer zone, however, changed depending on external O2 concentrations and reached up to 250 microm at 100% O2. A clear regional difference in tetramethyl rhodamine methyl ester (TMRM) staining was noticed between the outer zone and the inner zone: the inner zone was more strongly stained with TMRM than the outer zone, which faced the air. Using inhibitors of oxidative phosphorylation (dinitrophenol (DNP) or NaN3) and a specific inhibitor of CN-resistant respiration (benzohydroxamic acid (BHAM)), it has been demonstrated that the outer zone is basically formed by the O2 threshold for oxidative phosphorylation, while the inner cells mainly perform cyanide-resistant respiration. When cells around the early mound stage (just before prestalk and prespore differentiation) were cultured as 2-D cell masses, ecmA-expressing cells (pstA cells), ecmB-expressing cells (pstB cells) and D19-expressing cells (prespore; psp cells), arose in a position-dependent manner in the outer zone. In the inner zone, cell motility seemed to be markedly impaired and neither prestalk nor prespore differentiation occurred. In addition, once-differentiated prespore cells were found to dedifferentiate rapidly in the inner zone. The reason for dedifferentiation as well as for failure of cells to differentiate in the inner zone is discussed with reference to O2 radicals.     

Keratocyte-like locomotion in amiB-null Dictyostelium cells

Starved Dictyostelium amoebae continuously change their shape and they are elongated along the front-rear axis during locomotion. In contrast, we found that disruption of the amiB gene, which had been identified as a gene required for the aggregation process during development, caused these cells to move in a manner similar to fish keratocytes. Starved amiB- cells were elongated laterally and had one large lamellipodium along the front side arc of the cell. These cells moved unidirectionally for long distances maintaining the half-moon shape, and this movement followed the predictions of the graded radial extension model, which was originally developed to describe the keratocyte movements. Furthermore, the distributions of actin, Arp2, and myosin II in amiB- cells were similar to those in keratocytes. Therefore, locomotion by keratocytes and amiB- cells appears to be driven by similar mechanisms of cytoskeletal regulation. Double knockout cells lacking both AmiB and myosin II were still able to move unidirectionally in a keratocyte-like manner, although the frequency of those movements was lower. Thus, myosin II is dispensable for the unidirectional movement, though it likely functions in the maintenance of the characteristic half-moon shape. This mutant cell can be a useful tool for further molecular genetic analysis of the mechanism of cell locomotion.     

Bleb-driven chemotaxis of Dictyostelium cells

Blebs and F-actin–driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.     

Stochastic signal inputs for chemotactic response in Dictyostelium cells revealed by single molecule imaging techniques

Chemotactic cells can exhibit extreme sensitivity to chemical gradients. Theoretical estimations of the signal inputs required for chemotaxis suggest that the response can be achieved under the strong influence of stochastic input noise generated by the receptors during the transmembrane signaling. This arises a fundamental question regarding the mechanisms for directional sensing: how do cells obtain reliable information regarding gradient direction by using stochastically operating receptors and the downstream molecules? To address this question, we have developed single molecule imaging techniques to visualize signaling molecules responsible for chemotaxis in living Dictyostelium cells, allowing us to monitor the stochastic signaling processes directly. Single molecule imaging of a chemoattractant bound to a receptor demonstrates that signal inputs fluctuate with time and space. Downstream signaling molecules, such as PTEN and a PH domain-containing protein that are constituent parts of chemotactic signaling system, can also be followed at single molecule level in living cells, illuminating the stochastic nature of chemotactic signaling processes. In this report, we start with a brief introduction of chemotactic response of the eukaryotic cells, followed by an explanation for single molecule imaging techniques, and finally discuss these applications to chemotactic signaling system of Dictyostelium cells.     

Involvement of the glucose-regulated protein 94 (Dd-GRP94) in starvation response of Dictyostelium discoideum cells

Upon deprivation of nutrients, Dictyostelium discoideum Ax-2 cells arrest proliferation and initiate a metamorphosed developmental program including induction of altered gene expressions which are necessary for differentiation. In Ax-2 cells, we found out a member of Hsp90 family usually contained in the endoplasmic reticulum (ER), Dd-GRP94 (Dictyostelium discoideum glucose-regulated protein 94). In general, GRP94 are induced either by glucose-depletion or by depletion of Ca(2+) in intracellular Ca(2+) stores. Unexpectedly, however, the expression of Dd-grp94 was greatly reduced within 60 min of starvation. Dd-grp94-overexpressing cells (GRP94(OE) cells) collected without forming distinct aggregation streams, and never formed normal fruiting bodies. Also, prespore differentiation as well as maturation into spores and stalk cells were particularly impaired in the GRP94(OE) cells. Thus Dd-GRP94 seems to be crucial in late differentiation as well as in starvation response.     

A talin homologue of Dictyostelium rapidly assembles at the leading edge of cells in response to chemoattractant

In an attempt to identify unknown actin-binding proteins in cells of Dictyostelium discoideum that may be involved in the control of cell motility and chemotaxis, monoclonal antibodies were raised against proteins that had been enriched on an F-actin affinity matrix. One antibody recognized a protein distinguished by its strong accumulation at the tips of filopods. These cell-surface extensions containing a core of bundled actin filaments are rapidly protruded and retracted by cells in the growth-phase stage. The protein of 269 kD turned out to resemble mouse fibroblast talin (Rees et al., 1990) in its primary structure. The fit is best among the first 400-amino acid residues of the NH2-terminal region where identity between the two proteins is 44% and the last 200-amino acid residues of the COOH-terminal region with 36% identity. In the elongated cells of the aggregation stage the Dictyostelium talin is accumulated at the entire front where also F- actin is enriched. Since this protein exists in a soluble state in the cytoplasm, mechanisms are predicted that cause accumulation at sites of the cell where a front is established. Evidence for receptor-mediated accumulation was obtained by local stimulation of cells with cAMP. When a new front was induced by the chemoattractant, the talin accumulated there within half a minute, indicating a signal cascade in Dictyostelium responsible for assembly of the talin beneath sites of the plasma membrane where chemoattractant receptors are strongly activated. The ordered assembly of the talin homologue together with actin and a series of other proteins is considered to play a key role in chemotactic orientation.     

Protein phosphatases in developing Dictyostelium discoideum cells

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities (M_r 170 000 and 520 000) and one kemptide phosphatase activity (M_r 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.     

Osmotic stress response in Dictyostelium is mediated by cAMP

DokA, a homolog of bacterial hybrid histidine kinases, is essential for hyperosmotic stress resistance in Dictyostelium: We show that a transient intracellular cAMP signal, dependent on the presence of DokA, is generated in response to an osmotic shock. This variation of cAMP levels contributes to survival under hypertonic conditions. In contrast to the low cAMP levels observed in dokA(-) strains, overexpression of the receiver domain of DokA causes an increase in cAMP levels, resulting in a rapidly developing phenotype. We present biochemical and cell biological data indicating that the DokA receiver domain is a dominant-negative regulator of a phosphorelay, which controls the intracellular cAMP phosphodiesterase RegA. The activity of the DokA receiver domain depends on a conserved aspartate, mutation of which reverses the developmental phenotype, as well as the deregulation of cAMP metabolism.     

Differentiation of Dictyostelium discoideum cells in suspension culture

Differentiation of Dictyostelium discoideum cells in suspension culture is reported, using a medium containing glucose, albumin, cyclic AMP, EDTA and streptomycin in a phosphate buffer. Production of UDPgalactose:polysaccharide transferase, an enzyme specifically present in prespore cells, and the formation of prespore-specific antigens in more than 60% of the cells, are demonstrated. Differentiation in this medium differs from that previously reported with other suspension systems in that (a) cells form only small, amorphous agglomerates, (b) there is an absolute requirement for cyclic AMP and (c) prior formation of loose cell mounds on a solid substratum is essential for subsequent differentiation in this medium. This last requirement indicates that the differentiation process, giving rise to the prespore-specific enzyme and antigen, can be resolved into two distinct stages, one requiring cell contact on a solid substratum and the other proceeding in small agglomerates incubated in the medium. This medium may be useful for elucidating the role of cyclic AMP and cell contact in slime mould development.     

Reduction of nucleolar size in reaggregated Dictyostelium cells

The ultra-structure of the nucleolus in Dictyostelium discoideum cells was studied by electron microscopy. Large nucleoli on the periphery of the nucleus in cells of the multi-cellular pseudoplasmodium (slug) were maintained during long migration. Disaggregation of the slug cells induced a reduction in the size of the large nucleoli. The size of the reduced nucleoli in the reaggregated cells were maintained during the long migration and culmination of reconstructed slug. The electron density of the cytoplasm clearly distinguishes the prespore from the prestalk region, and it takes about 6 h for the complete recovery of cell-to-cell contact after reaggregation.