Development of the simple cellular slime mold Dictyostelium minutum

An ultrastructural study has been made of the life cycle of the cellular slime mold Dictyostelium minutum. The development of D. minutum is rather simple if compared with Dictyostelium discoideum. After 2 hr of starvation, amoebas move in a nonpulsatile manner towards an acrasin-secreting founder cell. The chemotactic signal is not relayed by the amoebas and stream formation toward primary aggregation centers does not occur. Usually, more than one fruiting body arises from one pseudoplasmodium. No migration of the pseudoplasmodium takes place. The first signs of spore differentiation are found in late aggregates, where prespore cells can be distinguished from the surrounding undifferentiated cells by the increased electron density of their cytoplasm. Vacuoles comparable with the prespore vacuole of D. discoideum appear in both cell types; they fuse with the plasma membrane during sporulation of electron-dense cells and are lysed in electron-light cells, which eventually form the stalk. In contrast with D. discoideum no spatial separation between prespore and prestalk cells is found until very late in fruiting body development.     

A study on the amoebo-flagellate transformation in the slime moldEchinostelium minutum de Bary

Summary The structure and behavior of the non-flagellate and flagellate phases of the slime moldEchinostelium minutum de Bary are here described from living cultures examined with phasecontrast microscopy. The ultrastructure of the myxamoebal and swarm cell phases was studied in sectioned material fixed sequentially with glutaraldehyde-acrolein and OsO₄.The nearly spherical myxamoeba has two pairs of juxtanuclear centrioles with associated microtubular arrays. During the amoebo-flagellate transformation each pair of centrioles assumes an anterior position in the cell and becomes arranged at right angles to one another within a cone of microtubules. This microtubular cytoskeleton extending under the plasmalemma establishes the twisted, narrowly ovoid form of the swarm cell. Each centriole functions potentially as a basal body. When transformed in phosphate buffer pH 6.8 at 12 °C and a cell concentration of 5×10⁵/ml, the myxamoebae develop 1 to 8 flagella. The average number of flagella per swarm cell is 2.7. After approximately 2 hours the swarm cells begin to revert to myxamoebae by resorption of their flagella. The phylogenetic implications of these light and ultrastructural observations are discussed with regard to possible evolutionary relationships between theProtostelia andMyxomycetes.     

Laser light-scattering analysis of protoplasmic streaming in the slime mold Physarum polycephalum

Laser light scattering is shown to be an effective means of obtaining a rapid, objective assessment of dynamic changes in the intact plasmodium of the myxomycete Physarum polycephalum during bidirectional (shuttle) streaming. The motion of material in a 100 mum diameter region of a plasmodial vein was studied by following changes in the autocorrelation function of the fluctuations in the scattered light intensity. The autocorrelation function was recorded at 10 s intervals and analyzed to follow changes in the flow velocity of protoplasm associated with shuttle streaming. Rhythmic velocity changes and a "beating" pattern of velocity maxima were readily observed. In an attempt to locate the site of underlying structural changes in the vein responsible for the changing pattern of flow, the average scattered intensity was separated into components derived from moving and stationary scatterers. Periodic variations in the light intensity due to stationary scatterers are related to the streaming cycle and indicate the occurrence of important structural changes in the vein walls. Two possible interpretations of the data are offered; one involving gross dynamic changes in vein structure, the other involving the formation, contraction, or breakdown of fibrillar material in the vein wall during the streaming cycle.     

A light microscopical investigation of amoebal and plasmodial mitosis in the MyxomyceteEchinostelium minutum

Summary Light microscopical observations on mitosis in living material of the amoebal and plasmodial phases of the MyxomyceteEchinostelium minutum de Bary (orderEchinosteliales) are reported for the first time. The uninucleate amoebal cell undergoes centric, open spindle mitosis whereas the multinucleate plasmodium exhibits acentric, closed spindle mitosis.     

Mitosis in the cellular slime mold Polysphondylium violaceum

Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re- establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.     

High molecular weight phosphorus compound in nucleic acid extracts of the slime mold Physarum polycephalum

Orthophosphate labeled with (32)P was added to the growth medium of the plasmodia of Physarum polycephalum. The nucleic acid extracts of such plasmodia contained (32)P that was not removed by nuclease, protease, or amylase. This labeled material was shown to be separable from nucleic acids, could be eluted from a methylated albumin-kieselguhr column at 0.5 m NaCl, was of high molecular weight, and had several characteristics in common with polyphosphate. A fraction of this polyphosphate-like material was also found in extracts of isolated nuclei.     

Open spindle nuclear division in the amoebal phase of the acellular slime moldEchinostelium minutum with chromosomal movement related to the pronounced rearrangement of spindle microtubules

Summary Myxamoebae ofEchinostelium minutum exhibit extranuclear (open spindle) mitosis with centrioles present at the poles. Spindle microtubules are formed in association with a juxtanuclear MTOC which surrounds the cell's complement of centrioles. During late prophase or prometaphase the nuclear envelope breaks down and subsequently a metaphase plate is formed. Two anaphasic movements occur sequentially: firstly, the distance of the chromosomes to the poles shortens; secondly the distance between the spindle poles increases. The arrangement of spindle microtubules during anaphase is consistent with the hypothesis that chromosomal separation is due to lateral interaction (zippering) of microtubules. During telophase, reconstitution of the nuclear envelope usually takes place in the interzonal region prior to reformation in the polar region. Cytokinesis, which begins in anaphase or early telophase involves the participation of vesicles, microfilaments and microtubules.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Closed spindle nuclear division in the plasmodial phase of the acellular slime moldEchinostelium minutum

Summary Mitosis in the plasmodium ofEchinostelium minutum is intranuclear (closed spindle) and centrioles are not present at the spindle poles. The nuclear envelope remains essentially intact throughout mitosis with polar fenestrae appearing in anaphase and persisting through telophase. During anaphase there is a shortening in the distance of the chromosomes to the poles followed by a further separation of the poles. Zippering of microtubules may be the basis for these two anaphasic movements. During telophase the polar MTOCs are extruded into the cytoplasm through the polar fenestrae prior to reconstitution of the nuclear envelope. It is proposed that during sporulation such MTOCs are responsible for the differentiation of the centrioles which subsequently persist in the myxamoebal phase of this species.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Laser light-scattering analysis of protoplasmic streaming in the slime mold physarum polycephalum

Laser light scattering is shown to be an effective means of obtaining a rapid, objective assessment of dynamic changes in the intact plasmodium of the myxomycete Physarum polycephalum during bidirectional (shuttle) streaming. The motion of material in a 100 μm diameter region of a plasmodial vein was studied by following changes in the autocorrelation function of the fluctuations in the scattered light intensity. The autocorrelation function was recorded at 10 s intervals and analyzed to follow changes in the flow velocity of protoplasm associated with shuttle streaming. Rhythmic velocity changes and a “beating” pattern of velocity maxima were readily observed. In an attempt to locate the site of underlying structural changes in the vein responsible for the changing pattern of flow, the average scattered intensity was separated into components derived from moving and stationary scatterers. Periodic variations in the light intensity due to stationary scatterers are related to the streaming cycle and indicate the occurrence of important structural changes in the vein walls. Two possible interpretations of the data are offered; one involving gross dynamic changes in vein structure, the other involving the formation, contraction, or breakdown of fibrillar material in the vein wall during the streaming cycle.     

Immunocytochemistry of the acellular slime mold Physarum polycephalum

Abstract. The polygonal arrangement of actomyosin fibrils in different stages of the acellular slime mold Physarum polycephalum is correlated with morphogenetic processes at the cell surface. Light and electron microscopic investigations on both endoplasmic drops and thin-spread small plasmodia demonstrate that the differentiation of a polygonal pattern depends on a transient deficiency of plasma membrane invaginations.
Glycerol-extracted specimens show condensation and drastic spatial changes in the organization of the polygonal net after addition of ATP, thus indicating contractile properties of this system. Observations with the polarizing microscope reveal rhythmic changes in fibrillar birefringence intensity corresponding to the protoplasmic streaming activity, i.e., birefringence increases during contraction and decreases during relaxation. Cell fusion experiments, local irradiation with blue light (450 nm), and chemical treatment by impeding the mitochondria1 function with DNP (2,4-di-nitrophenol) demonstrate morphological as well as physiological interdependences of the actomyosin system, the motive force generation, and the expression of a locomotor polarity in plasmodia of Physarum polycephalum.     

Immunological analysis of histone H2A from the slime mold Physarum polycephalum

Specific antibodies against the histone H2A from calf thymus were generated by injecting rabbits with complexes: histone H2A-RNA with a protein to RNA ratio of 3:1. In the microcomplement fixation assay the antibodies against the histone H2A from calf thymus immuno-reacted with the histone H2A from calf thymus but not with H2A from Physarum polycephalum. The histone H2A from calf thymus therefore appears to have an immunological determinant(s) which does not exist in H2A from Physarum polycephalum.     

31P-nuclear magnetic resonance analysis of the intracellular pH in the slime mold Dictyostelium discoideum

Amoebae of the slime mold D. discoideum were studied by phosphorus nuclear magnetic resonance. Under aerobic conditions, major intracellular phosphate compounds included phosphomonoesters, inorganic phosphate (Pi), ADP and ATP. Nucleotides were essentially as magnesium complexes. Two intracellular Pi resonances were clearly resolved and the corresponding pHs determined by the chemical shifts characteristics were 7.7 and 6.7. These intracellular pHs were strictly constant over an extracellular pH range between 5.0 and 7.5. The two cellular compartments defined by the Pi resonances were assigned to mitochondria (pH 7.7) and cytosol (pH 6.7) on the basis of their response to anaerobiosis or to carbonylcyanide-m-chloro phenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, which equilibrate the two intracellular pHs.     

Single ion channels in the slime mold Dictyostelium discoideum

Single ion channels with different conductances and gating characteristics were observed in the plasma membrane of the slime mold Dictyostelium discoideum by means of the patch-clamp technique in the cell-attached mode. The predominant channel type shows outward current flow, probably carried by K+ ions. The slope conductance of this channel is 9 pS and its probability to be open increases with depolarization of the membrane. The channel is observed from 1 to 8 h after the beginning of starvation.     

Identification of mRNA in the slime mold Physarum polycephalum.

Using a differential extraction procedure which had previously been shown to yield one nucleic acid fraction enriched in cytoplasmic RNA and another enriched in nuclear RNA, we have been able to isolate two polyadenylated RNA populations from microplasmodia of Physarum polycephalum. The poly(A)-containing RNA from the cytoplasmic-enriched fraction accounts for approximately 1.2% of the cytoplasmic nucleic acid, has a number-average nucleotide size of 1339+/- 39 nucleotides, and has been shown, in a protein-synthesizing system in vitro, to be capable of directing the synthesis of peptides which have also been shown to be synthesized in vivo by microplasmodia. The poly(A)-containing RNA from the nuclear-enriched fraction has a number-average nucleotide size of 1533 +/- 104 nucleotides and represents a mixture of cytoplasmic and nuclear adenylated RNA molecules. Based upon these observations, we have identified the polyadenylated RNA isolated from the fraction enriched in cytoplasmic nuclei acid as Physarum poly(A)-containing messenger RNA.