Patterns of oscillation during mitosis in plasmodia ofPhysarum polycephalum

Summary The rhythmic contraction pattern in plasmodia ofPhysarum polycephalum was studied to determine whether characteristic changes occur during the synchronized nuclear division. An electrical method that measures the contraction rhythm in situ during several cell cycles was used. Biopsies of the plasmodia were taken at 17 min intervals for precise determination of the cell cycle stages and were correlated with the simultaneously measured contraction rhythm. All measurements were performed in a temperature controlled environment (27 °C) at 100% relative humidity with the plasmodia (less than 24 h old) growing on a semi-defined agar medium. A total of 14 different plasmodia have been examined, and on one occasion the plasmodium was followed through 3 subsequent mitoses. The mitotic stages were identified with aceto-orcein coloring techniques and by fluorescence methods. Except for a few cases where a mitotic asynchrony of 2–3 min was observed, the mitotic events occurred simultaneously in the nuclei within a single plasmodium. Both the occurrence of the first mitosis after inoculation and the intermitotic times were highly variable. Our study indicates that the contraction rhythm in plasmodia ofPhysarum is unperturbed during the synchronized nuclear division. However, in 5 of the 17 examined mitoses an amplitude decay was observed. We discuss possible explanations for the obtained results with emphasis on the applied techniques, interpretation of the oscillation patterns, and possible restrictions in the cell itself.     

Propagated waves induced by gradients of physiological factors within plasmodia ofPhysarum polycephalum

Plasmodia ofPhysarum polycephalum were analyzed with the aid of cinematography and the infrared reflection technique for characterization of the phase behavior of their oscillating contraction activity, with special emphasis placed on the effects of temperature gradients. In response to temperature gradients, phase gradients were documented cinematographically as well as by infrared registration. A quantitative evaluation of the cinematographically recorded phenomena was carried out with the aid of streak photography. The phase gradient is directed across the region of the temperature gradient with a delay in phase toward the colder side. The correspondingly generated waves are as short as 1 mm and are propagated toward the colder region. A comparison of these waves with the known flickering phenomena in cinematographic films reveals a common nature of both.Supported by Deutsche Forschungsgemeinschaft.     

Periodic events and cell cycle regulation in the plasmodia ofPhysarum polycephalum

The multinucleated plasmodia ofPhysarum polycephalum, a myxomycete, have been extensively used in cell cycle studies. The natural synchrony of mitosis and DNA synthesis, easy culture methods, the ready fusions obtainable between plasmodia, and the amenability to phase specific studies, employing physical and chemical perturbers, are some of the attractive features of this organism. Because of the absence of a Gl phase in the plasmodia, there is a crowding of cell cycle specific marker events at the G2/M boundary, which reflect features of both the G2/M and the Gl/S boundaries of a typical eukaryotic cell. Prominent among these are the synthesis and overall activity of thymidine kinase, the co-triggering of tubulin and histone genes, translation of their mRNA, the organization and duplication of the microtubular organizing centres of the mitotic spindle and the triggering of cdc 2 kinase activity. These above events have not only served as good markers to monitor the progress of the plasmodial cell cycle, but have also been fairly thoroughly analysed by means of specific perturbers such as DNA synthesis inhibitors, antimicrotubular drugs, UV-irradiation, heat-shock etc. Along with fusion studies, these perturbation studies have been helpful in the formulation of various models on regulation of mitosis. These above aspects as well as prospects for future studies employing this organism are discussed This paper is dedicated to the memory of the late Prof. S C K Nair, formerly University Professor of Physics.     

Centriole maturation in the amoebae ofPhysarum polycephalum

Summary The precise geometry of pro-centriole formation has been studied inPhysarum polycephalum amoebae. The spatial references used were the posterior and the anterior kinetosomes which are unequivocally defined by the presence of the posterior para-kinetosomal structure, the microtubular array 4 and the microtubular arrays 1, 2, and 3. The observations made suggest that pro-centrioles follow a maturation process. A pro-centriole formed during the n^th cell cycle becomes the posterior kinetosome during the (n + 1)^th cell cycle and the anterior one during all the following cell cycles. Pro-centriole formation occurs late in the cell cycle. This observation disagrees with a role of pro-centriole formation in the regulation of S phase in contrast to what has been suggested in other eucaryotic cells.     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

Events in the amoebal-plasmodial transition ofPhysarum polycephalum studied by enrichment for committed cells

Summary Amoebae of strain CLof Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebalplasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. Committed cells can be cultured to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. Uninucleate committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial specific tubulin isotypes.Abbrevations SDM Semi-defined medium - DSDM Dilute semidefined medium - LIA Liver infusion agar - SBS Standard bacterial suspension - IEF Isoelectric focussing - SDS Sodium dodecyl sulphate - PAUF Precommitted amoebae unable to flagellate (for the explanation of these cells see text).     

Stabilization of monoasters by taxol in the amoebae ofPhysarum polycephalum (Myxomycetes)

Summary Although the plasmodial stage of the MyxomycetePhysarum polycephalum was unaffected with 200 M taxol, the amoebal stage was sensitive to 10 M taxol. The first effect of taxol resulted in an accumulation of cells blocked as a monopolar centrosphere surrounded by condensed chromosomes. In 79% of cases these monoasters contained two pairs of centrioles. The mitotic block in a monopolar stage in the presence of taxol delayed the occurrence of late mitotic events such as chromosome decondensation and formation of the nuclear envelope. Escape from the monopolar centrosphere stage and formation of multinucleated amoebae involved a transient monopolar reconstruction stage in which a long microtubular bundle interacted with a small chromosomal mass outside the monoaster.     

Synchronization and signal transmission in protoplasmic strands ofPhysarum

Isolated protoplasmic strands ofPhysarum polycephalum, mounted as a trapeze, show synchronous contraction activities when the isometric tension development of both arms of the trapeze is measured independently of each other. This phase regulation can be experimentally disturbed by temperature changes. Within a permanent gradient, however, the phases become resynchronized. The maximal temperature gradient between both arms allowing a phase resynchronization was approximately 9° C along a distance of 25 mm. The transmission of the signal along the middle piece of the trapeze (which, as the connecting part of both arms, is responsible for signal transmission in phase synchronization) can be influenced by temperature changes. The minimal temperature allowing a signal transmission is 15° C, the maximal temperature approximately 29° C. A morphological investigation of protoplasmic strands mounted as trapezes revealed that the normal architecture of the objects is not influenced by the experimental trapeze arrangement. Permanent thermal gradients induce thermotactic reactions, i.e., a preferred protoplasmic mass transport into one arm of the trapeze. This leads, after several hours, to a morphological asymmetry of the trapeze. In spite of the fact that this reaction limits the temporal use of trapezes within thermal gradients to 2–3 h, the capacity of such strands for phase regulation is not hindered. Thermal gradients are suitable methods for studying the unknown phase-regulating factor and its transmission. As criteria for an intact pathway of signal transmission, the capacity of the trapeze arms to resynchronize as well as to maintain synchronization within a thermal gradient can be used.Dedicated to A. Frey-Wyssling on the occasion of his 80^th birthday on November 8^th, 1980     

Fine structure of plasmodial nuclei in the slime moldPhysarum polycephalum I. Comparison of diploid and haploid vegetative mitosis

Summary The same basic ultrastructural features of interphase and mitotic nuclei were found for both the haploid Colonia and the diploid wild type strains of the myxomycete,Physarum polycephalum. Differences in nuclear size and chromocenter numbers were observed, but the nucleolar cycle and the intranuclear and acentriolar type of mitosis characteristic of the plasmodial stage of the diploid is present in haploid plasmodia, ruling out any relation between ploidy level and type of mitotic figure.     

Studies on microplasmodia ofPhysarum polycephalum

Summary The new technique of fluorescent analog cytochemistry was used to investigate the cell surface morphology (RITC-WGA staining), the organization of the microfilament system (Rh-phalloidin staining) and the spatial distribution of mitochondria (Rh-123 staining) in the various growth stages of axenically cultured living and fixed microplasmodia ofPhysarum polycephalum. The differentiation degree of the cell surface is generally size- and age-dependent: the invagination system develops by degrees from small spherical stages (50–100 m) without invaginations to large vein-like or dumbbell-shaped specimens (300–1,000 (m long) with extensive invagination systems. The microfilaments are always organized in a cortical system along the entire cell surface and sometimes in a fibrillar system as well, extending throughout the cytoplasmic matrix. Results on living microplasmodia demonstrate that the cortical microfilament system is mainly involved in motive force generation for changes of cell surface morphology and protoplasmic streaming activity, whereas the fibrillar system rather serves a stabilizing and adhering function. Moreover, the functional differences of the two microfilament systems are indicated by the position of a large population of stationary mitochondria in close vicinity to the cell surface, thus pointing to a reasonable arrangement of the energy-supplying and energy-transforming system.     

Cell membrane isolation from plasmodium ofPhysarum polycephalum

Plasmodia were fractionated to isolate a cell membrane rich fraction by sucrose density-gradient centrifugation. The fractions were identified by electron microscopic observation, PTA-chromic acid staining and assays of marker enzymes, applying the methods for cell membranes of higher plants. The cell membranes were recovered on the density of 1.13 g·cm⁻³.     

Relationship between endoplasmic and ectoplasmic oscillations during chemotaxis ofPhysarum polycephalum

Summary The plasmodium ofPhysarum polycephalum usually migrates coordinately as one whole body even in a complicated environment. By measuring oscillation phenomena in endoplasm and ectoplasm separately during chemotactic process, we studied the mechanism of information processing to achieve such a coordination. (1) The interaction between endoplasmic oscillators was long-range, competitive according to the length of period, and fast (18 cm/min). Ectoplasmic one was short-range. (2) After a partial stimulation of attractant to the organism, the period at the stimulated portion decreased first, and a global phase gradient appeared in endoplasm. Then ectoplasm at the non-stimulated portion was entrained to the endoplasmic pattern, and the migration direction at each part changed in accordance with the phase gradient as a whole body. (3) When the endoplasmic interaction was interrupted, the above coordinated response was not observed. These facts suggest that two-layer coupled oscillator system composed of endoplasm and ectoplasm play important roles for such an information integration.     

Information processing for the organization of chemotactic behavior ofPhysarum polycephalum studied by micro-thermography

Summary The spatial and temporal pattern of oscillating temperatures on the cell surface of a plasmodial strand ofPhysarum polycephalum was measured with a sensitive thermal image camera. The longitudinal tension of the strand was studied simultaneously. In the absence of chemical stimulation, the phases of the temperature oscillation observed at various portions of the strand were entrained with almost coincidental phase. The temperature and tension oscillation were synchronized, although the phase difference between them was occasionally changed. With local chemical stimulation, the phase of the temperature oscillation advanced in the portion to which the plasmodium would be induced to migrate. The phases between temperature and tension oscillations then became constant. The mechanism by which the plasmodium processes local information of chemical stimulus to global information for the migration is discussed.     

Ca2+ oscillation in the homogenate ofPhysarum plasmodium

Summary Using aequorin luminescence, we observed a distinct oscillation in Ca²⁺ levels in the supernatant of the homogenate ofPhysarum plasmodium. Ca²⁺ oscillation continued for 10–120 minutes, with a period coinciding with that of the contraction rhythm of a plasmodium.Abbreviations EDTA Ethylenediaminetetraacetic acid - EGTA Ethyleneglycol-bis-(-aminoethylether)-N,N-tetraacetic acid - PIPES Piperazine-N,N-bis-(2-ethane sulfonic acid) - DTT Dithiothreitol The present work was supported by Grants-in Aid from the Ministry of Education, Science and Culture, Japan.     

Amino acid pool and protein turnover during differentiation (spherulation) ofPhysarum polycephalum

The composition of the amino acid pool during spherulation was determined. It changes in size and in composition, the concentration of each amino acid behaving individually. The first response to the onset of spherulation either by starvation or osmotic shock (0.5 M mannitol) always is a decrease of the pool's size, which during further starvation expands for a short period and then decreases again. During development induces by mannitol in the presence of external amino acids, the pool size increases continuously after the initial depletion.As shown by radioactive labeling, amino acids were actively released from the plasmodium into a medium containing amino acids, but retained by the microplasmodia in an amino acid-free medium. The kinetics of the uptake of radioactive amino acids from the medium is biphasic, indicating the existence of multiple pools. Even after a labeling period of 8 h the amino acid pool is not yet in equilibrium with the medium. The possibility of a compartimentation of the pool was confirmed by density labeling of two different enzymes.Whereas the turnover of total protein is only very low during growth, it is rather high in spherulating microplasmodia. At least 70% of the originally existing protein is degraded during this development, while, simultaneously, at least 50% of the protein present after 24 h starvation is newly synthesized during that period.     

The centriole cycle in the amoebae of the myxomycetePhysarum polycephalum

Summary In the amoebae of the myxomycetePhysarum polycephalum, procentrioles are formed on the anterior and posterior centrioles in early prophase. Although the relative position of the parental and procentrioles is fixed, all relative positions of the daughter and parental centrioles were observed. During the different stages of mitosis daughter centrioles elongate and acquire anterior satellites, one of the characteristic features of the anterior centrioles. All other anterior morphological characteristics appear only in telophase and early reconstruction stages. In contrast to the parental posterior centrioles, which do not change morphologically during the successive mitotic stages, the parental anterior centrioles lose their morphological characteristics in late prophase and early prometaphase and then acquire the morphological features characteristic of the posterior centrioles. Thus, the following maturation scheme is suggested: a procentriole becomes an anterior centriole during the first mitosis and a posterior centriole during the second mitosis. Since posterior features are maintained during mitosis, the posterior centriole corresponds to the final state of centriole maturation.     

Electrical properties of the plasma membrane of microplasmodia ofPhysarum polycephalum

Summary Microplasmodia ofPhysarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30mm K⁺, 4mm Ca⁺⁺, 3mm Mg⁺⁺, 18mm citrate buffer, pH 4.7, 22°C), the transmembrane potential differenceV _m is around –100 mV and the membrane resistance about 0.25 m².V _m is insensitive to light and changes of the Na⁺/K⁺ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN^–, N ₃ ^– ),V _m drops to about 0 mV. Under normal conditions,V _m is very sensitive to external pH (pH _o ), displaying an almost Nernstian slope at pH _o =3. However, when measured during metabolic inhibition,V _m shows no sensitivity to pH _o over the range 3 to 6, only rising (about 50 mV/pH) at pH _o =6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1mm of the sugar. Sugar-induced depolarization was insensitive to pH _o . The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981,J. Membrane Biol. 63:165–190). Three transport systems are characterized: 1) An electrogenic H⁺ extrusion pump with a stoichiometry of 2 H⁺ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K⁺ pathways, there is a passive H⁺ transport system; here the protonated form seems to be positively charged. 3) A tentative H⁺-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Optical isolation of individual mitochondria ofPhysarum polycephalum for PCR analysis

Summary We attempted to amplify a specific region of mitochondrial DNA (mtDNA) using the polymerase chain reaction (PCR) from fewer than ten mitochondria isolated individually by microdissection or use of an optical tweezer. We selected preliminarily isolated mitochondria fromPhysarum polycephalum as the model materials and tried to amplify the mtDNA region corresponding to the specific mitochondrial plasmid of this true slime mould. For separation of a few mitochondria from the mitochondrial population, we initially used a destruction method in which excluded mitochondria were disrupted by a UV laser. However, mtDNA was still amplified, although weakly, from mitochondria that had been destroyed by the UV laser. Therefore, we used an optical tweezer to trap individual mitochondria and separate them from the others. The required number of mitochondria were separated from the mitochondrial suspension through a narrow canal of isolation buffer and used directly for PCR amplification. The results showed that the mtDNA could be amplified from at least 9 mitochondria trapped by the optical tweezer.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EDTA ethylenediaminetetraacetic acid - mtDNA mitochondrial DNA - PCR polymerase chain reaction     

A polarized light and electron microscopic study of the birefringent fibrils inPhysarum plasmodia

Summary The birefringent fibrils in thin-spread plasmodium ofPhysarum polycephalum have been investigated with both polarizing and electron microscopes. The birefringent fibrils were classified into three groups by polarized light microscopy. The first type of fibril is observed in the advancing frontal region as a mutual orthogonal array. The birefringence changes rhythmically in accordance with the shuttle streaming. The second type of birefringent fibril is located in the strand region and runs parallel or somewhat oblique to the strand axis. The third type is observed in the strand region always perpendicular to the streaming axis. Electron microscopy confirmed that all these fibrils are composed of microfilaments, which range in densities in the cross view of the fibril from 1.2 to 1.7 × 10³/m² (1.5 × 10³/(xm² on the average).