Calcium accumulation in plasmodia of Physarum polycephalum

The total concentration of calcium in the endoplasm of plasmodia of was measured using a calcein fluorescence-quenching technique. The calcium concentration of the endoplasm increases with the time of cultivation on different substrates under culture conditions frequently used for routine experiments. Calcium accumulation within endo- and ectoplasm as well as in microplasmodia is most pronounced when plasmodia are starved and illuminated. Starvation and illumination lead to calcium concentrations frequently exceeding 200 mM in the case of macroplasmodia and 20 mM in the case of microplasmodia.     

Multiple effects of 5 mM sodium butyrate on Physarum polycephalum macroplasmodia

Summary In a Physarum polycephalum macroplasmodium, nuclei naturally divide synchronously. Thus, it offers an opportunity to study growth and mitosis within a true organism. The effects of 5 mM sodium-butyrate on these processes have been examined. When this material is added to the culture medium during mitosis, the butyrate acts like a fixative on condensed chromosomes. During interphase, this short fatty acid stops growth and immediately inhibits DNA synthesis. Furthermore, it prevents differentiation in macroplasmodia induced to spherulate. All these modifications are readily reversible after transfer to a medium lacking butyrate.     

Studies on microplasmodia ofPhysarum polycephalum: Endocytotic activity,morphology of the vacuolar apparatus and defecation mechanism

Summary The investigation of endocytotic processes in axenically cultured microplasmodia ofPhysarum polycephalum is considerably complicated by the development of an extensive cell membrane invagination system. Cross-sections through single channels of this system are difficult to distinguish from vacuoles formed endocytotically. Therefore the whole system was labelled by staining the extracellular slime with ruthenium red or lanthanum hydroxide. In this way endosomes produced during the incubation period could be clearly identified. Aerosil andThorotrast are suitable markers for food vacuoles because they can easily be detected with the electron microscope. The application of these substances revealed that submerged cultured microplasmodia are able to form endosomes which contain material of extracellular origin. However, the endocytotic uptake of food material is of much less intensity than in normal macroplasmodia. Microplasmodia seem to cover most of their requirements for metabolic substances by active trans-membrane transport.The intracellular digestive system of microplasmodia corresponds to the vacuolar apparatus of other cells. Preexisting lysosomes originating by autophagic processes play a central role in this system: They coalesce with endosomes or secondary lysosomes thus forming digestion vacuoles. Indigestible food components are extruded together withCa-containing granules into the cell surface invagination system by defecation. The physiological significance of theCa-granules is unknown.     

Identification of ubiquitinated histones 2A and 2B in Physarum polycephalum. Disappearance of these proteins at metaphase and reappearance at anaphase

Ubiquitinated histones uH2A.1, uH2A.Z, and uH2B have been identified in the basic nuclear proteins of the slime mold Physarum polycephalum by three methods: peptide mapping, cross-reaction with anti-ubiquitin antibody, and uH2A and uH2B isopeptidase cleavage. In microplasmodia, uH2A amounts to 7% of H2A and uH2B amounts to 6% of H2B. Detailed studies of mitosis in Physarum polycephalum macroplasmodia show that in early prophase, which last 15 min, the uH2As and uH2B are both strongly present, whereas minutes later in metaphase, which lasts 7 min, they disappear. When the nuclei enter anaphase, which lasts 3 min, both the uH2As and uH2B reappear. These precise studies suggest that cleavage of ubiquitin from the uH2As and uH2B is a very late, possibly final event in chromosome condensation to metaphase chromosomes and that ubiquitination is an early event in their decondensation. It is proposed that the uH2A and uH2B mark specific regions of the genome which have to be deubiquitinated prior to packaging into metaphase chromosomes; after metaphase these regions are the first to be decondensed and ubiquitinated. This modification, however, is not thought to be a general factor in chromosome condensation but labels a specific subcomponent of chromatin containing the expressed genes of a particular cell type or an important subset of these genes required by the cell to be available for activation, e.g. stress genes.     

Formation and division of binucleated cells in kidney cell cultures of microtus agrestis

Summary Epithelial kidney cell cultures of Microtus agrestis contain 10 to 25% binucleated cells. Observations of living cells under the phase contrast microscope showed that binucleated cells can arise by nuclear mitosis without cytoplasmic division. When binucleated cells divide the two nuclei are highly synchronized as they enter mitosis. In mitosis the chromosomes of both nuclei combine to a common metaphase plate leading to polyploid cells. In one case a tripolar spindle was seen after formation of a metaphase by the chromosomes of the two nuclei of a binucleated cell. This tripolar mitosis resulted in one binucleated and one mononucleated cell. The DNA-content (Feulgen photometry) and the distribution of heterochromatic bodies of the nuclei were corresponding to a tetraploid, a triploid and a haploid chromosome set. This suggests the possibility of somatic segregation of complete haploid sets.Supported by the Deutsche Forschungsgemeinschaft.     

CYTOLOGICAL STUDIES IN LACCARIA (AGARICALES). I. MEIOSIS AND POSTMEIOTIC MITOSIS

In the genus Laccaria, basidial formation, dikaryotic basidia, karyogamy, and meiosis were generally similar to structures and phenomena reported for other Agaricales. The haploid nuclei of dikaryotic basidia resided side by side in the basidium prior to karyogamy. Following karyogamy a single nucleolus was observed in L. montana (four-spored species); several nucleoli remained in the nucleus of L. tortilis (two-spored species). The haploid number of chromosomes for L. montana appeared to be n = 9. Postmeiotic mitosis typically occurred in the basidiospores resulting in binucleate basidiospores for four-spored species and quadrinucleate basidiospores for two-spored species. Postmeiotic mitosis sometimes occurred in the sterigmata and basidia proper. In instances where postmeiotic mitosis occurred in basidia, mature basidiospores were not formed and the basidia were collapsed, and contained up to eight nuclei.     

Identification of tubulin isoforms in the plasmodium of Physarum polycephalum by in vitro microtubule assembly

The tubulins of the plasmodium of Physarum polycephalum have been identified by in vitro microtubule assembly from partially purified extracts of asynchronous microplasmodia and late G2 macroplasmodia. The plasmodial tubulin group comprised of 2 alpha tubulins (app. m.w. 51000 daltons) and 2 beta tubulins (app. m.w. 58000 daltons and 55000 daltons) and appeared to be identical with a group of polypeptides which are synthesized periodically in late G2. Two of the plasmodial tubulin subunits (one alpha and one beta) were identical to the Physarum amoebal tubulin alpha and beta subunits as characterised by 2D gel positions.     

RNA Polymerase B levels during the cell cycle ofPhysarum polycephalum

Summary Tritiated -amanitin has been used as a specific and sensitive probe to estimate the number of RNA polymerase B molecules in isolated nuclei, chromatin and nucleoids, obtained from macroplasmodia ofPhysarum polycephalum. During mitosis (metaphase±10 min) there is at least 10-fold less RNA polymerase B than at all phases of the cell cycle, even if DNA replication has been blocked in vivo. It is concluded that many of the RNA polymerase B molecules leave the chromatin during decondensation of the chromosomes in telophase of the synchronous nuclear division ofPhysarum.     

Changes in diadenosine tetraphosphate levels in Physarum polycephalum with different oxygen concentrations.

Cellular levels of diadenosine tetraphosphate (Ap4A) were measured, by a specific high-pressure liquid chromatography method, in microplasmodia of Physarum polycephalum subjected to different degrees of hypoxia, hyperoxia, and treatment with H2O2. Ap4A levels increased three- to sevenfold under anaerobic conditions, and the microplasmodia remained viable after such treatment. Elevated levels of Ap4A returned to the basal level within 5 to 10 min upon reoxygenation of the microplasmodia. The increases in Ap4A levels were larger in stationary-phase or starved microplasmodia than in fed, log-phase microplasmodia. The maximal increase measured in log-phase microplasmodia was twofold. No significant changes in Ap4A levels occurred in microplasmodia subjected to mild hypoxia, hyperoxia, or treatment with 1 mM H2O2. These results indicate that in P. polycephalum, Ap4A may function in the metabolic response to anaerobic conditions rather than in the response to oxidative stress.     

The control of mitosis in Physarum polycephalum: The effect of lowering the DNA: Mass ratio by UV irradiation

A model for the control of mitosis is presented and, along with four other models described previously, is tested by the response of Physarum polycephalum to UV irradiation. Plasmodia were irradiated following the second mitosis (MII) after fusion of microplasmodia. As shown by other authors, the onset of the next mitosis (MIII) was delayed but the period MIII–MIV was shortened relative to control plasmodia. It is shown that the period MIII–MIV cannot be shortened beyond a minimum of 6 h despite increasing doses of UV. This minimum length is shown to be relatively independent of growth rate. If conditions were such that the length of MIII–MIV was shortened to this minimum value the length of MIV–MV was also shorter than the corresponding control period. If the period MII–MIV was longer than the minimum following irradiation then the length of MIV–MV was not shortened. It is argued that only the latter situation allows models to be tested and it is shown how the observed result is consistent with only two of the five models considered. A further test compared the length of MIII–MIV under these conditions with that predicted from the amount of DNA destroyed by the UV. This result was consistent only with the same two models.     

MITOSIS AND CYTOKINESIS IN THE PRASINOPHYCEAE. I. MANTONIELLA SQUAMATA (MANTON AND PARKE) DESIKACHARY

Mitosis in Mantoniella squamata (Manton and Parke) Desikachary, a small scale-covered green monad, is presented. Organelle replication precedes nuclear division and begins with the replication of the chloroplast. As the chloroplasts separate, the Golgi and flagellar apparatuses divide. The discoid microbody enlarges and becomes ‘V'-shaped, with the arms extending toward depressions in the pyrenoid stalks of the chloroplasts. At prophase, microtubules produced by an amorphous microtubule organizing center enter the nucleus via polar fenestre. The nuclear membrane remains intact. As the chloroplasts migrate further apart, the spindle pole-to-pole distance increases. By metaphase, daughter-cell lobes are discernible as a cleavage furrow, which appears as early as prophase, and begins to incise the cell. A single Golgi apparatus is situated near the spindle pole; the flagellar apparatus lies adjacent to the pole. The cleavage furrow continues to constrict the cell, resulting in a narrowing isthmus containing the elongate microbody, nucleus and a rootlet system connecting the basal bodies of the daughter flagella. At telophase, no extra-nuclear microtubular systems other than the previously observed rootlet are present and the nuclei remain separated from each other. In cells undergoing multiple divisions to produce more than two daughter cells, the orientation of organelles changes somewhat, with the basal bodies and the Golgi apparatus separating daughter nuclei prior to the onset of cytokinesis. The mechanics of mitosis in Mantoniella are compared with other green monads and the evolutionary implications discussed.     

Mitosis and autospore formation in the green algaKirchneriella lunaris

Summary Asexual reproduction inKirchneriella lunaris involves autospore formation. After an initial mitosis, the curved cell cleaves to a variable extent, and then the nuclei divide again; finally the cytoplasm is partitioned into four around each nucleus. Rudimentary centrioles appear prior to the first mitosis; centriole complexes then become associated with a developing sheath of extranuclear microtubules at prophase; fenestrae appear at the poles through which both microtubules and centrioles migrate, preceding intranuclear spindle formation. The nucleus meanwhile is enveloped by a perinuclear layer of endoplasmic reticulum which is also interposed between the golgi body and nuclear envelope. Chromosome separation is accompanied by considerable spindle elongation. Finally the reforming nuclear envelope excludes both centriole complex and interzonal spindle apparatus from daughter nuclei. Cleavage is preceded by i) nuclear movement to the cell center, ii) movement of centriole complexes around daughter nuclei until they are opposite one another, and iii) the concurrent formation of a system of transverse microtubules extending across the cell. Other microtubules encircle the cell predicting the cleavage plane. A septum then appears amongst these cytokinetic microtubules, possibly derived from the plasmalemma; it extends across the cell too, through the cleaving peripheral chloroplast. Secondary mitoses follow (as above) during which this septum may be partially resorbed. Finally this septum is reformed, if necessary, and two other septa appear (as above) to quadripartition the cell. Mitotic and cytokinetic structures in this algae are briefly compared with some others.     

ENDOMITOSIS IN TAPETAL CELLS OF EREMURUS (LILIACEAE)

True endomitosis in the anther tapetum of the liliaceous plant Eremurus is described. The nuclear membrane does not disappear, but during metaphase the chromosomes are condensed, often considerably more than in normal mitosis. When the pollen mother cells (PMCs) go through the last premeiotic mitosis, the tapetal cells have one diploid nucleus which divides while the cell remains undivided. The two diploid nuclei may undergo an endomitosis and the resulting tetraploid nuclei a second endomitosis. An alternative pathway is an ordinary mitosis—again without cell division—instead of one of the endomitotic cycles. The cytological picture in the tapetum is further complicated by restitution in anaphase and fusion of metaphase and anaphase groups during mitosis, processes which could give rise to cells with one, two, or three nuclei, instead of the expected two or four. No sign of the so-called “inhibited” mitosis is seen in these tapetal cells. When the PMCs are in leptotene-zygotene, very few tapetal nuclei are in endomitosis. When the PMCs have reached diplotene, almost 100% of cells which are not in interphase show an endomitotic stage.     

Mitosis in the Hemoflagellate Trypanosoma cyclops*

SYNOPSIS. The ultrastructure of interphase and mitotic nuclei of the epimastigote form of Trypanosoma cyclops Weinman is described. In the interphase nucleus the nucleolus is located centrally while at the periphery of the nucleus condensed chromatin is in contact with the nuclear envelope. The nucleolus fragments at the onset of mitosis, but granular material of presumptive nucleolar origin is often recognizable in the mitotic nucleus. Peripheral chromatin is in contact with the nuclear envelope throughout mitosis, and it seems reasonable to assume that the nuclear envelope is involved in its segregation to the daughter nuclei. Spindle microtubules extend between the poles of the dividing nucleus and terminate close to the nuclear envelope. The basal body and kinetoplast divide before the onset of mitosis and do not appear to have any morphologic involvement in that process. Spindle pole bodies, kinetochores, and chromosomal microtubules have not been observed.     

Effect of ADP-ribosylation on differentiation in Physarum polycephalum

Abstract. We studied ADP-ribosylation in the vegetative life cycle of the myxomycete Physarum polycephalum . Proliferating macroplasmodia are delayed in their progression through the cell cycle by the specific ADP-ribo-syltransferase inhibitor 3-methoxybenzamide. DNA and RNA synthesis is depressed. During the differentiation of microplasmodia into quiescent microsclerotia, ADP-ribosylation strongly increases in an early stage. The same stage is sensitive towards treatment with 3-methoxybenzamide, which delays the termination of the sclerotization process. The increase of ADP-ribosylation is not evenly distributed among all nuclear acceptor proteins. Histones H3 and H4 are modified to a lower extent in relation to H2A and H2B at the time of maximum ADP-ribosylation. Germination of microsclerotia into growing plasmodia is also repressed by 3-methoxybenzamide.     

Observations On Nuclear Division in vegetative hyphae of aquatic fungi

Summary Achlya aquatica, A. prolifera andA. racemosa are additional examples of fungi in whose vegetative mycelium, the nuclei do not divide in a manner directly comparable to ordinary mitosis. During division the entire nucleus, which consists of variously shaped chromatin and the nucleolus, becomes angular and elongates. The chromatin separates into two portions, which are situated at opposite ends of the elongating nucleus. As division proceeds the nuclei separate. Each sister nucleus sonsists of chromatin and a portion of the divided nucleolus. Spindless, chromosomal filaments or metaphase plates could not be observed during division.     

NUCLEAR BEHAVIOR IN THE BASIDIOMYCETE,VOLVARIELLA VOLVACEA

The basidiospores of the straw mushroom are typically uninucleate and its vegetative hyphae are generally multinucleate. There is a marked reduction of nuclear number in the trama and subhymenium. Interphase nuclei exist in two forms, each of which undertakes a particular mode of division. The “diffused” nuclei divide by conventional mitosis while the “constricted” ones divide amitotically. In metaphase of mitosis nine chromosomes were seen both in polar and lateral view. This haploid number confirms the nine bivalents found in basidia during meiosis. A unique characteristic of this fungus is that the diploid nucleus, the two postkaryotic nuclei and the four postkaryotic nuclei may be enclosed by a well-defined nuclear envelope during division.     

Divide and conquer: cytokinesis in plant cells

Plant cells divide in two by constructing a new cell wall (cell plate) between daughter nuclei after mitosis. Golgi-derived vesicles are transported to the equator of a cytoskeletal structure called a phragmoplast, where they fuse together to form the cell plate. Orientation of new cell walls involves actindependent guidance of phragmoplasts and associated cell plates to cortical sites established prior to mitosis. Recent work has provided new insights into how actin filaments and other proteins in the phragmoplast and cell plate contribute to cytokinesis. Newly discovered mutations have identified a variety of genes required for cytokinesis or its spatial regulation.     

LIGHT-DEPENDENT INHIBITION OF CELL DIVISION AND RHIZOID ELONGATION IN GEMMAE OF THE FERN,VITTARIA GRAMINIFOLIA

Gametophytes of the shoe-string fern Vittaria graminifolia produce linear, six-celled propagules called gemmae. The terminal cells of each gemma elongate into primary rhizoids in culture, and the inner body cells divide asymmetrically to produce prothallial or rhizoid initials. The initiation of both asymmetric cell division and rhizoid elongation is delayed by light intensities greater than 2 w/m². The maximal rates of cell division and rhizoid elongation are unaltered. A 24-hr pulse of high light intensity delays cell division and rhizoid elongation to the same extent, whenever applied during the first 3 d of culture. The model we propose for cell division hypothesizes the existence of a preparatory phase of finite duration prior to mitosis that is sensitive to light intensity. If a cell is irradiated by light intensities greater than 2 w/m² while in the preparatory phase, its entrance into mitosis is delayed. A similar model is proposed for the initiation of rhizoid elongation. Despite the fact that both cell division and rhizoid elongation are dependent on photosynthesis, direct measurements of CO₂-uptake rates show that the inhibitory effects of high light intensities are not due to an inhibition of photosynthesis.