The role of microtubules in establishing nuclear spatial patterns in multinucleate green algae

Summary In uninucleate cells, cytokinesis follows karyokinesis, thereby reestablishing a specific nucleus-to-cytoplasm ratio. In multinucleate cells, cytokinesis is absent or infrequent; no plasmalemma boundary defines the cytoplasmic territory of an individual nucleus. Several genera of large multinucleate green algae were examined with epifluorescence light microscopy to determine whether the patterns of cytoplasmic organization establish nuclear cytoplasmic domains. Randomly spaced nuclei, singular mitotic events and cytoplasmic streaming characterize the organization of two genera,Derbesia andBryopsis (Caulerpales). The cells ofValonia, Valoniopsis, Boergesenia, Ventricaria (Siphonocladales), andHydrodictyon (Chlorococcales) display regularly spaced nuclei which undergo synchronous divisions in a stationary cytoplasm. In the cytoplasm of genera with regularly spaced nuclei, microtubules radiate from all nuclei in late telophase-early interphase. These internuclear microtubule arrays are not found in algal genera with randomly spaced nuclei. It is hypothesized that these microtubule arrays play a role in establishing the cytoplasmic domain of each nucleus in genera with regularly spaced nuclei. Loss of microtubule arrays during the events of mitosis correlated positively with the increasing randomization of nuclear patterns in algae grown in microtubule inhibitors. Cytoplasmic domains were maintained when cells were grown in the same media in the dark. This suggests that, after a round of division, regular nuclear spacing in certain multinucleate algae is reestablished by internuclear microtubule arrays, which are not, however, required to maintain spacing during interphase.Dedicated to the memory of Professor Oswald Kiermayer     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium     

Über einen intrazellulären Parasiten beiCryptomonas (Cryptophyceae), II.

A new intracellular parasite inCryptomonas spp. div. previously known is described asProteromonas steinii spec. nova. The flagellate (infectious) stage isBodo-like, with a distinct kinetoplast. The immobile parasitic stage occurs in the cells of Cryptomonads as a multinucleate trophocyst with a thin mucilage envelope (pseudocyst), while the hypnocyst is uninucleate, walled and bearing two horned projections.

     

A developmental mutation (npfL1) resulting in cell death in Physarum polycephalum.

In Physarum, microscopic uninucleate amoebae develop into macroscopic multinucleate plasmodia. In the mutant strain, RA614, plasmodium development is blocked. RA614 carries a recessive mutation (npfL1) in a gene that functions in sexual as well as apogamic development. In npfL+ apogamic development, binucleate cells arise from uninucleate cells by mitosis without cytokinesis at the end of an extended cell cycle. In npfL1 cultures, apogamic development became abnormal at the end of the extended cell cycle. The cells developed a characteristic rounded, vacuolated appearance, nuclear fusion and vigorous cytoplasmic motion occurred, and the cells eventually died. Nuclei were not visible by phase-contrast microscopy in most of the abnormally developing cells, but fluorescence microscopy after DAPI staining revealed intensely staining, condensed nuclei without nucleoli. Studies of tubulin organization during npfL1 development indicated a high frequency of abnormal mitotic spindles and, in some interphase cells, abnormally thick microtubules. Some of these features were observed at low frequency in the parental npfL+ strain and may represent a pathway of cell death, resembling apoptosis, that may be triggered in more than one way. Nuclear fusion occurred during interphase and mitosis in npfL1 cells, and multipolar spindles were also observed. None of these features were observed in npfL+ cells, suggesting that a specific effect of the npfL1 mutation may be an incomplete alteration of nuclear structure from the amoebal to the plasmodial state.     

Ultrastructural studies of the cell cycle in a multicentriolar form ofBracteacoccus minor (Chlorophyceae,Chlorellales)

Summary The ultrastructure of mitosis and cytokinesis is studied in the typical and a multicentriolar form of the multinucleate green algaBracteacoccus minor (Chodat) Petrovà. These processes are essentially identical in both forms, and are similar to those in other uni- and multinucleate chlorellalean algae. The mitotic spindle is closed and centric, and a fragmentary perinuclear envelope is present. In multinuclear cells mitosis is synchronous and may occur at the same time as cytokinesis. Cleavage is simultaneous and centrifugal, starting near the nucleus-associated centrioles and apparently mediated by phycoplast microtubules of the trochoplast type. Flagellated wall-less spores are usually formed. In the typical form ofB. minor, each interphase nucleus is associated with two mature centrioles (= one set) which function as centrosomal markers. At the onset of mitosis these centrioles duplicate and segregate and eventually establish the two poles of the spindle, where polar fenestrae develop in the nuclear envelope. In the multicentriolar form, however, each interphase nucleus generally is associated with two or three sets of centrioles. Consequently, during mitosis each half-spindle is associated with two or three sets. These centrioles are not necessarily all associated with the fenestrae at the spindle poles, but one or more sets are frequently associated with the nuclear membrane, more or less remote from the nuclear poles. However, the spindle in this multicentriolar form remains essentially bipolar. Cleavage generally results in zoospores with two, four or six flagella. The behaviour of the extra centrioles during the cell cycle and their possible relationship with centrosomes are discussed.     

Building a plasmodium: Development in the acellular slime mould Physarum polycephalum

The two vegetative cell types of the acellular slime mould Physarum polycephalum - amoebae and plasmodia - differ greatly in cellular organisation and behaviour as a result of differences in gene expression. The development of uninucleate amoebae into multinucleate, syncytial plasmodia is under the control of the mating-type locus matA, which is a complex, multi-functional locus. A key period during plasmodium development is the extended cell cycle, which occurs in the developing uninucleate cell. During this long cell cycle, many of the changes in cellular organisation that accompany development into the multinucleate stage are initiated including, for example, alterations in microtubule organisation. Genes have been identified that show cell-type specific expression in either amoebae or plasmodia and many of these genes alter their pattern of expression during the extended cell cycle. With the introduction of a DNA transformation system for P. polycephalum, it is now possible to investigate the functions of genes in the vegetative cell types and their roles in the cellular reorganisations accompanying development.     

Multinucleate plant cells: I. Aneuploidy in a proliferating population

A new method for the production of multinucleate plant cells in meristematic populations is described. This method involves the aneuploidy induction of nuclei of a same cell. Allium cepa root tips were chemically treated, and the multinucleate cells obtained were scored at interphase and mitosis. When meristematic cells are treated with γ-hexachlorocyclohexane in appropriate culture conditions, the anaphase chromatids are distributed into discrete unbalanced groups. This phenomenon has been profited for inducing viable multinucleate cells with aneuploid nuclei after cytokinesis inhibition with caffeine.     

Multinucleate cyst formation in Acetabularia mediterranea after x-irradiation

Cells of Acetabularia mediterranea were irradiated with increasing doses of X-rays (64.5–258·10^-4 kC kg^-1). The cells are radioresistant up to 193.5·10^-4 kC kg^-1 in terms of growth and progression through he life cycle but the morphogenesis of whorls, caps, and cysts is accompanied by morphological alterations. Microscopical examination of cyst bearing caps in irradiated cells has shown the presence of giant cysts neighboring particularly small ones. Photographic recording of cyst development showed that the multinucleate cap cytoplasm partitions into multinucleate portions rather than uninucleate ones as in the control cells. After complete cleavage a cyst wall is deposited onto the multinucleate cytoplasm. In contrast to uninucleate cysts with one lid the wall contains multiple lids. Their number appears to correspond to the number of nuclei in the cytoplasm compartment during cleavage. The results indicate that X-rays preferentially inhibit the synthesis of a factor which plays a role in establishing the normal spatial morphogenetic pattern necessary for cyst formation.     

Variants of asynchronous DNA synthesis and mitoses in multinucleate cells induced by cytochalasin B

Cases of asynchronous progression with separate nuclei of S-period and initial mitotic stages in multinucleate cells were discovered in Chinese hamster cell cultures during a prolonged action of cytochalasin B (7 days) and after its stopping (7 days of cell cultivation without drug). The interphase asynchrony under experimental conditions vary in value corresponding to the level of interphase asynchrony in spontaneous multinucleate cells in control cultures. So, the interphase asynchrony in cytochalasin B-induced multinucleate cells is suggested not to be connected with the drug action. Fusion of heterophase cells and a high level of proliferation activity of multinucleate cells seem to be the main reason of interphase asynchrony both in control cultures and in experimental conditions. Unlike the interphase asynchrony, the appearance of the mitotic asynchrony in multinucleate cells is shown to be connected with the action of cytochalasin B. The high level of the mitotic asynchrony remains after the stopping of drug action. A conclusion is made that mitotic asynchrony of nuclei, along with multipolar mitosis and cytokinesis inhibition, is one more display of the cytotoxic action of cytochalasin B on mitosis.     

Apogamic development of plasmodia in the myxomycetePhysarum polycephalum: a cinematographic analysis

Summary Strain CL ofPhysarum polycephalum forms multinucleate plasmodia within clones of uninucleate amoebae. The plasmodia have the same nuclear DNA content as the amoebae. Analysis of plasmodial development, using time-lapse cinematography, showed that binucleate cells were formed as a result of nuclear division in uninucleate cells. Binucleate cells developed into plasmodia by further nuclear divisions and cell fusions. No fusions involving uninucleate cells were observed. A temporary increase in cell and nuclear size occurred at the time of binucleate cell formation.     

Karyology and hyphal characters as taxonomic criteria in Ascomycetous black yeasts and related fungi

Mycelial development of seventy-three strains of black yeasts and related fungi were studied, and numbers of nuclei per hyphal cell were counted. Two main patterns were apparent in expanding hyphae, viz. (1) uninucleate expanding hyphal cells, septum formation strictly following mitosis, and (2) multinucleate, branched, aseptate hyphal tips, septa being formed in a later stage, leading to oligo- or uninucleate mature cells. Characteristic genera in the two groups areExophiala andAureobasidium, respectively. InZasmidium and in someRamichloridium species all mycelial cells are oligonucleate. The character is indicative for relationships at the family level in black yeasts.     

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.     

CHROMOSOME NUMBERS IN THE HYPOCREALES. II. ASCUS DEVELOPMENT IN NECTRIA CINNABARINA

The cytology of ascus development in Nectria cinnabarina was investigated with the orceinsmear technique, from crozier formation to ascospore maturation. At prophase I synapsis occurs while the chromosomes are still contracted, and the nucleus passes through dictyotene, a diffuse stage rarely seen in plants. A haploid complement of five chromosomes has been precisely determined. The first two divisions in the ascus constitute meiosis, and the third (mitotic) is followed by ascospore delimitation. A fourth division takes place in the ascospore, which is subsequently divided by a septum into two uninucleate cells. Of all species of Nectria thus far investigated N. cinnabarina is the only species in which additional nuclear divisions in the ascus do occur, accounting for the multinucleate condition in the ascospore cells. The bearing that this distinctive nuclear condition has on phylogeny and evolution in the Hypocreales is discussed.     

Cytokinesis of the binucleate zoosporangia of Allomyces macrogynus

Allomyces macrogynus, a true fungus, produces zoosporangia which discharge uninucleate zoospores after cytoplasmic cleavage. Binucleate zoosporangia of A. macrogynus were induced and examined to understand the basic principles of cytokinesis associated with the multinucleate zoosporangia. Development of cleavage membranes was visualized by constructing three dimensional models based on electron micrographs and confocal images. Cleavage membranes on the cleavage plane showed asymmetric ingression from the cortex, but cleavage of cytoplasm was completed by the fusion of cleavage membranes with plasma membrane. Also, the position of the cleavage plane was continuously rotated until settled at the last stage. These studies suggest that the positions of the numerous cleavage planes within a multinucleate zoosporangium are continuously adjusted during development of cleavage membranes. The final settlement of cleavage planes would define the exact boundary of cleavage planes and the expansion of cleavage membranes toward the boundary could complete the cleavage of cytoplasm.     

CELL DIVISION AND COLONY FORMATION IN THE GREEN ALGA COELASTRUM (CHLOROCOCCALES)1

Multinucleate cells of Coelastrum undergo precisely directed cytokinesis, guided by phycoplast microtubules, to form a number of uninucleate daughter cells which subsequently adhere to form characteristically patterned aggregates. As there is no movement of the daughter cells relative to one another before their adhesion, the disposition of cells in daughter colonies reflects the pattern of cytokinesis of parent cells. Centrioles lie at the poles of the mitotic nuclei which are partially enclosed by a perinuclear envelope of endoplasmic reticulum. The centrioles disappear at the time of cytokinesis of the parental cell and apparently reform de novo once the daughter cells have acquired a cell wall following their adhesion. The trilaminar layer of cell wall, often termed the pectic layer, does not stain with ruthenium red and resists acetolysis suggesting that it contains sporopollenin rather than pectin.     

Heterovesicula cowani N. G., N. Sp. (Heterovesiculidae N. Fam.), a Microsporidian Parasite of Mormon Crickets, Anabrus simplex Haldeman, 1852 (Orthoptera: Tettigoniidae)

ABSTRACT. Heterovesicula cowani , n. g., n. sp., is a dimorphic microsporidium described from the adipose tissue of the Mormon cricket, Anabrus simplex Haldeman. Proliferation of the microsporidium is by karyokinesis of uninucleate and binucleate cells to form binucleate and tetranucleate cells, respectively. These cells will undergo binary fission (merogony). Ultimately, the meronts undergo karyokinesis without subsequent cytokinesis producing spherical multinucleate plasmodia that are transitional to 2 types of sporogony. Transitional to disporoblastic sporogony, a fragile interfacial envelope delaminates from the plasmodium with morphogenesis to a monfiliform plasmodium consisting of fusiform binucleate diplokaryotic sporonts. These undergo karyokinesis to form tetranucleate diplokaryotic sporonts that undergo cytokinesis during disintegration of the plasmodium into isolated binucleate sporonts. Transitional to octosporoblastic sporogony, multinucleate plasmodia disintegrate into short monofiliform plasmodia of diplokaryotic sporonts which then segregate while undergoing gradual nuclear dissociation (haplosis by nuclear dissociation). These undergo two sequences of karyokinesis and subsequent multiple fission to form eight uninucleate (haploid) sporoblasts in a fusiform arrangement within a persistent envelope. Binucleate spores are ovocylindrical, about 5.4 × 1.7 μm (fresh), with an isofilar polar filament singly coiled about 11 turns. Uninucleate spores are ovoid to slightly pyriform, 4.0 × 1.7 μm (fresh), with an isofilar filament singly coiled about 9 turns. A new family, Heterovesculidae, is proposed for the new genus.     

Mapping the jp (jumbo pollen) gene and QTLs involved in multinucleate microspore formation in diploid alfalfa

The objective of this research was to map the jumbo-pollen trait in diploid alfalfa. Homozygous recessive (jpjp) plants are characterized by the complete failure of post-meiotic cytokinesis during microsporogenesis resulting in 100% 4n-pollen formation. Three F₁ segregating populations were produced and analyzed for pollen-grain production and the segregation of RFLP markers. The first cross combination did not segregate for the jumbo-pollen trait, but showed a clear segregation for multinucleate (bi-, tri- and tetra-nucleate)-microspore formation. Cytological analysis showed that few plants produced 100% normal (uninucleate) microspores, whereas most of them produced multinucleate microspores at a variable frequency (0–75%). Plants with multinucleate microspores always showed a prevalence of binucleated microspores, even though some plants showed a background presence of tri- and tetra-nucleate microspores. QTL analysis based on ANOVA I and Stepwise Multiple Regression identified three QTLs with a highly significant effect on multinucleate-microspore formation. Two cross combinations, subsequently executed, showed Mendelian segregation for the jumbo-pollen trait and were effective in locating the jp gene on linkage group 6 close to the Vg1G1b RFLP locus. Interestingly, this RFLP locus was also linked to one QTL for multinucleate-microspore formation. Genetic models are discussed concerning the presence in linkage group 6 of a cluster of genes involved in multinucleate-microspore formation together with possible relationships between the jp gene and the Vg1G1b QTL. Received: 28 January 1999 / Accepted: 16 December 1999     

MORPHOLOGY OF HYPOMYCES TRICHOTHECOIDES

Large, spirally coiled initials embedded in a subiculum develop into multicellular, multinucleate ascogonia. Hyphae grow up around them to form a prosenchymatous perithecial wall. The ascogonia give rise to multinucleate ascogenous cells from which croziers and asci form. As the ascocarp develops, an apical meristem produces uninucleate cells that elongate downward into long, slender filaments, the apical paraphyses. From a basal layer of ascogenous cells, asci grow up among the apical paraphyses, which disintegrate as the ascocarp matures. Ascospores are verrucose, with obtuse apiculi. This pattern of development is typical of the Nectria-type of Luttrell.     

Reproduction by Binary and Multiple Fission in Gigantomonas*

SYNOPSIS. Gigantomonas usually exists in the multinucleate stage. Hence, multiple fission is more common than binary fission. In its flagellate as well as its aflagellate state it is always an amoeboid cell which possesses from one to several large, clear pseudopodia. Fairly often in the multinucleate, as well as the uninucleate, stage no extranuclear organelles are present except large plain centrioles, two of which are always closely associated with each nucleus. During nuclear reproduction four centrioles, two old ones and two new ones, are always present with each nucleus. During all nuclear reproductions, regardless of the number of nuclei present, extranuclear organelles, such as flagella, axostyle, undulating membrane, and costa when present are discarded. If they are renewed, it is by the new centrioles at the same time that the old ones produce new central spindles, two always cooperating in the process. Thus, Gigantomonas, like other genera of the Devescovinidae, the family to which it belongs, never has for each nucleus present more than one set of extranuclear organelles, a characteristic which Devescovinidae and Lophomonadidae have in common. It is the only genus of Devescovinidae without a parabasal body. Owing to the liquidness of the cytoplasm, the central spindle, which becomes very long indeed, often extends beyond the cytoplasm and thus pushes the nucleus fastened to this end of it completely out of the cell. Mainly because of this situation, multinucleate forms with an odd number of nuclei occur often; otherwise the nuclear numbers would be 2, 4, 8, 16, 32, etc., because, no matter how many nuclei are present, they all reproduce simultaneously or nearly so. An unusual situation occurs in which Gigantomonas ingests and digests a small species of Holomastigotoides, while several hundred individuals of the latter become attached to and destroy Gigantomonas.