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.     

Use of enlarged cells and nuclei for studying mitosis inNeurospora

Summary Mitotic division stages studied by light microscopy in differentNeurospora crassa cell types clearly resemble prophase, metaphase, anaphase, and telophase stages of higher eukaryotes. 1. When conidia are cultured in liquid medium containing 3.22 M ethylene glycol, they grow without cell division, forming giant spheres with multiple nuclei. In a few giant cells, nuclear numbers remain small (1 to 3) but the nuclei become very large. Seven large chromosomes are seen in some nuclei suggesting polyteny, 14 or more chromosomes are seen in other, very large nuclei, indicating polyploidy. Cell volume and nuclear volume are positively correlated in giant cells. Nuclear divisions are not synchronous within individual multinucleate giant cells. 2. Nuclear division stages were also observed in crosses heterozygous for the dominant mutant banana where haploid prefusion nuclei in late-forming croziers revert to mitosis. Swollen ascogenous hyphae become highly multinucleate after several rounds of mitosis. Mitosis is completely synchronous in nuclei of the same crozier cyst, providing replicate information for unambiguous identification of division stage. 3. Observations are also reported of mitosis in a cell-wall deficient slime strain. Previous observations on mitosis in large nuclei of the ascus are summarized for comparison. The nucleolus persists throughout mitosis in the giant cells, multinucleate reverted croziers, and in the cell-wall deficient slime strain. It is expelled from the dividing nuclei in the ascus. Spindles and spindle pole bodies, which are normally conspicuous in asci, are also seen in normal and reverted croziers, but they have not been clearly identified in the ethylene glycol-induced giant cells.     

Morphology,development and cytology of Taphrina maculans Butler

Morphology, development and nuclear behavior of the ascogenous stroma and asci in the infection spots have been described inTaphrina maculans Butler. The fungus forms subcuticular and intercellular mycelium in the leaf tissues and the ascogenous layers originate through division of the subcuticular hyphal cells in the infection sites. Germination of ascogenous cells starts with their elongation in the uppermost layer forming asci and ascospores without formation of stalk cells. Meiosis of the fusion (diploid) nucleus occurs in the young ascus as in otherTaphrina species devoid of stalk cells. The haploid chromosome complement in this species consists of 3 chromosomes (n=3). All the cells in the stromatic layer are potential ascogenous cells and ascus formation continues, until all of them are exhausted in the infection spot. Eight ascospores are normally formed in each ascus, but multi-plication of ascospores may occurin situ later. Three morphologically distinct types of ascus opening are encountered, which are apparently not correlated with prevalent environment. Multiplication of ascospores after their discharge from mature asci occurs by budding proceded by a mitotic division of the spore nucleus. Blastospores (budded cells) germinate into short hyphae and binucleate condition of cells originates by mitotic division of the nucleus. Occurrence of giant cells containing 2 nuclei is often observed. Possible origin of Uredinales fromTaphrina-like ancestors has been indicated due to their close resemblance.     

The ultrastructure of septal pores and associated structures in the ascogenous hyphae and asci ofSordaria humana

Summary Septal pores and associated structures have been studied in ascogenous hyphae, croziers and asci ofSordaria humana by means of electron microscopy of serial and random sections. Pores exhibit variable structures from relatively simple pore caps to complex swollen rims with associated membrane cisternae. The simple types are found at the base of the ascogenous hyphae while the complex forms occur at the apex, in the croziers and in very young, presporulation asci. Post-sporulation asci contain a relatively simple type of pore structure. Cells which subtend the ascogenous hyphae exhibit both open and capped pores in their cross walls. Pore structures may be asymmetric in which case they show greater complexity on the side of the cross wall nearest to the apex or crozier. Membranous components of the complex pores are continuous with the endomembrane systems of the two adjacent cells and thus with the outer membranes of the nuclear envelopes. Membrane continuities may connect prefusion nuclei or fusion nuclei in penultimate cells, with nuclei of the stalk and terminal cells of croziers. Some speculation is presented as to the implication and possible roles of these structures in relation to cell differentiation within the ascogenous hyphae and croziers.     

Meiosis and ascospore development in Cochliobolus heterostrophus.

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

Meiosis and ascospore development in Cochliobolus heterostrophus

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

Studies on the development of perithecium of Ceratocystis stenoceras

The development of the perithecium of Ceratocystis stenoceras was observed by a light microscope and by a scanning electron microscope.The fungus has developed dark brown perithecia on wheat agar medium in three days of incubation. Perithecial primordia appeared as tightly knotted coils. At the center of it an oval ascogonium was observed. The ascogonium was developed from a lateral wall of a hypha, and the hyphae covering the ascogonium branched at the basal part where the ascogonium was attached. These hyphae branched repeatedly in the developmental growth to cover the ascogonium, and it was finally covered tightly. The plasmogamy of this fungus is much probably performed by the gametangial contact. As the stage proceeded, the ascogonium elongated, the terminal and the basal portions of it swelled and cleavage of the ascogonium resulted. Each of the cleaved ascogonia germinated continuously and stretched out the ascogenous hyphae. About that time the cells consisting of perithecia were vacuolated from the center and successively dissolved, so that a space was formed in the center of the body. Ascogenous hyphae continued to develop downwards, and their end were fixed to the inner wall of the body.The upper portion of the hyphae converged to the center of the body and the ascogenous hyphae became the supporting tissue for ascus formation.Hook formation was observed prior to the ascus formation. After completion of karyogamy by hook formation, the fissure appeared on the ascus and the end portion was released. The released portion included eight ascospores. The ascus had a smooth surface and no special structure was seen on the top. As the asci were matured, they evanesced by themselves and concurrently ascospores came out. Finally the body was massively filled with ascospores.     

MORPHOLOGY OF CHAETOMIUM ERRATICUM

Development of perithecia from single, uninucleate ascospores disclosed a homothallic condition for Chaetomium erraticum. This species was found to produce sessile ascogonial coil initials from uninucleate vegetative cells that become enveloped by hyphae formed at the base of the ascogonium. The ascogonium consists of several cells that are uninucleate or binucleate. A perithecium forms from numerous divisions and enlargement of the surrounding uninucleate cells. Differentiation of the perithecial cells results in the formation of a carbonaceous wall, perithecial hairs, and an ostiole lined with periphyses. A convex hymenial cluster of ascogenous cells forms in the lower half of the centrum from which typical croziers develop. Asci push up into the pseudoparenchyma cells of the centrum. The growth of the ascogenous system is in part responsible for increase in perithecial size. The breakdown of the pseudoparenchyma cells around the developing asci results in the formation of a central cavity in which ascospores are released when the asci deliquesce. No paraphyses are present. The type of development and features of the centrum of C. erraticum and other species of Chaetomium indicate a distinct Xylaria-type centrum.     

STUDIES IN THE GENUS NECTRIA. II. MORPHOLOGY OF N. GLIOCLADIOIDES

Hanlin , Richard T. (Georgia Experiment Station, Experiment.) Studies in the genus Nectria. II. Morphology of N. gliocladioides. Amer. Jour. Bot. 48(10): 900–908. Illus. 1961.—Swollen tips of vegetative hyphae develop into multicellular archicarps from which multinucleate ascogonia form. From basal cells of each archicarp arise hyphae which grow up into a prosenchymatous, true perithecial wall; around this wall is formed a thin pseudoparenchymatous stroma of compacted hyphae. The ascogonia give rise to ascogenous cells from which croziers and asci form directly. At the same time, an apical meristem forms cells that grow downward into the centrum. These are pseudoparaphyses. Asci grow up among the pseudoparaphyses, which deliquesce as the ascocarp matures. The ascus tip contains a thick ring with a pore and lateral thickening of the ascus wall. Ascospores are forcibly ejected. The chromosome number is 4. This species conforms to the Nectria Developmental Type of Luttrell.     

Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora

Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.     

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.     

THE DEVELOPMENT AND CYTOLOGY OF PYCNIDIOPHORA DISPERSA

Ascocarp development in Pycnidiophora dispersa is similar to that in Phaeotrichum. A stroma originates in an intercalary position on a hypha. It increases in size, and the outer cell layer differentiates to form the wall. The ascogenous system forms from a mass of fertile cells in the center of the centrum. These become enlarged and multinucleate and give rise to ascogenous hyphae which form asci at their tips by means of croziers. In time, most of the cells of the centrum become fertile and give rise to ascogenous hyphae. There are no sterile threads in the centrum and no hymenium is present, the asci being scattered throughout the locule. The haploid chromosome number is n = 6.     

Hypoxylon cohaerens: Cytology of the ascus

The haploid chromosome number ofHypoxylon cohaerens apparently is 5, based on counts made at meiotic prophase and meiotic and mitotic metaphases. Newly formed ascospores are at first uninucleate, becoming binucleate following mitosis in the ascospore. Subsequently, one of the two nuclei disappears. Maturing ascospores are uninucleate.Scientific Paper No. 3732 Washington State University. College of Agriculture, Project 1767. This study was supported in part by National Science Foundation Grant GB-19924.     

Immunofluorescence microscopy of the microtubule cytoskeleton during conjugate division in the dikaryon Pleurotus ostreatus N001

Fluorescence microscopy was used to describe the distribution of nuclei and the organization of the microtubule network in hyphae of Pleurotus ostreatus. Dikaryotic hyphae of P. ostreatus N001 grow by tip extension with two closely spaced nuclei moving slowly forward with the growing hyphal tip. During vegetative growth of the hyphae, cytoplasmic microtubules are found as long filaments oriented longitudinally within fungal hyphae. When the apical cell reaches a length of approximately 150 μm, the two nuclei divide synchronously. Mitosis occurs in association with clamp connection formation, with one of the nuclei dividing in the hook of the developing clamp connection and the other in the main hypha. After mitosis, two daughter nuclei move forward to approximately the center of the apical cell, while the other two move backward to a central position in the subapical cell. Two septa are formed, one in the clamp and the other across the main axis of the hypha to delimit the apical cell. The use of fluorescence microscopy made it possible to examine the changes in the cytoplasmic microtubules, the configuration of the mitotic apparatus, the site of septation and the post-mitotic nuclear migrations during conjugate division in P. ostreatus dikaryotic hyphae.     

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.     

THE MORPHOLOGY AND CYTOLOGY OF PREUSSIA FUNICULATA

The vegetative nuclei of Preussia funiculata (Preuss) Fuckel appear to divide in two ways. One is very similar to mitosis in higher plants except that no typical metaphase is present. The other consists of elongated nuclei splitting longitudinally into two halves. Ascocarp development is similar to that found in the Pleosporales. A stroma originates in an intercalary position on a hypha. It increases in size, and the outer cell layers differentiate to form the wall. The ascogenous system arises from multinucleate ascogonial cells scattered throughout the centrum. These give rise to large, lobate, multinucleate cells which in time form asci by means of croziers. The mature centrum contains a distinct hymenium and paraphysoids. The haploid chromosome number appears to be 12.     

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     

MORPHOLOGICAL STUDIES IN PODOSPORA ANSERINA

Perithecium development in Podospora anserina begins with the formation of a coiled ascogonial initial that arises as a lateral branch from a vegetative hypha. Hyphae grow up around the initial, forming an envelope that will become the ascocarp wall. As the ascocarp increases in size, several layers of thin-walled pseudoparenchyma cells form inside the wall, especially at the apex of the ascocarp. Paraphyses arise both from the base of the ascocarp and from the innermost layer of pseudoparenchyma cells and grow inward and upward, completely filling the centrum with tightly packed filaments. During development of the ascocarp the ascogonium proliferates to form ascogenous hyphae along the base of the centrum. Asci arise from the ascogenous hyphae and grow up among the paraphyses. Meristematic growth at the ascocarp apex results in the formation of an ostiole lined with periphyses. Centrum structure in P. anserina could be interpreted as intermediate between the Xylaria and Diaporthe types.     

Hypoxylon rubiginosum: Cytology of the ascus and surface morphology of the ascospore

Summary The haploid chromosome number ofHypoxylon rubiginosum is 5. The ascospore is uninucleate when formed, becoming binucleate following a mitosis. One of the nuclei subsequently disintegrates. Maturing ascospores are uninucleate.The morphology of the ascospore, as revealed by the scanning electron microscope, is described. The outer wall layer — the perisporium — shows heretofore undescribed surface fibrils. The possible significance of the fibrils is discussed.Paper No. 3205. Washington State University College of Agriculture Project 1767. This study was supported in part by National Science Foundation Grants GB-5219 and GB-8004.     

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica

Summary In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one binucleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascospore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny on non-allelic auxotrophs since random spore isolates are made without distinguishing between mono-and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.