Griseofulvin-induced alterations in site of dividing nuclei and structure of septa in a dikaryon ofSchizophyllum commune

Summary Ultrastructural study of a dikaryon of the basidiomyceteSchizophyllum commune showed that treatment with griseofulvin affected the site of the dividing nuclei and the location and structure of the septa. The microtubules were considered to be the primary target of griseofulvin, since they participate in nuclear division and movement in the hyphae, and their assembly is known to be in other organisms than fungi inhibited by griseofulvin. It is pointed out that dikaryotic hyphae with two nuclei and a clamp connection per cell are more sensitive indicators of the effect of griseofulvin than homokaryotic hyphae, whose structure is less complex.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.     

MITOSIS AND CLAMP FORMATION IN THE FUNGUS PORIA MONTICOLA

Nuclear division in P. monticola is in general similar to mitosis in higher organisms. Synchronous division of the nuclei in the dikaryon progresses with clamp development. Mitosis begins with the movement of the centriolar plaques into and under the forming clamp. The pull of the centriolar plaque on the attached nucleolus forms a long strand of nucleolar material. Chromosomes now appear as dense granules at the end of the nucleus proximal to the clamp. At this time the nucleolus moves adjacent to the centriolar plaque and contracted chromosomes. The nuclear membrane at least partially disintegrates, and the nucleolus is released into the cytoplasm where it may persist through telophase. A faintly staining spindle is often observed, and it produces a “double bridge” effect in separating chromatin. Somatic chromosomes are attached together forming strings that appear double and at least partially separated before metaphase.     

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.     

Intranucleäre Mitosen in homokaryotischen und dikaryotischen Mycelien der Basidiomyceten

Zusammenfassung In homokaryotischen und in dikaryotischen Mycelien der Basidiomyceten wird das Volumen des prämitotischen Kernes dadurch reduziert, daß der eigentliche Teilungskern von einem membranumgebenen Restkern abgetrennt wird. Da die Kernmembran während der gesamten Mitose persistiert, arbeitet der Spindelapparat intranucleär.

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Intranuclear mitosis in homokaryotic and dikaryotic mycelia of basidiomycetes

Summary In homokaryotic as well as in dikaryotic mycelia of Basidiomycetes the volume of the premitotic nucleus is reduced by separating the division nucleus from a residual nucleus which is surrounded by a membrane. For the nuclear membrane persists throughout the mitosis the spindle apparatus acts intranuclear.

     

Occurrence of microtubules in the hyphae of Schizophyllum commune during intercellular nuclear migration

Summary During the intercellular nuclear migration of the basidiomycete Schizophyllum commune cytoplasmic microtubules were frequently observed scattered in the hyphae around interphase nuclei and connected with a semiglobular structure at the poles of mitotic and postmitotic nuclei. Thus it seems possible that microtubules, which have been demonstrated to participate in the intracellular nuclear movements in the dikaryotic hyphae of the basidiomycetes, are also involved in the intercellular nuclear movements of these fungi. During hyphal fusion microtubules close to an interphase nucleus were connected with electron-dense structures. It is suggested that these structures are centers for the assembly of microtubules necessary for nuclear movements not associated with nuclear divisions.Abbreviations KCE kinetochore equivalent - ch chromatin - cw cross wall of septum - ge semiglobular end of KCE - gm grey material - m mitochondrion - mp middle plate of KCE - mt microtubules - n nucleus - ne nuclear envelope - nu nucleolus - s electron-dense structure connected with microtubules     

Direct studies of dikaryotization in Schizophyllum commune

The growth, duplication and fate of multikaryotic hyphae bearing true clamp connections, as derived from compatible matings of Schizophyllum commune, were studied by phase contrast microscopy. The nuclei (N) of multikaryotic apices maintained a near central position during hyphal growth. True clamp connection formation occurred with near synchronous mitosis followed by septal synthesis across the clamp neck and main hyphal axis. Nuclear progeny after mitosis in a hexakaryon included 6 N in the apex, 1 N in the clamp and 5 N in the penultimate cell; the solitary nucleus in the clamp later entered the penultimate cell. Similar events occurred for clamp connection formation and mitosis in the trikaryon, quadrikaryon or pentakaryon, whether in the apex or primary branches. Nuclear content of the multikaryotic apex (2 N through 10 N) had no apparent effect on the rate of individual hyphal growth. Reduction of the nuclear number in a trikaryon occurred by long-term entrappment of a solitary nucleus in the clamp and subsequent outgrowth of the dikaryotic penultimate cell. Occasionally, more than one nucleus became entrapped in the clamp cell. The ephemeral nature of the multikaryon was indicated by the fact that older cultures appeared to be exclusively dikaryotic hyphae at the colony periphery.     

Nuclear division cycle in Neurospora crassa hyphae under different growth conditions.

Treatment with picolinic acid blocked Neurospora crassa nuclei in G1, and recovery from the treatment allowed a synchronous wave of deoxyribonucleic acid synthesis to occur. Nuclei, which appeared as compact globular bodies during the period of blockage, assumed a ring shape during the following S phase, which was also maintained in the G2 phase. The proportion of compact globular nuclei was much higher in hyphae growing at lower rates, whereas that of ring nuclei increased when the hyphae were growing at higher rates. Horseshoe nuclei (probably mitotic nuclei) and double ring nuclei were also observed in growing hyphae, but their frequencies were low and fairly independent of the rate of growth. The length of the S phase of the Neurospora nuclear division cycle was determined to be about 30 min. From the frequencies of the phase-specific nuclear shapes, the durations of the G1 phase and the combined S plus G2 phases were calculated. The results showed that variations in the growth rates of the mycelia were mainly coupled with variations in the G1 phase of the nuclear division cycle. For mycelia growing in minimal sucrose, the lengths of all of the phases of the nuclear division cycle were estimated.     

Expression of Proteins and Glycoproteins Encoded by the Haploid Nuclei in the Dikaryotic State in the Basidiomycete Agrocybe aegerita

The total proteins and concanavalin A-binding glycoproteins of the cultivated mushroom Agrocybe aegerita were studied in homokaryotic siblings and in dikaryotic strains. The glycoproteins exhibited considerable variability compared with the proteins; the genetic diversity detected in homokaryons in the glycoprotein analysis was 30-fold higher than the genetic diversity revealed by protein analysis, and the glycoprotein patterns could be used to characterize individual genotypes. We found that the expression of glycoproteins in haploid nuclei was significantly asymmetric when the nuclei were paired in dikaryons. The expression levels of the two component nuclei depended on their genotypes, and each haploid nucleus was characterized by its level of expression. Furthermore, some specific glycoproteins that were not detected in all of the homokaryons were newly synthesized in the dikaryotic strains. Among these was a glycoprotein designated gpAa-65, which was identified in all of the dikaryotic strains and appeared to be a good molecular marker of the dikaryotic state.     

Direct studies of nuclear movements in Schizophyllum commune

Summary Measurements of nuclear positions in apical cells of homokaryotic mycelia and dikaryotic mycelium of Schizophyllum commune showed that nuclei occupied a near central position in most cases. Forward nuclear movements observed in living hyphal apices occurred at rates within the range of hyphal growth and could account for the maintenance of centrally located nuclei. Opposed nuclear movements followed mitosis and greatly exceeded the rate of hyphal growth. Septum disruption and rapid nuclear movements characterized an A _xB_mut homokaryon. Neither cytoplasmic streaming nor actively participating granules or filaments could account for any of these nuclear movements.     

Nuclear fusion,meiosis and the origin of dikaryotic hyphae in Armillariella mellea

The behaviour of nuclei during the growth and differentiation of basidiocarp primordia of Armillariella mellea (Vahl) Karst. is described. The primordial initials which arose from monokaryotic rhizomorphs were also monokaryotic. In older primordia, at the site of initiation of gill folds, multinucleate cells formed at the tips of monokaryotic hyphae and gave rise to the dikaryotic hyphae bearing clamp connections. These formed the gills of the older primordia. Cytological studies suggested that the nuclei in monokaryotic cells were diploid. In young basidial primordia haploidization occurred in the cells which were to become multinucleate prior to giving rise to dikaryotic hyphae of the gills. In mature basidia after nuclear fusion and meiosis had occurred, each of the four haploid daughter nucleic migrated into a basidiospore and then divided mitotically. One of the mitotic daughter nucleic migrated from each spore back into the basidium so that mature spores were uninucleate.Abbreviations M.T.O.C. microtubule organizing centre     

Nuclear and Non-nuclear Factors influencing Clamp Connection Formation in Coprinus disseminatus

Matings between sister single spore lines of Coprinus disseminatusshowed a cryptic tetrapolar pattern. The two groups of matingsthat resulted in formation of mycelia with clamp connections(apparent dikaryons) differed in rate of nuclear penetrationduring mating. In one group penetration occurred at rates comparablewith nuclear migration in other species and in the other groupit was often extensive but at a rate similar to, or less than,the dikaryon growth-rate. No differences were detected betweenthese two groups in stability, colony extension rate, frequencyof clamp connections, proportions of true clamp connectionsand pseudoclamps, or number of nuclei per hyphal tip cell. Cytological studies and the isolation of hyphal tips showedthat both groups of apparent dikaryons were heterokaryotic di-or trikaryons. The di- and trikaryotic conditions co-existedin the same mycelium, but adjacent cells of individual branchingsystems usually contained equal numbers of nuclei. Within apparentdikaryons the number and kinds of nuclei per cell were similarin hyphae with clamp connections and those with simple septa.Treatments that prevented clamp connection formation did notalter the nuclear status of most of the hyphae. Irregularities in nuclear distribution were infrequent and mostwere associated with pseudoclamps. Forty per cent of nodes withprobable pseudoclamps yielded homokaryotic branches, which wereof either constituent mating type. There was some indicationthat irregularities in nuclear distribution could also occurduring divisions associated with simple septa.     

Internuclear Genetic Transfer in Vegetative Dikaryons of SCHIZOPHYLLUM COMMUNE: I. Di-Mon Mating Analysis

A series of hemi-compatible dikaryon x monokaryon (di-mon) matings was designed to determine whether there was any genetic interaction between the dikaryotic nuclei. One of the nuclei in each dikaryon was known to carry a recessive gene (mnd) that promoted the development of an abnormal growth form, mound. Dikaryons containing both mnd ⁺ and mnd nuclei produced mosaic colonies that consisted of three distinct kinds of hyphae: mound, fruiting body, and vegetative (devoid of any multihyphal structure). When dikaryotic hyphae from each of these morphological regions were used in di-mon matings, the genetic and developmental characteristics of the selected nuclear types were examined in the resulting derived dikaryons. The results showed that fruiting-body and vegetative cells contained the expected mnd and mnd⁺ nuclei. Dikaryotic mound hyphae, however, contained only mnd nuclei. In a manner as yet unresolved, but clearly dependent on the presence of the mnd allele, the mnd ⁺ allele of a wild nucleus was altered to, or acquired, the mnd allele. A number of hypotheses were considered to explain the genetic event(s) that generates [mnd + mnd*] dikaryotic cells from [mnd⁺ + mnd] cells, but none was found to be entirely satisfactory.     

THE BASIDIA OF EXIDIA NUCLEATA. II. DEVELOPMENT

The basidia of Exidia nucleate, were studied by light and electron microscopy. The basidium arises from a basal clamp-connection as a subcylindrical, dikaryotic structure but soon becomes sphaeropedunculate. Mitochondria, endoplasmic reticulum, and compact lamellar systems are more abundant and more evenly dispersed in diploid probasidia than in dikaryotic probasidia, whereas vacuoles in the apical regions are larger in dikaryotic probasidia. Oil globules are few or absent in the earlier stages but increase in size and number throughout basidial development. Karyogamy results in a diploid nucleus approximately twice the volume of haploid nuclei. Although indications were noted of reorganization of the nuclear envelope during the terminal stages of reduction division, available evidence does not suggest a prolonged loss of the nuclear envelope during division. Internal wall formation is centripetal, at least in those stages observed. In areas of active wall formation, a conspicuous zone devoid of mitochondria, oil globules, and endoplasmic reticulum was noted consistently. The circumscribing wall of a hypobasidial segment is composed of 2 lamellae of contrasting electron-density and is separated from the walls of adjacent hypobasidial segments and the wall delimiting the enucleate stalk by a median electron-transparent lamella. Although extensive vacuolation occurs in the hypobasidial segments as the nuclei and most of the cytoplasm pass through the epibasidia into the basidiospores, evidence of active nuclear migration was observed. Each basidiospore contains a nucleus, mitochondria, endoplasmic reticulum, and numerous oil globules.     

Fragmentation of polyploid nuclei in the giant cells of the rat trophoblast. I. The ultrastructure of the nuclear fragments

Electron microscope study of the nuclear fragments in the rat trophoblast has demonstrated that the division of the trophoblast giant nucleus results first in the formation of a multinuclear cell. Each nuclear fragment is covered with its own nuclear envelope made of two membranes with numerous pore complexes. The chromatin in these nuclear fragments is condenced with various degrees of condensation, which depends on the step of placenta development, cell differentiation and the degree of nuclear fragmentation. The nuclear ultrastructure in nuclear fragments also depends on the degree of nuclear fragmentation and on the level of chromatin condensation. The nucleolus has no granular component. On large fragments, with lower chromatin condensation the nucleolus is not homogenous being made of fragments of more and of less electron dense fibrilles. Small light lacunae are seen in the nucleolus where chromatin threads and strands pass on. With a high chromatin condensation in the nucleus, round small nucleoli look homogenous being made of moderately electron dense fibrilles. Products of chromosome activity have been found in the nuclear fragments: accumulations of minute granules (d = 15--20 nm), perichromatinous granules (d = 35--40 nm), and fibrillar nucleolus-like bodies. In the multinuclear cell, made as the result of fragmentation of the initially giant nucleus, all the small nuclei are first arranged very close to each other, so that the contours of the neighbouring nuclei coincide.     

CYTOLOGY OF HELMINTHOSPORIUM TURCICUM AND ITS ASCIGEROUS STAGE,TRICHOMETASPHAERIA TURCICA

Knox- Davies , P. S., and J. G. Dickson . (U. Wisconsin, Madison.) Cytology of Helmintho sporium turcicum and its ascigerous stage, Trichometasphaeria turcica . Amer. Jour. Bot. 47(5) : 328—339. Illus. 1960.–The cells of the vegetative hyphae were generally multinucleate. Interphase nuclei resembled those of higher organisms, with a matrix of thread-like chromatin material surrounding a spherical nucleolus. “Beaked” nuclei frequently associated with anastomosing hyphae were interpreted as migrating nuclei. Nuclear division in the vegetative hyphae was rapid. Various division stages were distinguished but it was difficult to make accurate chromosome counts. The nucleoli were discarded at prophase or prometaphase and were reorganized in daughter nuclei at telophase. An outstanding feature of nuclear division was that all the nuclei in a cell divided simultaneously. Conidiophores and conidia were occasionally joined by wide cytoplasmic connections. They were multinucleate throughout their development. Mechanisms therefore exist for the perpetuation of heterokaryons through the conidium. Ascus development was studied in a hybrid between a dark and an albino isolate. Crozier formation was typical and nuclear fusion occurred in the young ascus. Four nuclear divisions were completed in the ascus before there was evidence of ascospore delimitation. Further nuclear division took place in the ascospores whose cells were multinucleate. The occurrence of less than 8 ascospores in an ascus appeared to follow degeneration of nuclei rather than the incorporation of a number of division-Ill nuclei in a single ascopore. Chromosome counts and irregularities in the appearance and behavior of nuclei and chromosomes in the asci indicate that aneuploidy occurs in Trichometasphaeria turcica. It is suggested that aneuploidy is a common phenomenon in the conidial stage of the fungus H. turcicum, and possibly also in other imperfect fungi.     

Reversion of protoplasts from dikaryotic mycelium ofSchizophyllum commune

Summary Morphological observations on the reversion of homokaryotic protoplasts obtained from dikaryotic mycelium ofSchizophyllum commune reveal that part of the morphogenetic processes operative in clamp formation are transiently expressed in the absence of heterokaryotic conditions. Often the homokaryotic revertants start off to produce simple septa and then 21form pseudoclamps for some time before they revert permanently to the normal monokaryotic morphology with simple septa. Heterokaryotic revertants often form pseudoclamps before reverting to the normal dikaryotic morphology with true clamps.     

Occurence of microtubules and microfilaments,and origin of septa in dikaryotic hyphae ofSchizophyllum commune

Summary In an electron microscopic study on the dikaryotic hyphae ofSchizophyllum commune, microtubules were observed during the nuclear division, and close to the non-dividing nuclei of apical cells and older cells. Microtubules of the spindle were connected with semicircular bodies at nuclear poles. Microfilaments were detected in the distal part of the apical cells. Vesicles similar to those in the tips of the hyphae occured also at the sites of septa formation. The occurrence of microtubules and the structure of semicircular bodies are compared with those in other basidiomycetes. It is suggested that vesicles are involved in the primary growth of the septal cross wall.     

Cytologic Observations on the Double Fertilization in Maize

1.The double fertilization is the type of the premitotic syngamy. 2. In 21 to 24 hours after pollination, most of the female nuclei fuse with the male nuclei. When the female nucleus fuses with the male nucleus, there are two situations in the appearance of the male nucleolus: one is that while the chromatin of the sperm nucleus relaxes gradually, a male nucleolus appears; the other is that after the chromatin of the sperm nucleus relaxes gradually, the male nucleus just appears in about 2 to 4 hours. 3. Generally, the fusion of the female nucleolus with the male nucleolus takes place before the division of the nygote. The nygote enters the stage of division when it has a jarge nucieojus; the zygote, in which the female nucleoius does not fuse with the male nucleolus, also can enter the stage of division. In 30 to 33 hours after pollination, the first division of the zygote occurs. The resting period of the zygote is about 9 hours 4. In the primary endosperm nucleus, the female nucleolus does not fuse with the male nucleolus. 24 to 26 hours after pollination, the primary endosperm nucleus begins the first division. The resting period of the primary endosperm nucleus is 2 to 4 hours. 5. Under the condition of artificial pollination, the fertilization of the fruit ears of maize proceeds sequencely, i.e. from the upper of the fruit ears to the lower the fertilization is fulfilled gradually.