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.     

Direct microscopic studies of clamp connection formation in growing hyphae of Schizophyllum commune

Summary Photomicroscopic studies of clamp connection formation were collated with microscopic measurements of apical extension and mitosis in rapidly growing dikaryotic hyphae of Schizophyllum commune. Intercalary clamp connection formation was described in sub-terminal regions of the dikaryon. Conventional (i.e., rearward) clamp initiation was compared to forward clamp connection formation. Primary branch emergence was observed from clamp connections in growing hyphae and contrasted to sub-basidial branching in the hymenium of dikaryotic fruit-bodies.     

Dikaryotic arthroconidiation of Pleurotus subgenus Coremiopleurotus

This study was carried out to elucidate the poorly understood processes of arthroconidiation through coremium formation using Pleurotus cystidiosus subsp. abalonus. The coremia exclusively produced dikaryotic arthroconidia with the remnant of a clamp connection. Cells in the subapical zone of the hyphal bundle reduced their length by division before arthroconidiation. Approximately 400 000 arthroconidia were produced by a coremium in 1 day, with constant productivity over a 2-week period. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). Maintenance of moisture with coremioliquid was necessary for arthroconidiation. The coremioliquid formation was performed by active liquid transportation in the hyphae, a process that was blocked by the microtubule depolymerization agent thiabendazole.     

Enviromentally Induced Changes in Clamp Connection Incidence in Hyphal Branching Systems of Coprinus disseminatus

The incidence of clamp connections among surface hyphae at thecolony margin of apparent dikaryons from four stocks of Coprinusdisseminatus has been investigated under several environmentalconditions. On 2 per cent malt agar, clamp connections are formed at allnodes of the main leader hyphae, but they are absent from theearlier-formed nodes of primary branch hyphae. Most primarybranches have begun to form clamp connections by their fifthcell division, and continue to do so subsequently. Onset ofclamp connection formation in primary branches is delayed whenthe concentration of malt in the medium is reduced. The occurrenceof clamp connections on main leader hyphae is reduced or preventedwhen nutrient supply in the medium is reduced, or when the mediumis ‘pre-staled’ by previous growth of C. disseminatus.Clamp-free main hyphal tips revert to formation of clamp connectionswhen the intact hyphal system is transferred to more favourableenvironmental conditions. Presence of clamp connections on bothmain and branch hyphae is associated with high hyphal diameter,and in some instances, also with high hyphal extension rate. The significance of these observations to mechanisms of growthof hyphal branching systems and their relevance to other speciesthat form clamp connections intermittently, are discussed.     

Antifungal antibiotic of the basidiomyceteOudemansiella mucida

Nuclear ratios were studied in terminal and subterminal cells of various mycelia of the basidiomycete Oudemansiella mucida, the producer of the antifungal antibiotic mucidin (MuciderminR Spofa). The dikaryon, the monokaryon, and the mucidin-producing strain that had been cultivated for a long time under submerged conditions were compared. Dedikaryotization was found to have taken place in the producing strain. The originally dikaryotic culture with characteristic clamp connections on the mycelium and with two nuclei in every hyphal cell lost permanently the clamp connections, probably owing to continuous intense agitation. The hyphae contained solely mononuclear cells. Mating with a compatible monokaryon yielded a dikaryon capable of normal fructification.     

Morphology and molecular phylogeny ofTretopileus sphaerophorus, a synnematous hyphomycete with basidiomycetous affinities

Tretopileus sphaerophorus, a synnematous hyphomycete with basidiomycetous affinities was newly isolated from the decaying petiole and peduncle ofCocos nucifera collected in Depok, Indonesia. The species produced first a bulbil as a propagule on the top of a synnema. After the bulbil had fallen, the synnema proliferated about seven times to produce new bulbils, each time making conspicuous nodes at the upper part. By careful morphological observation, clamp connections were confirmed on the hyphae in the specimens and culture. In culture, each hyphal cell with or without a clamp was found to be dikaryotic by DAPI nuclear staining. Germination of the bulbils occurred first from projecting hyphal tips on their upper surface, which have been treated as germ pores. The inner structure of the bulbils, the hyaline mucus of the bulbils, and conidium-like hyphal fragments were also examined. Phylogenetically,T. sphaerophorus was inferred to be related to the Aphyllophorales based on the nuclear encoded small subunit (18S) rDNA using the homology search system (FASTA) and the neighbour-joining method. Part 10 in a series on the taxonomy of synnematous fungi.     

Morphological and cytological aspects of oidium formation in a basidiomycete,Pholiota nameko

Pholiota nameko produced abundant oidia on aerial hyphae from monokaryotic and dikaryotic test stocks, but oidia were rare on submerged hyphae. The oidia from the former stocks had a layer of hydrophobic protein between the cell wall and the inner cell membrane which was absent in the oidia from the latter. The only remarkable differences in the morphological features of the oidia from monokaryotic and dikaryotic mycelia was the slightly larger size of the latter. Observation of various test stocks on slide cultures revealed that about 80% of oidia were produced from the secondary branched hypha, and about 20% from the terminal hyphal, cell of the main hypha. In the former, the secondary hyphae were segmented to form several oidium cells; in the latter, a single or several oidia were formed at the terminal end of the main hypha. Most oidia from monokaryons and dikaryons had only one haploid nucleus, while the remainders were multinucleate. Among the stocks tested, most oidia had a DNA content with a haploid amount at the G1 phase of the cell cycle, but a few contained twice that amount corresponding to the G2 phase     

Kinetic studies of septum synthesis,erosion and nuclear migration in a growing B-mutant of Schizophyllum commune

Summary Microscopic measurements of apical growth and primary branch elongation were compared with nuclear movements, septum synthesis and erosion in a growing B-mutant of Schizophyllum commune. Apical growth, mitosis, septum formation, and intercalary cell division were similar to wild-type hyphae. Nuclear replication and new cross-wall formation also occurred in either apical cells bounded by eroding septa or in subterminal cells adjoined by eroded septa. An anucleate subterminal unit of the B-mutant hypha was invaded by a migrant nucleus which subsequently replicated and laid down a new septum in this region. Septum erosion occurred as early as 1 h following synthesis. Cellular granules and filaments were implicated in both septum synthesis and erosion.     

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     

Vegetative nuclear structures and their mode of division inCyathus species

Vegetative nuclear division in the homokaryotic and dikaryotic hyphae ofCyathus olla Brodie,C. setosus Brodie andC. bulleri Brodie was investigated. In the homokaryotic hyphae a nucleolus develops within a globular condensed nucleus consisting of a folded up filament. As the nucleolus increases in size, the nucleus unfolds and can assume a ring, horseshoe or filament configuration. The filament duplicates and (usually when unwound from the nucleolus) divides longitudinally. Occasionally, strand separation occurs while the filament is wrapped in the form of a ring around the nucleolus. The daughter nuclei may condense before the next division. In the dikaryotic hyphae the same nuclear cycle occurs as in the homokaryons except that an extra nuclear condensation to the globular form can occur in both the clamp and tube nuclei. The division of these two nuclei is not always synchronous and, moreover, the stage of karyokinesis of the clamp nucleus is not closely synchronized with the formation of the clamp connection. A deeply stained granule is associated with the nucleus. Some granules can be observed to be connected to the nucleus by a faintly Feulgen positive thread-like structure but other granules are sessile. The granule or centriole-like body is thought to direct the nuclear unfolding process. It may divide prior to, or after nuclear division.     

Dynamics of the actin cytoskeleton,hyphal tip growth and the movement of the two nuclei in the dikaryon ofCoprinus cinereus

We first examined the changes in distribution of F-actin during conjugate division in the apical cells of the dikaryon ofCoprinus cinereus using indirect immunofluorescence microscopy, then followed hyphal tip growth and the movement of the two nuclei in the apical cells using differential interference contrast microscopy (DIC). In apical cells with interphase nuclei, F-actin occurred solely as peripheral plaques, which were distributed along the whole length of the cell and were more concentrated at the tips, where they formed caps. In the early prophase of conjugate division, F-actin was transiently concentrated, as diffused form and plaques, at hyphal regions where the two nuclei sit, and this was accompanied by transient disappearance of the actin cap at the hyphal tip in the majority of cells. The actin cap was also present at the tips of growing clamp cells from late prophase through metaphase and disintegrated during anaphase. In telophase, actin rings formed at the future septa. DIC revealed that, in early prophase, when the F-actin array occurs around the two nuclei and the actin cap is absent at hyphal tips, hyphae kept growing and the second nucleus accelerated its forward movement to catch up with the leading nucleus, which was still moving forward.     

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.     

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.     

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.     

Nuclear behavior during branch formation in a centrifugedAdiantum protonema and the nuclear polarity

A new branch was induced on the side wall of fern protonema by cell centrifugation and subsequent polarized red light irradiation after the induction of cell division under white light. Nuclear behavior during the branch formation was analyzed. Immediately after cell division, the two daughter nuclei moved away from the division site in both red and dark conditions. Under continuous irradiation with polarized red light, cell swelling occurred as an early step of branching near the cell dividing wall, even though the nucleus was localized far from the branching site at the beginning of the swelling. After a new branch started to grow, the nucleus returned to the branching site and moved into the new branch from its basipetal end. When a protonema incubated in the dark was centrifuged again acropetally or basipetally just before the irradiation of polarized red light, the rate of apical growth or branch formation was increased, respectively. Moreover, growth of a branched protonema was altered from its former apex or from the branch again by dislocating the nucleus acropetally or basipetally by centrifugation, respectively. These facts suggest that the nucleus has no polarity physiologically, i.e. head and tail, namely either end of the spindle-shaped nucleus can be the nuclear front in a tip-growing protonema.     

Oldest fossil basidiomycete clamp connections

A rachis of the fossil filicalean fern Botryopteris antiqua containing abundant septate hyphae with clamp connections is preserved in a late Visean (Mississippian; ~330 Ma) chert from Esnost (Autun Basin) in central France. Largely unbranched tubular hyphae pass from cell to cell, but may sometimes produce a branch from a clamp connection. Other clamp-bearing hyphae occur clustered in individual cells or small groups of adjacent host cells. These hyphae may be tubular, catenulate with numerous hyphal swellings, or they may display a combination of both. The Visean hyphae with clamp connections predate Palaeancistrus martinii, the heretofore oldest direct fossil evidence of Basidiomycota, by some 25 Ma.     

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.     

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.     

Satellite Spitzenkörper in growing hyphal tips

Summary Growing hyphal tips of higher fungi contain an organized assemblage of secretory vesicles and other cell components collectively known as the Spitzenkörper. Until now, the Spitzenkörper has been portrayed as a single spheroid complex located near the apical cell wall. This study demonstrates the occurrence of multiple Spitzenkörper in growing hyphal apices imaged by video-enhanced phase-contrast microscopy. In addition to the main Spitzenkörper, smaller satellite Spitzenkörper arise a few micrometers behind the apical pole. Four developmental stages were identified: (a) the satellites first appeared as faint phase-dark plaques next to the plasma membrane, (b) gradually increased in size and assumed an ovoid profile, (c) they migrated to the hyphal apex, and (d) finally they merged with the main Spitzenkörper. After the merger, the main Spitzenkörper temporarily increased in size. Satellites were observed in 14 fungi, most of which had relatively large (5–10 m diam.), fast-growing hyphae (2–33 m/min elongation rate). The average frequency of in-focus satellites was 7+/min forFusarium culmorum and 11+/min forTrichoderma viride. As with the main Spitzenkörper, satellites were present only in growing cells. They were transient and remained visible for 3–8 s before merging with the main Spitzenkörper. Within the hyphae, satellites travelled up to six times faster than the average cell elongation rate. Multiple satellites sometimes occurred simultaneously; up to three were seen within a hyphal apex at the same time. Localized cell enlargement occurred next to stationary satellites, suggesting that satellite Spitzenkörper are functional as sources of new cell surface before they reach the main Spitzenkörper; therefore, they account for some variations in the profiles of the growing hyphae. By electron microscopy, satellites consisted of small clusters of apical vesicles surrounding a group of microvesicles located next to the plasma membrane. The identification and behavior of the satellites represent clear evidence of directional mass transport of vesicles toward the hyphal apex. Our observations indicate that satellites are a common phenomenon in growing hyphal apices of septate fungi and that they contribute to growth of the hyphal apex.Abbreviations VSC vesicle supply center