The cytoplasmic organization of hyphal tip cells in the fungusAllomyces macrogynus

Summary Light and transmission electron microscopy were used to examine hyphal tip cells of the fungusAllomyces macrogynus (Chytridiomycetes). A well defined apical body, i.e., Spitzenkörper, was observed at the extreme apex of hyphal cells. This distinctive, spherical cytoplasmic region consisted of a granular matrix devoid of ribosomes and most organelles. To our knowledge this is the first report describing such a structure in hyphae of an aseptate fungus. Vesicles (45–65 nm diameter) were concentrated in the peripheral cytoplasm of the apex, while relatively few were observed within the Spitzenkörper. Filasomes, spherical patches of dense fibrillar material containing a microvesicle core, were abundant in the apical regions near the plasma membrane. Microtubules traversed the Spitzenkörper at various angles and were in close association with the plasma membrane. Microfilaments were observed as individual elements in the cytoplasm or were organized into bundles. Individual microfilaments were frequently in close association with the plasma membrane, vesicles and microtubules. In the immediate subapical region mitochondria, multivesicular bodies, microbodies, Golgi equivalents and nuclei were abundant.Abbreviations CW cell wall - F filasome - M mitochondria - N nucleus - PM plasma membrane - TEM transmission electron microscopy     

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     

Diversity and dynamics of the Spitzenkörper in growing hyphal tips of higher fungi

Summary The Spitzenkörper, located in the apex of growing hyphae of septate fungi, has been portrayed previously as a spheroid complex containing a cluster of apical (secretory) vesicles which sometimes encloses a differentiated core area. With the aid of computer-enhanced video microscopy and phase-contrast optics, we studied 32 fungi in the Ascomycetes, Deuteromycetes, Hyphomycetes, Basidiomycetes, and Agonomycetes. The Spitzenkörper appeared as a highly dynamic and pleomorphic multicomponent complex capable of changing shape, size, and position within the hyphal apex during growth. The main theme of this study is to demonstrate two kinds of morphological diversity/variation in Spitzenkörper from diverse fungi: (a) inherent diversity — Spitzenkörper features characteristic of particular fungi, and (b) dynamic pleomorphism — gradual or rapid changes in size, shape, and position of the Spitzenkörper within a single hyphal tip. Several components associated with the Spitzenkörper were identified: (a) vesicle cluster, (b) vesicle cloud, (c) differentiated core region(s) within the Spitzenkörper, (d) apical granules, (e) cytoplasmic filaments. Eight morphological patterns of Spitzenkörper organization are described in the higher fungi based on the shape and distribution of their components. An additional (ninth) pattern was recognized in the chytridiomyceteAllomyces macrogynous from recent work by others. All these patterns appeared to be conserved at the genus level. In all patterns but one, a core region was observed by light microscopy. The Spitzenkörper not only exhibited spontaneous dynamic pleomorphism but also reacted to stress conditions (light, mechanical, and electrical fields). These reactions include migration of the Spitzenkörper back into the subapical zone and/or disassembly of its components. The understanding and conceptualization of this dynamic complex is problematic and should remain flexible enough to encompass the diversity of Spitzenkörper patterns and the dynamic pleomorphism of this specialized apical apparatus which appears to drive hyphal tip growth in the higher fungi.Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Hyphal tip ultrastructure ofAspergillus nidulans andAspergillus giganteus and possible implications of woronin bodies close to the hyphal apex of the latter species

Summary The hyphal tip ultrastructure ofAspergillus nidulans andAspergillus giganteus indicates that their apical organization is very similar to that found in other filamentous fungi. Both species have an area immediately behind the hyphal apex free of all large organelles and containing a high concentration of vesicles. InA. giganteus only one size class of vesicle is clearly evident, with a mean diameter of 72 nm. InA. nidulans two size classes of vesicle were found, with mean diameters of 75 nm and 31 nm. A Spitzenkörper is evident inA. nidulans as an area very close to the tip containing only the smaller vesicles. InA. giganteus one or more apparently mature Woronin bodies were found within the first 1 m of some hyphal apices. The possible significance of their presence is discussed.     

Apical Branching in a Temperature Sensitive Mutant ofAspergillus niger

An apical branching, temperature-sensitive, mutant ofAspergillus niger(ramosa-1) was isolated by UV mutagenesis.Ramosa-1has a wild type morphology at 23°C, but branches apically when shifted to 34°C. The cytological events leading to apical branching were recorded by video-enhanced phase contrast microscopy. The first event was a momentary, localized, cytoplasmic contraction lasting approximately 1 s. This contraction was seen as a sudden unidirectional movement of visible organelles (mitochondria, spheroid bodies) toward the hyphal apex. During the contraction, there was a transitory sharp increase in refractive index in a localized area of cytoplasm in the apex or subapex of the cell. Within 5 s, the Spitzenkörper retracted from its normal position next to the apical pole and disappeared from view 20 to 50 s later. Hyphal elongation rate diminished sharply, and the typical distribution of organelles at the hyphal tip was disturbed. After 210–240 s, organelle distribution returned to normal, polarized growth resumed, but instead of one Spitzenkörper two new Spitzenkörper appeared, each giving rise to an apical branch. The second branch Spitzenkörper appeared with a 60- to 100-s delay. We did not observe the original Spitzenkörper dividing in two; instead, the new Spitzenkörper arosede novofrom vesicle clouds that formed in the apical region next to the future site of branch emergence. In all instances that we examined, the dislocation and disappearance of the Spitzenkörper was preceded by cytoplasmic contractions. We therefore suspect the existence of an intimate connection between the cytoskeletal network and the Spitzenkörper. Accordingly, we propose that the apical branching phenotype inramosa-1is triggered by a molecular event that induces a transient alteration in cytoskeleton organization.     

Protoplasmic Organization of Hyphal Tips Among Fungi: Vesicles and Spitzenkörper

Hyphal tips of fungi representing Oömycetes, Zygomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes were examined by light and electron microscopy and compared with respect to their protoplasmic organization. In all fungi studied, there is a zone at the hyphal apex which is rich in cytoplasmic vesicles but nearly devoid of other cell components. Some vesicle profiles are continuous with the plasma membrane at the apices of these tip-growing cells. The subapical zones of hyphae contain an endomembrane system which includes smooth-surfaced cisternae associated with small clusters of vesicles. The findings are consistent with the hypothesis that vesicles produced by the endomembrane system in the subapical region become concentrated in the apex where they are incorporated at the expanding surface. Septate fungi (Ascomycetes, Basidiomycetes, and Deuteromycetes) have an apical body (Spitzenkörper) which is associated with growing hyphal tips. In electron micrographs of these fungi, an additional specialized region within the accumulation of apical vesicles is shown for the first time. This region corresponds on the bases of distribution among fungi, location in hyphae, size, shape and boundary characteristics to the Spitzenkörper seen by light microscopy. This structure is not universally associated with tip growth, whereas apical vesicles are widespread among tip-growing systems.     

Ultrastructure of freeze-substituted hyphae of the basidiomyceteLaetisaria arvalis

Summary The ultrastructure of freeze-substituted (FS) hyphae ofLaetisaria arvalis is described and compared to that of similar hyphae preserved by conventional chemical fixation (CF). The outline of membrane-bound organelles as well as the plasma membrane was smooth in FS cells. In contrast, hyphae preserved by CF exhibited membrane profiles that were extremely irregular. Centers of presumed Golgi activity were best preserved by FS. Microvesicles, 27–45 nm diameter and hexagonal in transverse section, were observed most readily in FS cells. Filasomes (= microvesicles within a filamentous matrix) were only observed in FS cells. Apical vesicles, 70–120 nm diameter, associated with the centers of Golgi activity and within the Spitzenkörper region exhibited finely granular matrices in FS hyphae, whereas in CF hyphae the contents were coarsely fibrous and less electron-dense. Microvesicles were present at hyphal apices and regions of septa formation. Filasomes were also found at regions of septa formation as well as along lateral hyphal tip cell walls. Microvesicles, but not filasomes, were observed in membrane-bound vesicles (= multivesicular bodies) and in larger vacuoles. Filaments, 5.2–5.4 nm wide, were juxtaposed with centripetally developing septa. Cytoplasmic inclusions, 20–40 m in length, composed of bundles of 6.7–8.0 nm wide filaments were observed in both FS and CF hyphae.     

Tip-localized actin polymerization and remodeling,reflected by the localization of ADF,profilin and villin,are fundamental for gravity-sensing and polar growth in characean rhizoids

Polar organization and gravity-oriented, polarized growth of characean rhizoids are dependent on the actin cytoskeleton. In this report, we demonstrate that the prominent center of the Spitzenkörper serves as the apical actin polymerization site in the extending tip. After cytochalasin D-induced disruption of the actin cytoskeleton, the regeneration of actin microfilaments (MFs) starts with the reappearance of a flat, brightly fluorescing actin array in the outermost tip. The actin array rounds up, produces actin MFs that radiate in all directions and is then relocated into its original central position in the center of the Spitzenkörper. The emerging actin MFs rearrange and cross-link to form the delicate, subapical meshwork, which then controls the statolith positioning, re-establishes the tip-high calcium gradient and mediates the reorganization of the Spitzenkörper with its central ER aggregate and the accumulation of secretory vesicles. Tip growth and gravitropic sensing, which includes control of statolith positioning and gravity-induced sedimentation, are not resumed until the original polar actin organization is completely restored. Immunolocalization of the actin-binding proteins, actin-depolymerizing factor (ADF) and profilin, which both accumulate in the center of the Spitzenkörper, indicates high actin turnover and gives additional support for the actin-polymerizing function of this central, apical area. Association of villin immunofluorescence with two populations of thick undulating actin cables with uniform polarity underlying rotational cytoplasmic streaming in the basal region suggests that villin is the major actin-bundling protein in rhizoids. Our results provide evidence that the precise coordination of apical actin polymerization and dynamic remodeling of actin MFs by actin-binding proteins play a fundamental role in cell polarization, gravity sensing and gravity-oriented polarized growth of characean rhizoids.Abbreviations ADF Actin-depolymerizing factor - CD Cytochalasin D - MF Microfilament     

In-vivo observations of a spherical aggregate of endoplasmic reticulum and of Golgi vesicles in the tip of fast-growing Chara rhizoids

In-vivo differential interference contrast microscopy was used to detect individual Golgi vesicles and a new structure in the tip of fast-growing rhizoids of Chara fragilis Desvaux. This structure is a spherical clear zone which is free of Golgi vesicles, has a diameter of 5 m and is positioned in the center of the apical Golgi-vesicle accumulation (Spitzenkörper). After glutaraldehyde fixation and osmium tetroxide-potassium ferricyanide staining of the rhizoid, followed by serial sectioning and three-dimensional reconstruction, the spherical zone shows a tight accumulation of anastomosing endoplasmic reticulum (ER) membranes. The ER membranes radiate from this aggregate towards the apical plasmalemma and to the membranes of the statolith compartments. Upon gravistimulation the ER aggregate changes its position according to the new growth direction, indicating its participation in growth determination. After treatment of the rhizoid with cytochalasin B or phalloidin the ER aggregate disappears and the statoliths sediment. It is concluded that the integrity of the ER aggregate is actin-dependent and that it is related to the polar organisation of the gravitropically growing cell tip.Abbreviations CB cytochalasin B - DIC differential interference contrast microscopy - DMSO dimethyl sulfoxide - ER endoplasmic reticulum     

Confocal microscopy of Spitzenkörper dynamics during growth and differentiation of rust fungi

Summary. The membrane-selective fluorescent dye FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridium dibromide, was used to stain the apical vesicle cluster within the specialized Spitzenkörper of the germ tube of the rust fungi Uromyces vignae and Puccinia graminis f. sp. tritici grown on glass surfaces. The Spitzenkörper stained within 15 min following addition of the dye. Optical sectioning by confocal microscopy of stained hyphal tips showed that the Spitzenkörper was asymmetrically positioned close to the cell–substratum interface during germ tube growth. The Spitzenkörper showed variations in shape and positioning over short (5 s) time intervals. The movement to a new location in the hyphal dome was followed by new growth in that region, consistent with the view that the Spitzenkörper supplies secretory vesicles for germ tube growth. A pronounced Spitzenkörper disappeared at the onset of appressorium differentiation during swelling of the germ tube. However, a stained structure, similar in appearance to a Spitzenkörper, was again observed during the formation of the highly polarized penetration peg.Correspondence and reprints: Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.Received October 25, 2002; accepted February 26, 2003; published online August 26, 2003     

Die Ultrastruktur der Apikalregion von Pilzhyphen

Zusammenfassung Alle Apikalregionen vegetativer Hyphen von 24 untersuchten Pilzarten unterschiedlicher systematischer Zugehörigkeit besitzen einen Spitzenkörper, der kalottenförmig oder sphÄroid sein kann. Ultrastrukturell reprÄsentiert der Spitzenkörper ein Grundplasmaareal unterschiedlicher Grö\e und Form, das Apexvesikel, Mikrovesikel und Ribosomen enthÄlt. Die Membran der Vesikel besitzt eine unkontrastierte Mittelschicht, die von gleicher Dicke (41,5 å) ist wie die des Plasmalemmas und sich signifikant von der des ER (28,1 å) unterscheidet.Echte Golgi-Dictyosomen fehlen. Morphologisch charakterisierte Einzelcisternen, die in unterschiedlicher Dichte im gesamten Hyphenplasma auftreten, scheinen die sekretorische Funktion des Golgi-Apparates zu übernehmen. Sie bilden peripher die Apexvesikel, in denen wahrscheinlich Exoenzyme apikalwÄrts transportiert und am Apex ausgeschleust werden.Bei Sistierung des Wachstums wird die Akkumulation der vesikulÄren Komponenten des Spitzenkörpers aufgehoben. Die Einzelvesikel erfahren dabei keine strukturelle VerÄnderung. Bei Krümmungen erfolgt eine Translokation des gesamten Vesikelaggregates. Es wird ein Modell vorgelegt, dessen Grundlage die Rekonstruktion einer Schnittserie ist, in dem jedoch Details, die das Ergebnis der Gesamtuntersuchungen sind, korrigiert wurden.

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Ultrastructure of the apical region of fungal hyphae

Summary All apical regions of vegetative hyphae of 24 fungal species of different systematical origin are possessing an apical body the appearance of which may be caplike or spheroidal. Ultrastructurally the apical body represents an area of ground cytoplasm of different size and shape, containing apical vesicles, microvesicles and ribosomes. The vesicles-membrane has an uncontrasted middle-layer of the same thickness (41,5 å) as the plasmalemma and is significantly different from that of the ER (28,1 å).True Golgi-dictyosomes are lacking. Morphologically characterized single cisternae, appearing in graded density in the whole hyphal cytoplasm seem to take over secretory function of the Golgi-apparatus. They produce Apex vesicles peripherically in which probably are transported exoenzymes apically and are extruded at the apex.Accumulation of the vesicular components of the apical body is destroyed as stopping of growth is induced. No structural changes of single vesicles occur. Translocation of the whole vesicles-aggregation takes place if the apex is bent.A model is constructed, based on reconstruction of serial sections and corrected due to results of the whole investigations.

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Dem Elmi-Kollektiv unter der Leitung von FrÄleinFritsche und Herrn Ing.Wachsmuth sowie FrauBÄhring und FrauPohl sei für ihre gro\e Einsatzfreudigkeit bestens gedankt.     

The Neurospora crassa exocyst complex tethers Spitzenk?rper vesicles to the apical plasma membrane during polarized growth

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.     

Aggregation and collapse of fungal wall vesicles in hyphal tips: a model for the origin of the Spitzenk?rper

The intracellular origins of polarity and branch initiation in fungi centre upon a localization in the supply of fungal wall constituents to specific regions on the hyphal wall. Polarity is achieved and maintained by accumulating secretory vesicles, prior to incorporation into the wall, in the form of an apical body or Spitzenkörper. However, neither the mechanisms leading to this accumulation nor the initiation of branching, are as yet understood. We propose a mechanism, based on experimental evidence, which considers the mechanical properties of the cytoskeleton in order to explain these phenomena. Cytoskeletal viscoelastic forces are hypothesized to be responsible for biasing vesicles in their motion, and a mathematical model is derived to take these considerations into account. We find that, as a natural consequence of the assumed interactions between vesicles and cytoskeleton, wall vesicles aggregate in a localized region close to the tip apex. These results are used to interpret the origin of the Spitzenkörper. The model also shows that an aggregation peak can collapse and give rise to two new centres of aggregation coexisting near the tip. We interpret this as a mechanism for apical branching, in agreement with published observations. We also investigate the consequences and presumptive role of vesicle–cytoskeleton interactions in the migration of satellite Spitzenkörper. The results of this work strongly suggest that the formation of the Spitzenkörper and the series of dynamical events leading to hyphal branching arise as a consequence of the bias in vesicle motion resulting from interactions with the cytoskeleton.     

The tubulin cytoskeleton and its sites of nucleation in hyphal tips ofAllomyces macrogynus

Summary The tubulin cytoskeleton in hyphal tip cells ofAllomyces macrogynus was detected with an -tubulin monoclonal antibody and analyzed with microscopic and immunoblot techniques. The -tubulin antibody identified a 52 kilodalton polypeptide band on immunoblots. Immunfluorescence data were collected from formaldehyde-and cryofixed hyphae. Both methods provided similar images of tubulin localization. However, cryofixation yielded more consistent labeling and did not require detergent extraction or cell-wall lytic treatments. Tubulin was primarily localized as microtubules observed in the peripheral and central cytoplasmic regions and in mitotic spindles. Cytoplasmic microtubules were oriented parallel to the cells' longitudinal axis, with central microtubules more often varied in their alignment, and emanated from a region in the hyphal apex resulting in an apical zone of bright fluorescence. A thin layer of microtubules appearing as bands of fluorescence encircled many nuclei. Discrete spots of fluorescence were also associated with nuclei. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins that may be involved in the regulation of microtubule nucleation, stained centrosomes but not apical regions of hyphae. Nocodazole was used to depolymerize the microtubule network and reveal its regions of origin. A hocodazole concentration of 0.01 g/ ml (3.3× 10^–8M) provided a 70 to 75% inhibition of hyphal tip growth and was used throughout this study. The number of cells having an apical zone of fluorescence declined by 15 min of exposure. This zone was present in only a few cells after 60 min. After 30 min, the central cytoplasm consisted of small microtubule fragments and nuclear-associated spots. A small number of peripheral microtubules and nuclear-associated spots persisted throughout nocodazole treatments. Spindle microtubules were restored by 30 min after removal of nocodazole. This was followed by the reappearance of the apical zone of fluorescence and then by central and peripheral cytoplasmic microtubules. Apical fluorescence coincided with the presence of a Spitzenkörper. The results suggest that the Spitzenkörper and centrosome function as centers of microtubule nucleation and organization during hyphal tip growth in this fungus.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - IB incubation buffer - LN₂ liquid nitrogen - LSCM laser scanning confocal microscopy - MTOCs microtubule-organizing centers - PBS phosphate buffered saline - PIPES 1,4-piperazinedietha-nesulfonic acid - PFB PIPES fixation buffer - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SPB spindle pole body - TEM transmission electron microscopy - YpSs yeast extract-inorganic phosphate-soluble starch     

How hyphae grow: Morphogenesis explained?

Summary Apical growth of fungal hyphae represents a relatively simple instance of cellular morphogenesis. Thanks to the polarized transport and exocytosis of precursor vesicles, new cell wall and plasma membrane are continuously deposited at the hyphal apex; the question is how the characteristic shape of tube and tapered tip comes about. Recent experiments lend support to a model whose central feature is a mobile vesicle supply center corresponding to the Spitzenkörper (apical body) visible in growing hyphae. Shapes predicted by the model agree remarkably well with those of actual hyphae. Nevertheless, critical examination of the model's premises suggests that it requires extension so as to incorporate both a driving force for expansion and a gradient of cell wall plasticity. I propose that a mobile vesicle supply center may be one, but only one, of a range of physiological devices employed by tip-growing organisms to localize the exocytosis of precursor vesicles. Apical growth should ensue whenever the loci of exocytosis advance vectorially, and nascent cell wall expands in a graded manner.Abbrevations VSC vesicle supply center - SPK Spitzenkörper     

A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.     

The effects of ropy-1 mutation on cytoplasmic organization and intracellular motility in mature hyphae of Neurospora crassa

We have used light and electron microscopy to document the cytoplasmic effects of the ropy (ro-1) mutation in mature hyphae of Neurospora crassa and to better understand the role(s) of dynein during hyphal tip growth. Based on video-enhanced DIC light microscopy, the mature, growing hyphae of N. crassa wild type could be divided into four regions according to cytoplasmic organization and behavior: the apical region (I) and three subapical regions (II, III, and IV). A well-defined Spitzenk?rper dominated the cytoplasm of region I. In region II, vesicles ( approximately 0.48 micro m diameter) and mitochondria maintained primarily a constant location within the advancing cytoplasm. This region was typically void of nuclei. Vesicles exhibited anterograde and retrograde motility in regions III and IV and followed generally parallel paths along the longitudinal axis of the cell. A small population of mitochondria displayed rapid anterograde and retrograde movements, while most maintained a constant position in the advancing cytoplasm in regions III and IV. Many nuclei occupied the cytoplasm of regions III and IV. In ro-1 hyphae, discrete cytoplasmic regions were not recognized and the motility and/or positioning of vesicles, mitochondria, and nuclei were altered to varying degrees, relative to the wild type cells. Immunofluorescence microscopy revealed that the microtubule cytoskeleton was severely disrupted in ro-1 cells. Transmission electron microscopy of cryofixed cells confirmed that region I of wild-type hyphae contained a Spitzenk?rper composed of an aggregation of small apical vesicles that surrounded entirely or partially a central core composed, in part, of microvesicles embedded in a dense granular to fibrillar matrix. The apex of ro-1 the hypha contained a Spitzenk?rper with reduced numbers of apical vesicles but maintained a defined central core. Clearly, dynein deficiency in the mutant caused profound perturbation in microtubule organization and function and, consequently, organelle dynamics and positioning. These perturbations impact negatively on the organization and stability of the Spitzenk?rper, which, in turn, led to severe reduction in growth rate and altered hyphal morphology.     

γ-Tubulin is a component of the Spitzenkörper and centrosomes in hyphal-tip cells ofAllomyces macrogynus

Summary A monoclonal antibody was used to localize -tubulin in hyphal tip cells of the chytridiomycete fungusAllomyces macrogynus, and its distribution determined with standard epifluorescence and laser scanning confocal microscopy. The results demonstrate that -tubulin is a component of the Spitzenkörper and centrosomes. Immunoblot analysis of total soluble protein extracts separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified a single 56 kDa -tubulin-related polypeptide. Localization of -tubulin to the Spitzenkörper ofA. macrogynus provides evidence that the Spitzenkörper in this fungus functions as a microtubule-organizing center.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - LSCM laser scanning confocal microscopy - MTOCs microtubule-organizing centers - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SPB spindle pole body - YpSs yeast extract-inorganic phosphate-soluble starch     

New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicateMetandrocarpa uedai

Summary Subcellular compartments comprising the endomembrane system in filamentous fungi are poorly characterized with most showing significant morphological differences from eukaryotic cells. For example, many filamentous fungi lack stacked Golgi-body cisternae, but contain Golgi equivalents — single cisternae or tubules which appear to serve the same functions. To help identify fungal endomembrane compartments and interrelationships between them we used a pharmacological agent, brefeldin A, known to affect specific endomembrane organelles in other organisms, most prominently the Golgi apparatus. At 10 g/ml brefeldin A, radial hyphal growth of the rice blast pathogenMagnaporthe grisea on solid agar medium was reduced by 96% over an initial 48 h, but recovered and was reduced by only 20% over a subsequent 72 h exposure. Light microscopic examination of individual living hyphae showed that apical elongation generally halted within 1 min after exposure to brefeldin A. Acute effects of 14 g/ml brefeldin A were characterized ultrasiructurally in cells prepared by freeze substitution. These included the appearance of two types of cisternae with unusual morphology, associated with ca. 45 nm diameter vesicles, as well as the unexpected persistence and increase in complexity of the Golgi equivalents. Also observed were (1) reduced numbers of apicale vesicles and disruption of Spitzenkörper organization, (2) apical clusters of 30–35 nm diameter microvesicles and associated tubular arrays, (3) dilation of rough endoplasmic reticulum, (4) packets of membrane-bounded electron-opaque cell wall inclusions, and (5) altered morphology of some vacuolar compartments. The distribution of concanavalin A binding sites, previously mapped to particular endomembrane compartments, was documented to aid the interpretation of these results. We conclude that brefeldin A effects on cells ofM. grisea differ from those reported with plant and animal cells, perhaps reflecting underlying differences in the endomembrane systems among these eukaryotes.Abbreviations BFA brefeldin A - ConA concanavalin A - ER endoplasmic reticulum - PDA potato dextrose agar - RER rough endoplasmic reticulum     

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3)

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC₆(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.