Hyphal Tip Cells Research Articles

Subcellular structure and behaviour in fungal hyphae.

This work briefly surveys the diversity of selected subcellular characteristics in hyphal tip cells of the fungal kingdom (Mycota). Hyphae are filamentous cells that grow by tip extension. It is a highly polarised mechanism that requires a robust secretory system for the delivery of materials (e.g. membrane, proteins, cell wall materials) to sites of cell growth. These events result it the self-assembly of a Spitzenkörper (Spk), found most often in the Basidiomycota, Ascomycota, and Blastocladiomycota, or an apical vesicle crescent (AVC), present in the most Mucoromycota and Zoopagomycota. The Spk is a complex apical body composed of secretory vesicles, cytoskeletal elements, and signaling proteins. The AVC appears less complex, though little is known of its composition other than secretory vesicles. Both bodies influence hyphal growth and morphogenesis. Other factors such as cytoskeletal functions, endocytosis, cytoplasmic flow, and turgor pressure are also important in sustaining hyphal growth. Clarifying subcellular structures, functions, and behaviours through bioimagining analysis are providing a better understanding of the cell biology and phylogenetic relationships of fungi. LAY DESCRIPTION: Fungi are most familiar to the public as yeast, molds, and mushrooms. They are eukaryotic organisms that inhabit diverse ecological niches around the world and are critical to the health of ecosystems performing roles in decomposition of organic matter and nutrient recycling (Heath, 1990). Fungi are heterotrophs, unlike plants, and comprise the most successful and diverse phyla of eukaryotic microbes, interacting with all other forms of life in associations that range from beneficial (e.g., mycorrhizae) to antagonistic (e.g., pathogens). Some fungi can be parasitic or pathogenic on plants (e.g., Cryphonectria parasitica, Magnaporthe grisea), insects (e.g., Beauveria bassiana, Cordyceps sp.), invertebrates (e.g., Drechslerella anchonia), vertebrates (e.g., Coccidioides immitis, Candia albicans) and other fungi (e.g., Trichoderma viride, Ampelomyces quisqualis). The majority of fungi, however, are saprophytes, obtaining nutrition through the brake down of non-living organic matter.

Genetic dissection of the secretory route followed by a fungal extracellular glycosyl hydrolase.

Hyphal tip cells of Aspergillus nidulans are > 100 µm-long, which challenges intracellular traffic. In spite of the basic and applied interest of the secretory pathway of filamentous fungi, only recently has it been investigated in detail. We used InuA, an inducible and highly glycosylated inulinase, and mutations affecting different intracellular membranous compartments, to investigate the route by which the enzyme traffics to the extracellular medium. InuA is core-N-glycosylated in the ER and hyperglycosylated during transit across the Golgi. Hyperglycosylation was prevented by ts mutations in sarASAR1 impeding ER exit, and in sedVSED5 and rabORAB1 dissipating the early Golgi, but not by mutations in the TGN regulators hypATRS120 and hypBSEC7 , implicating the early Golgi in cargo glycosylation. podB1ts (cog2ts ) affecting the COG complex also prevents glycosylation, without disassembling early Golgi cisternae. That InuA exocytosis is prevented by inactivation of any of the above genes shows that it follows a conventional secretory pathway. However, ablation of RabBRAB5 regulating early endosomes (EEs), but not of RabSRAB7 , its equivalent in late endosomes, also prevents InuA accumulation in the medium, indicating that EEs are specifically required for InuA exocytosis. This work provides a framework to understand the secretion of enzyme cargoes by industrial filamentous fungi.

Endocytic recycling via the TGN underlies the polarized hyphal mode of life.

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.

Spatial heterogeneity of glycogen and its metabolizing enzymes in Aspergillus nidulans hyphal tip cells

Glycogen is a homopolymer of glucose and a ubiquitous cellular-storage carbon. This study investigated which Aspergillus nidulans genes are involved in glycogen metabolism. Gene disruptants of predicted glycogen synthase (gsyA) and glycogenin (glgA) genes accumulated less cellular glycogen than the wild type strain, indicating that GsyA and GlgA synthesize glycogen similarly to other eukaryotes. Meanwhile, gene disruption of gphA encoding glycogen phosphorylase increased the amount of glycogen to a higher degree than wild type during the stationary phase that accompanies carbon-source limitation. GFP-tagged GsyA and GphA were distributed in the cytosol and formed punctate and filamentous structures, respectively. Carbon starvation resulted in elongated GphA-GFP filaments and increased numbers of filaments. These structures were more frequently located in the basal regions of tip cells and adjacent cells than in the apical regions of tip cells. Cellular glycogen visualized by incorporation of a fluorescent glucose analog accumulated in cytoplasmic puncta that were more prevalent in the basal regions, a pattern similar to that seen for GsyA. The colocalization of glycogen and GsyA at punctate structures in tip and sub-apical cells likely represents the cellular machinery for synthesizing glycogen. More frequent colocalization in the basal, rather than tip cell apical regions indicated that tip cells have differentiated subcellular regions for glycogen synthesis. Our findings regarding glycogen, GsyA and GphA distribution evoke the spatial heterogeneity of glycogen metabolism in fungal hyphae.

The aquatic hyphomycete Heliscus lugdunensis protects its hyphae tip cells from cadmium: A micro X-ray fluorescence and X-ray absorption near edge structure spectroscopy study

Aquatic fungi can be used to evaluate the functioning of natural ecosystems. Heliscus lugdunensis is an early colonizer of allochthone leafs. Since this aquatic hyphomycete is able to develop in metal contaminated habitats and tolerates cadmium, it appears to be a good candidate to investigate adaptation to metal pollution. This study aimed at examining the sequestration of Cd in the hyphae of H. lugdunensis, and particularly the role of the tip cells. For that, H. lugdunensis growth was evaluated under various Cd concentrations, and a combination of synchrotron micro X-ray fluorescence and X-ray absorption near edge structure spectroscopy was carried out to determine the compartments of Cd accumulation and the Cd chemical species, respectively. Results showed that the hyphal tip cells were depleted in Cd, and that the metal was stored in older cells. Cd was mainly associated with sulfur ligands and to a lesser extent bound to phosphates and carboxyl/hydroxyl groups from cell wall and/or organic acids. Finally, the aquatic fungus was able to maintain the tip cell as a functional system, thus allowing the colonization of contaminated environments.

Transport of fungal RAB11 secretory vesicles involves myosin-5, dynein/dynactin/p25, and kinesin-1 and is independent of kinesin-3.

Hyphal tip cells of the fungus Aspergillus nidulans are useful for studying long-range intracellular traffic. Post-Golgi secretory vesicles (SVs) containing the RAB11 orthologue RabE engage myosin-5 as well as plus end- and minus end-directed microtubule motors, providing an experimental system with which to investigate the interplay between microtubule and actin motors acting on the same cargo. By exploiting the fact that depolymerization of F-actin unleashes SVs focused at the apex by myosin-5 to microtubule-dependent motors, we establish that the minus end-directed transport of SVs requires the dynein/dynactin supercomplex. This minus end-directed transport is largely unaffected by genetic ablation of the Hook complex adapting early endosomes (EEs) to dynein but absolutely requires p25 in dynactin. Thus dynein recruitment to two different membranous cargoes, namely EEs and SVs, requires p25, highlighting the importance of the dynactin pointed-end complex to scaffold cargoes. Finally, by studying the behavior of SVs and EEs in null and rigor mutants of kinesin-3 and kinesin-1 (UncA and KinA, respectively), we demonstrate that KinA is the major kinesin mediating the anterograde transport of SVs. Therefore SVs arrive at the apex of A. nidulans by anterograde transport involving cooperation of kinesin-1 with myosin-5 and can move away from the apex powered by dynein.

Golgi organization and the apical extension of fungal hyphae: an essential relationship

The Golgi apparatus performs crucial functions in the sorting and processing of proteins destined for secretion from eukaryotic cells. In filamentous fungi, organization of the Golgi apparatus reflects the unique challenges brought about by the highly polarized nature of hyphal growth. Recent results show that Golgi compartments are spatially segregated within hyphal tip cells in a manner that depends upon the integrity of the cytoskeleton. Moreover, loss of normal Golgi organization stops polarized hyphal extension and triggers de-polarization of the hyphal tip. These results emphasize the point that a spatially organized and dynamic Golgi apparatus represents an adaptation that is as important for hyphal extension as is the presence of a Spitzenkörper. In addition, they also identify regulatory mechanisms that could enable controlled de-polarization of hyphae during development or infection-related morphogenesis.

Nonlethal ectoparasitism of the mycophagous nematode Filenchus discrepans (Nematoda: Tylenchidae)

Filenchus discrepans ingested hyphal contents of Botrytis cinerea without killing the hyphal cells that they fed on. Individuals of this nematode penetrated B. cinerea hyphae with their stylets, after which intense pulsation of the median bulb and dorsal gland duct followed (indicating salivation). In addition, light pumping of the median bulb (indicating food ingestion) was also observed. Fungal cells continued to show cytoplasmic streaming while the nematode ingested hyphal contents. Moreover, actively growing tips of the hyphae usually continued their growth during and after cytoplasm ingestion by the nematode. In less active, older hyphae the penetrated tip cells stopped growing during nematode feeding. However, the majority of the tip cells resumed growth after feeding by the nematode. The hyphal tips often exhibited an abnormal shape during nematode feeding and gradually regained normal shape and growth afterwards. The growth rate of hyphal tip cells decreased to less than 10% compared with the growth of intact hyphal tip cells during attack by F. discrepans, which often fed on a hyphal cell for up to 3 h. Filenchus discrepans propagated well in B. cinerea cultures and their population growth rate ranged between six- and 11-fold 1 month after incubation. This is the first report of nonlethal ectoparasitism by a mycophagous nematode, showing that the nematode does not kill fungal cells during and after feeding.

Searching for gold beyond mitosis: Mining intracellular membrane traffic in Aspergillus nidulans.

The genetically tractable filamentous ascomycete fungus Aspergillus nidulans has been successfully exploited to gain major insight into the eukaryotic cell cycle. More recently, its amenability to in vivo multidimensional microscopy has fueled a potentially gilded second age of A. nidulans cell biology studies. This review specifically deals with studies on intracellular membrane traffic in A. nidulans. The cellular logistics are subordinated to the needs imposed by the polarized mode of growth of the multinucleated hyphal tip cells, whereas membrane traffic is adapted to the large intracellular distances. Recent work illustrates the usefulness of this fungus for morphological and biochemical studies on endosome and Golgi maturation, and on the role of microtubule-dependent motors in the long-distance movement of endosomes. The fungus is ideally suited for genetic studies on the secretory pathway, as mutations impairing secretion reduce apical extension rates, resulting in phenotypes detectable by visual inspection of colonies.

The hyphal tip structure of Basidiobolus sp.: A zygomycete fungus of uncertain phylogeny

To date, among the zygomycete fungi that have been examined, a Spitzenkörper has not been reported. In this paper, the cytoplasmic order of hyphal tip cells of Basidiobolus sp., a zygomycete genus of uncertain phylogeny, has been examined using light microscopy and transmission electron microscopy methods. With phase-contrast light optics, a phase-dark body was observed at the tips of growing hyphae of Basidiobolus sp. The hyphal apex also showed high affinity for FM4-64 labelling resulting in an intense fluorescence signal. The phase-dark inclusion exhibited independent motility within the hyphal apex and its presence and position were correlated to the rate and direction of hyphal growth. The hyphal apex of Basidiobolus sp. did not contain γ-tubulin. Ultrastructural observations revealed a dense cluster of vesicles at the hyphal apex. These results suggest that the growing hypha of Basidiobolus sp. contains a Spitzenkörper, a character generally attributed to members of the ascomycete and basidiomycete fungi and not to zygomycete fungi.

Macroautophagy-mediated degradation of whole nuclei in the filamentous fungus Aspergillus oryzae.

Filamentous fungi consist of continuum of multinucleate cells called hyphae, and proliferate by means of hyphal tip growth. Accordingly, research interest has been focusing on hyphal tip cells, but little is known about basal cells in colony interior that do not directly contribute to proliferation. Here, we show that autophagy mediates degradation of basal cell components in the filamentous fungus Aspergillus oryzae. In basal cells, enhanced green fluorescent protein (EGFP)-labeled peroxisomes, mitochondria, and even nuclei were taken up into vacuoles in an autophagy-dependent manner. During this process, crescents of autophagosome precursors matured into ring-like autophagosomes to encircle apparently whole nuclei. The ring-like autophagosomes then disappeared, followed by dispersal of the nuclear material throughout the vacuoles, suggesting the autophagy-mediated degradation of whole nuclei. We also demonstrated that colony growth in a nutrient-depleted medium was significantly inhibited in the absence of functional autophagy. This is a first report describing autophagy-mediated degradation of whole nuclei, as well as suggesting a novel strategy of filamentous fungi to degrade components of existing hyphae for use as nutrients to support mycelial growth in order to counteract starvation.

Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans

The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical "comets" of AbpA.

Analysis of the cytochrome distribution via linear and nonlinear Raman spectroscopy

The cytochrome distribution in hyphal tip cells of Schizophyllum commune was visualized using resonance Raman mapping and CARS microscopy. For comparison, resonance Raman mapping and CARS imaging of cytochrome was also performed during branch formation and in completely developed central hyphae. Cytochrome, as an essential component of the electron transport chain in mitochondria, plays an important role in providing energy to actively growing mycelia. It could be shown that mitochondria at the growing hyphal tips and at branching regions are more active, i.e. contain more cytochrome, as compared to those in older parts of the hyphae. This finding is compatible with the idea of high energy consumption for biosynthesis and intracellular transport at the growing tip, while older hyphae have lower needs for ATP generated via the respiratory chain in mitochondria. To the best of our knowledge this is the first study reporting about the localization and distribution of cytochrome, as an indirect mitochondria localization within S. commune or other basidiomycetous fungi, by means of resonance Raman microspectroscopy and CARS microscopy. These Raman methods bear the potential of label-free in vivo mitochondria localization and investigation.

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC 6(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 DiOC 6(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.

Effect of nickel, copper, and zinc on emulsifier production and saturation of cellular fatty acids in the filamentous fungus Curvularia lunata

In the first step of this investigation the toxicity of Ni 2+, Cu 2+, and Zn 2+ ions to the emulsifier producing strain of Curvularia lunata was assessed. Among all the heavy metals studied, Ni 2+ ions were found to be the most toxic to C. lunata, whereas Zn 2+ ions exhibited the lowest toxicity. Moreover, only Ni 2+, when used at sublethal concentration (5 mM) caused lysis of some hyphal tip cells after a short-term exposure (5 h). In the next step, emulsifier production, accumulation of heavy metals by mycelia and emulsifier as well as saturation of cellular fatty acids were examined in 48-h-old cultures where fungal growth intensity was not inhibited by heavy metals (in the presence of Ni 2+, Cu 2+, and Zn 2+ ions at the initial concentration of 1, 5, and 15 mM, respectively) and in cultures where approximately 50% biomass inhibition occurred (in the presence of Ni 2+, Cu 2+, and Zn 2+ ions at the initial concentrations of 3, 10, and 17.5 mM, respectively). Among all the heavy metals studied only Ni 2+ ions did not induce emulsifier production. As compared with the control, only biomass treated with Ni 2+ ions displayed an increase in total lipid saturation. This effect resulted mainly from the decrease in linoleic acid (18:2) content correlated with the increase in the amount of stearic acid (18:0). The possible mechanisms by which Ni 2+ ions could alter the fatty acid profile of C. lunata and the protective role of the emulsifier were also discussed.

Antifungal activity of oligochitosan against Phytophthora capsici and other plant pathogenic fungi in vitro

Antifungal activity of oligochitosan against nine phytopathogens was investigated in vitro. Oligochitosan was more effective than chitosan in inhibiting mycelial growth of Phytophthora capsici and its inhibition on different stages in life cycle of P. capsici was observed. Rupture of released zoospores induced by oligochitosan was reduced by addition of 100 mM glucose. The effects of oligochitosan on mycelial growth and zoospore release, but not zoospore rupture, were reduced largely when pH value was above 7. The ultrastructural study showed that oligochitosan caused distortion and disruption of most vacuoles, thickening of plasmalemma, and appearance of unique tubular materials. Plasmalemmasomes in hyphal tip cells were not found in the presence of oligochitosan. These results suggest polycationic nature of oligochitosan contributes only partly to its antifungal activity and multiple modes of action of oligochitosan exist including the disruption of endomembrane system.

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.

Live-cell imaging of tubulin in the filamentous fungus Magnaporthe grisea treated with anti-microtubule and anti-microfilament agents

Microtubule dynamics were examined in live cells of the fungal plant pathogen Magnaporthe grisea transformed for constitutive expression of a fusion protein containing enhanced yellow-fluorescent protein and a Neurospora crassa benomyl-resistant allele of beta-tubulin. Transformants retained their ability to differentiate appressoria and cause disease but remained sensitive to benomyl. Linear microtubule arrays and low-level cytoplasmic fluorescence were observed in vegetative hyphae, conidia, germ tubes, and developing appressoria. Fluorescence within nuclei was conspicuously absent during interphase but increased rapidly at the onset of mitosis. Treatment with either benomyl or griseofulvin resulted in the appearance of prominent brightly fluorescent aggregates, including a large aggregate near the apex, with the concomitant disappearance of most cytoplasmic microtubules. Electron microscope imaging of treated cells indicated that the aggregates lacked any obvious profiles of intact microtubules. During these treatments, hyphal tip cells continued to elongate in a nonlinear and aerial fashion at a much slower rate than untreated cells. With subsequent removal of griseofulvin, distal aggregates disappeared rapidly but the apical aggregates persisted longer. Treatment with latrunculin A caused hyphal tip swelling without apparent effect on linear microtubule arrays. Simultaneous treatment with griseofulvin and latrunculin A resulted in depolymerization of microtubules and a cessation of growth, but near-apical fluorescent aggregates were not observed.

The Role of Microtubules in Rapid Hyphal Tip Growth ofAspergillus nidulans

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.