The role of microtubules in rapid hyphal tip growth of Aspergillus 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.     

The microtubule cytoskeleton in hyphae ofUromyces phaseoli germlings: Its relationship to the region of nucleation and to the F-actin cytoskeleton

Summary The microtubule and F-actin cytoskeleton of nondifferentiated germlings ofUromyces phaseoli was studied using immunofluorescence methodologies. The microtubules were oriented mostly parallel to the longitudinal axis of the hypha. Microtubule depolymerizing agents, such as cold, demecolcine, griseofulvin and nocodazole, were effective in destroying the microtubule network, but not the F-actin system. Repolymerization of microtubules, following release from these agents, occurred first in the hyphal apices and not near the nuclei or spindle pole bodies. It was concluded that the microtubule nucleating region in such fungal cells is located in the apical regions. Enhanced microtubule arrays were visualized following incubation of the cells in taxol, an agent known to favor microtubule polymerization.     

Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1

Cytoplasmic dynein is a minus-end-directed microtubule motor that participates in multiple cellular activities such as organelle transport and mitotic spindle assembly [1]. To study the dynamic behavior of cytoplasmic dynein in the filamentous fungus Aspergillus nidulans, we replaced the gene for the cytoplasmic dynein heavy chain, nudA, with a gene encoding a green fluorescent protein (GFP)-tagged chimera, GFP-nudA. The GFP-NUDA fusion protein is fully functional in vivo: strains expressing only the GFP-tagged nudA grow as well as wild-type strains. Fluorescence microscopy showed GFP-NUDA to be in comet-like structures that moved in the hyphae toward the growing tip. Retrograde movement of some GFP-NUDA comets after they arrived at the tip was also observed. These dynamics of GFP-NUDA were not observed in cells treated with a microtubule-destabilizing drug, benomyl, suggesting they are microtubule-dependent. The rate of GFP-NUDA tip-ward movement is similar to the rate of cytoplasmic microtubule polymerization toward the hyphal tip, suggesting that GFP-NUDA is associated and moving with the polymerizing ends of microtubules. A mutation in actin-related protein Arp1 of the dynactin complex abolishes the presence of these dynamic GFP-NUDA structures near the hyphal tip, suggesting a targeting role of the dynactin complex.     

Growth rate of Aspergillus nidulans hyphae is independent of a prominent array of microtubules

Roles for the microtubule (MT) cytoskeleton in fungal growth include mitosis and nuclear migration but otherwise are less clearly understood. Confocal microscopy was used to quantify MT abundance and growth rate in hyphae of a haploid Aspergillus nidulans strain containing green fluorescent protein (GFP)-α-tubulin. There was no correlation between growth rate and MT abundance for 112 growing hyphae in an untreated population. However, 109 nongrowing hyphae from the same group had lower average MT abundance. Results for untreated cells were compared with cells treated for 30–120 min with the MT drugs benomyl and taxol, the actin drug latrunculin B, and with solvents used for the drug treatments. Compared with their respective controls, MT abundance was significantly increased by dimethyl sulfoxide (DMSO), significantly reduced by benomyl, and moderately increased by latrunculin, but was unaffected by ethanol. In the same cells, growth rates were significantly increased by ethanol and taxol, significantly reduced by latrunculin, and unaffected by DMSO. Average hyphal growth rate in the first 60 min following 1 μg/ml benomyl treatment was statistically similar to untreated cells, despite the absence of visible MTs after 2 min of treatment. However, growth rate was significantly reduced by 2.5 μg/ml benomyl over the same time period, implying additional effects at the higher concentration. For individual hyphae in each treatment, growth rates varied over short time periods; treatment with 0.1% ethanol substantially increased this variability. Growth rates of taxol-treated hyphae decreased following fluorescence observation, suggesting a possible application to cancer chemotherapy. Overall, there was no correlation between cytoplasmic MT abundance and A. nidulans growth rate within 2 h of cytoskeletal drug or solvent treatment.     

Involvement of the cytoskeleton in the intracellular distribution of mannoproteins in hyphal cells ofCandida albicans

Cylindrical growth of fungal hyphae requires spatial organization of secretion to the growing tip. In order to better understand the involvement of the cytoskeleton in the spatial control of the secretion, we examined the effects of two anti-cytoskeletal drugs, benomyl and cytochalasin A, on the intracellular distribution of mannoproteins, a major secreted component of the cell wall, in hyphal cells of the dimorphic yeastCandida albicans. The distribution of the mannoproteins was assessed by epifluorescence microscopy with a fluorescence-labelled lentil lectin (FITC-LCA). Brefeldin A, an inhibitor of secretory transport, induced a localized accumulation of the mannopolysaccharides near the tip as previously reported (Akashiet al. 1997). Benomyl, an inhibitor of microtubules, disrupted the localized accumulation of the polysaccharides. Cytochalasin A, an inhibitor of actin, caused a localized accumulation of the polysaccharides near the tip, where Golgi-like cisternae were also accumulated. Both cytochalasin A and brefeldin A caused some modifications of the actinnnetwork, but neither disturbed the polarization of actin and neither affected the microtubule network. Our results suggested that the microtubules are involved in membrane trafficking in hyphal growth as well as the cell polarity of the hyphae.     

Plasma membrane-adjacent actin filaments, but not microtubules, are essential for both polarization and hyphal tip morphogenesis in Saprolegnia ferax and Neurospora crassa

The organization and roles of F-actin and microtubules in the maintenance and initiation of hyphal tip growth have been analyzed in Saprolegnia ferax and Neurospora crassa. In hyphae of both species, the apex is depleted of microtubules relative to subapical regions and near-normal morphogenesis occurs in concentrations of nocodazole or MBC which remove microtubules, slow growth, and disrupt nuclear positioning. In contrast, each species contains characteristic tip-high arrays of plasma membrane-adjacent F-actin, whose organization is largely unaltered by the loss of microtubules but disruption of which by latrunculin B disrupts tip morphology. Hyphal initiation and subsequent normal morphogenesis from protoplasts of both species and spores of S. ferax are independent of microtubules, but at least in S. ferax obligatorily involve the formation of F-actin caps adjacent to the hyphal tip plasma membrane. These observations indicate an obligatory role for F-actin in hyphal polarization and tip morphogenesis and only an indirect role for microtubules.     

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     

Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

An InCytes from MBC Selection: The Aspergillus nidulans Kinesin-3 UncA Motor Moves Vesicles along a Subpopulation of Microtubules

The extremely polarized growth form of filamentous fungi imposes a huge challenge on the cellular transport machinery, because proteins and lipids required for hyphal extension need to be continuously transported to the growing tip. Recently, it was shown that endocytosis is also important for hyphal growth. Here, we found that the Aspergillus nidulans kinesin-3 motor protein UncA transports vesicles and is required for fast hyphal extension. Most surprisingly, UncA-dependent vesicle movement occurred along a subpopulation of microtubules. Green fluorescent protein (GFP)-labeled UncA^rigor decorated a single microtubule, which remained intact during mitosis, whereas other cytoplasmic microtubules were depolymerized. Mitotic spindles were not labeled with GFP-UncA^rigor but reacted with a specific antibody against tyrosinated α-tubulin. Hence, UncA binds preferentially to detyrosinated microtubules. In contrast, kinesin-1 (conventional kinesin) and kinesin-7 (KipA) did not show a preference for certain microtubules. This is the first example for different microtubule subpopulations in filamentous fungi and the first example for the preference of a kinesin-3 motor for detyrosinated microtubules.     

Response of ectomycorrhizal fungi to benomyl and nocodazole: growth inhibition and microtubule depolymerization

The growth of seven ectomycorrhizal fungi was tested in the presence of the antimicrotubule drugs benomyl and nocodazole. The polymerization stage of the cytoplasmic microtubules in the hyphal cells was visualized by indirect immunofluorescence microscopy after a 3-h drug treatment. Nocodazole reduced the growth of all the fungi tested at concentrations of 2 and 4 g ml^-1 and caused strong depolymerization of microtubules in all other species except Hebeloma cylindrosporum. Benomyl inhibited the growth and depolymerized the microtubules in the ascomycete Cenococcum geophilum, while in the basidiomycetes it reduced the growth and depolymerized the microtubules only in H. cylindrosporum. The role of the microtubule cytoskeleton and the target of the benzimidazole-derived drugs in fungal cells are discussed.     

CLIP-170 homologue and NUDE play overlapping roles in NUDF localization in Aspergillus nidulans

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32 degrees C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150(Glued) dynactin, and NUDF were all seen as plus-end comets at 32 degrees C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32 degrees C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42 degrees C).     

Microtubule Cycle in Root Tip Cells of Allium fistulosum L.

Using immunofluorescent localization techniques and TEM methods, the organization of microtubule arrays during the cell cycle of root tip cells of Allium fistulosum L. was studied. There are four basic types of microtubule organization, namely, interphase cortical microtubule, pre-prophase band microtubule, spindle microtubule and phragmoplast microtubule, which constitute the typical microtubule cycle in dividing cells of higher plants. The fluorescent figures of microtubules observed under fluorescent microscope were explained and analysed by the ultrastractural informations of microtubules obtained from TEM.     

The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics

Cytoplasmic dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged cytoplasmic dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that cytoplasmic dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.     

Role of microtubules in tip growth of fungi

Polarized cell growth is observed ubiquitously in all living organisms. Tip growth of filamentous fungi serves as a typical model for polar growth. It is well known that the actin cytoskeleton plays a central role in cellular growth. In contrast, the role of microtubules in polar growth of fungal tip cells has not been critically addressed. Our recent study, using a green fluorescent protein (GFP)-labeled tubulin-expressing strain of the filamentous fungus Aspergillus nidulans and treatment with an anti-microtubule reagent, revealed that microtubules are essential for rapid hyphal growth. Our results indicated that microtubule organization contributes to continuous tip growth throughout the cell cycle, which in turn enables the maintenance of an appropriate mass of cytoplasm for the multinucleate system. In filamentous fungi, the microtubule is an essential component of the tip growth machinery that enables continuous and rapid growth. Recent research developments are starting to elucidate the components of the tip growth machinery and their functions in many organisms. This recent knowledge, in turn, is starting to enhance the importance of fungal systems as simple model systems to understand the polar growth of cells.     

Hyphal tip growth and nuclear migration

Recent molecular and cytological studies have greatly advanced our understanding of hyphal tip growth and nuclear migration in filamentous fungi. Mutants involved in various aspects of hyphal tip growth have been isolated. Genes involved in nuclear migration continue to be identified, including putative regulators. The role of microtubules and microtubule motor proteins in hyphal tip growth has also been studied.     

Hypersensitivity to cytoskeletal antagonists demonstrates microtubule-microfilament cross-talk in the control of root elongation in Arabidopsis thaliana

Elongation of diffusely expanding plant cells is thought to be mainly under the control of cortical microtubules. Drug treatments that disrupt actin microfilaments, however, can reduce elongation and induce radial swelling. To understand how microfilaments assist growth anisotropy, we explored their functional interactions with microtubules by measuring how microtubule disruption affects the sensitivity of cells to microfilament-targeted drugs. We assessed the sensitivity to actin-targeted drugs by measuring the lengths and diameters of expanding roots and by analysing microtubule and microfilament patterns in the temperature-sensitive Arabidopsis thaliana mutant microtubule organization 1 (mor1-1), along with other mutants that constitutively alter microtubule arrays. At the restrictive temperature of mor1-1, root expansion was hypersensitive to the microfilament-disrupting drugs latrunculin B and cytochalasin D, while immunofluorescence microscopy showed that low doses of latrunculin B exacerbated microtubule disruption. Root expansion studies also showed that the botero and spiral1 mutants were hypersensitive to latrunculin B. Hypersensitivity to actin-targeted drugs is a direct consequence of altered microtubule polymer status, demonstrating that cross-talk between microfilaments and microtubules is critical for regulating anisotropic cell expansion.     

Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-m-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations APM Amiprophosmethyl - FITC Fluorescein isothiocyanate - LATB Latrunculin B     

Tracks for traffic: microtubules in the plant pathogen Ustilago maydis

Pathogenic development of the corn smut fungus Ustilago maydis depends on the ability of the hypha to grow invasively. Extended hyphal growth and mitosis require microtubules, as revealed by recent studies on the microtubule cytoskeleton. Surprisingly, hyphal tip growth involves only two out of 10 kinesins. Kinesin-3 is responsible for tip-directed (anterograde) endosome motility of early endosomes, which are thought to support hyphal elongation by apical membrane recycling. In addition, kinesin-3, together with kinesin-1 and myosin-5, appear to deliver secretory vesicles to the hyphal tip. Kinesin-1 also affects endosome motility by targeting cytoplasmic dynein to microtubule plus ends. This plus-end localization of dynein is essential for cell body-directed (retrograde) endosome motility, but also allows force generation during spindle elongation in mitosis. Furthermore, kinesin-1 and dynein participate in the organization of the microtubule array, thereby building their own network of tracks for intracellular motility. The recent progress in understanding microtubule-based processes in U. maydis has revealed an unexpected complexity of motor functions essential for the virulence of this pathogen. Further studies on structural and regulatory requirements for motor activity should help identify novel targets for fungicide development.     

THE EFFECT OF ENVIRONMENTAL ANEUPLOIDY-INDUCING AGENTS ON THE MICROTUBULE ARCHITECTURE OF MITOTIC MERISTEMATIC ROOT CELLS INHORDEUM VULGARE

Microtubules are the prime components involved in chromosomal segregation, their functional accuracy ensuring maintenance of the normal karyotype in the progeny. Chemically-induced disruption of microtubules during mitosis can lead to aneuploidy. In this study, seven environmental chemicals, i.e. cadmium chloride (CD), econazole nitrate (EZ), benomyl (BM), thiabendazole (TB), griseofulvin (GF), thimerosal (TM) and hydroquinone (HQ), were tested for their ability to induce microtubule disruption in mitotic meristematic root cells of the higher plantHordeum vulgarewith the use of anti-tubulin indirect immunofluorescence microscopy. All chemicals tested in this study, with the exception of TB and HQ, produced modifications in the morphology of microtubule organization and reduced the fidelity of the spindle apparatus inHordeum vulgare