Aspergillus myosin-V supports polarized growth in the absence of microtubule-based transport

In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV) in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA) and two kinesin-3s (UNCA and UNCB) undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1) rather than UNCA (kinesin-3) is the major kinesin motor that supports polarized growth in the absence of MYOV.     

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.     

Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. deltakin1 and deltakin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in deltakin1rkin3 double mutants, but polarity was lost in deltamyo5rkin1 and deltamyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenk?rper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.     

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.     

Microtubules in Candida albicans hyphae drive nuclear dynamics and connect cell cycle progression to morphogenesis

Candida albicans is an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicans hyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 microm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicans nuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.     

A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis

In the early stages of plant infection, yeast-like haploid sporidia of Ustilago maydis respond to pheromone secreted by compatible partners by forming conjugation tubes. These then fuse to generate a dikaryotic hypha that forms appressoria to penetrate the host plant. As a first step toward understanding the structural requirements for these transitions, we have identified myo5, which encodes a class-V myosin. Analysis of conditional and null mutants revealed that Myo5 plays nonessential roles in cytokinesis and morphogenesis in sporidia and is required for hyphal morphology. Consistent with a role in morphogenesis, a functional green fluorescent protein-Myo5 fusion protein localized to the bud tip and the hyphal apex as well as to the septa and the spore wall during later stages of infection. However, the loss of Myo5 did not affect the tip growth of hyphae and sporidia. By contrast, Myo5 was indispensable for conjugation tube formation. Furthermore, myo5 mutants were impaired in the perception of pheromones, which indicates a particular importance of Myo5 in the mating process. Consequently, few mutant hyphae were formed that penetrated the plant epidermis but did not continue invasive growth. These results indicate a crucial role of Myo5 in the morphogenesis, dimorphic switch, and pathogenicity of U. maydis.     

Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus,Ustilago maydis

Ustilago maydis is a dimorphic Basidiomycete fungus with a yeast-like form and a hyphal form. Here we present a comprehensive analysis of bud formation and the actin and microtubule cytoskeletons of the yeast-like form during the cell cycle. We show that bud morphogenesis entails a series of shape changes, initially a tubular or conical structure, culminating in a cigar-shaped cell connected to the mother cell by a narrow neck. Labelling of cells with concanavalin A demonstrated that growth occurs at bud tip. Indirect immunofluorescence studies revealed that the actin cytoskeleton consists of patches and cables that polarize to the presumptive bud site and the bud tip and an actin ring that forms at the neck region. Because the bud tip corresponds to the site of active cell wall growth, we hypothesize that actin is involved in secretion of cell wall components. The microtubule cytoskeleton has recently been shown to consist of a cytoplasmic network during interphase that disassembles at mitosis when a spindle and astral microtubules are formed. We have carried out studies of U. maydis cells synchronized by the microtubule-depolymerizing drug thiabendazole which allow us to construct a temporal sequence of steps in spindle formation and spindle elongation during the cell cycle. These studies suggest that astral microtubules may be involved in early stages of spindle orientation and migration of the nucleus into the bud and that the spindle pole bodies may be involved in reestablishment of the cytoplasmic microtubule network.     

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.     

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

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

The role of +TIPs in directional tip expansion

Aspergillus nidulans is an ideal model to study nuclear migration and intracellular transport by dynein and kinesin owing to its long neuron‐like hyphae, conserved transport mechanisms, and powerful genetics. In this organism, as in other filamentous fungi, microtubules have been implicated in patterning cell shape through polarized tip growth – the hallmark mode of growth that generates the elongated hyphae. Exactly how microtubules regulate tip growth is incompletely understood and remains a fascinating question for various cell types, such as pollen tubes and root hairs. Zeng et al. (2014) describe important new findings in A. nidulans regarding the role of EBA, the master regulator of microtubule plus end‐tracking proteins, in specifying microtubule dynamics required for directional tip growth at the hyphal tip.     

The myosin ATPase inhibitor 2,3-butanedione-2-monoxime disorganizes microtubules as well as F-actin in Saccharomyces cerevisiae

Interactions between microtubules and filamentous actin (F-actin) are essential to many cellular processes, but their mechanisms are poorly understood. We investigated possible roles of the myosin family of proteins in the interactions between filamentous actin (F-actin) and microtubules of budding yeast Saccharomyces cerevisiae with the general myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM). The growth of S. cerevisiae was completely inhibited by BDM at 20 mmol/L and the effect of BDM on cell growth was reversible. In more than 80% of BDM-treated budding yeast cells, the polarized distribution of F-actin was lost and fewer F-actin dots were observed. When cells were synchronized in G₁ with α-factor and released in the presence of BDM, cell number did not increase and cells were mainly arrested in G₁ DNA content without any bud, suggesting that myosin activity is required for new bud formation and the start of a new cell cycle. More than 10% of the BDM-treated cells also revealed defects in nuclear migration to the bud neck as well as in nuclear shape. Consistent with these defects, the orientation of mitotic spindles was random in the 57% of cells treated with 20 mmol/L BDM and immunostained with anti-tubulin antibody. Furthermore, microtubule structures were completely disorganized in most of the cells incubated in 50 mmol/L BDM, while similar amounts of tubulin proteins were present in both BDM-treated and untreated cells. These results show that the general myosin inhibitor BDM disorganizes microtubule structures as well as F-actin, and suggest that BDM-sensitive myosin activities are necessary for the interaction of F-actin and microtubules to coordinate polarized bud growth and the shape and migration of the nucleus in S. cerevisiae. This revised version was published online in July 2006 with corrections to the Cover Date.     

MYO2 is not essential for viability,but is required for polarized growth and dimorphic switches in Candida albicans

The human fungal pathogen Candida albicans changes from a budding yeast form to a polarized hyphal form in response to various external conditions. Dimorphic switching of C. albicans has been implicated in the development of pathogenicity. Morphogenic transformation requires polarized cell growth and rearrangement of the cytoskeleton. We previously showed that myosins play key roles in the conversion from the bud to the hyphal form of C. albicans by inhibiting myosin activities with 2,3-butanedione-2-monoxime (BDM), a general myosin ATPase inhibitor. In this study we investigated the function of MYO2 in C. albicans using deletion mutants. The amino acid sequence of CaMYO2 shows 60% identity and 77% homology with MYO2 and 54% identity and 70% homology with MYO4 of budding yeast Saccharomyces cerevisiae, suggesting that CaMYO2 is the only class V myosin in C. albicans. Cells in which both CaMYO2 alleles were deleted were viable, suggesting that MYO2 is nonessential in C. albicans. The proliferation of CaMYO2delta cells, however, was sharply decreased. In addition, CaMYO2delta cells showed defects in assembly and polarized localization of F-actin as well as an inability to induce germ tube formation and hyphal growth. The deletion of CaMYO2 also disrupted the shape and migration of the nucleus. These results strongly suggest that CaMYO2 is essential for polarized growth and hyphal transition in C. albicans.     

Somatic nuclear division in the sporidia of Ustilago violacea. IV. Microtubules and the spindle-pole body.

In unbudded cells of the anther smut fungus Ustilago violacea there is a dome-shaped spindle-pole body (SPB) consisting of a core 0.1 mum in diameter surrounded by a ribosome-free region 0.3-0.4 mum in diameter lying in a pocket of the nuclear membrane. After budding the nucleus moves towards the bud and begins to rotate rapidly. At about this stage the SPB divides into two parallel bars each about 0.1-0.15 mum in diameter and 0.3 mum long, separated by a distance of about 0.3 mum. Microtubules associated with the nuclear membrane but not with the SPB are present at the time of nuclear rotation. These microtubules disappear when rotation stops. Microtubules attached to the SPB are found during migration of the chromatinic portion of the nucleus into the bud cell. These microtubules disappear when migration stops and the nuclear membrand begins to break down. The twin SPB bars appear to move into the nucleus through a break in the membrane and begin to move apart forming a spindle about 1 mum long. Chromosomal microtubules (one per kinetochore) were found in several serial sections, and in addition there appeared to be several continuous microtubules present. The separation of the two chromatinic masses appeared to result from elongation of the continuous microtubules to about 3 mum long. Cytoplasmic microtubules and spindle microtubules were both found attached to the SPB as it elongated and one nucleus returned to the mother cell. The paper concludes with a discussion of the SPB as a multifuncitonal control center affecting nuclear migration, spindle formation, membrane breakdown and synthesis, karyogamy, conjugation, budding, chromosomal movement, replication, and disjunction.     

Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis

It is well established that polarized exocytosis is essential for fungal virulence. By contrast, the contribution of endocytosis is unknown. We made use of a temperature-sensitive mutant in the endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptor Yup1 and demonstrate that endocytosis in Ustilago maydis is essential for the initial steps of pathogenic development, including pheromone perception and cell-cell fusion. Furthermore, spore formation and germination were drastically reduced, whereas colonization of the plant was only slightly inhibited. The function of endocytosis in the recognition of mating pheromone through the G protein-coupled pheromone receptor Pra1 was analyzed in greater detail. Biologically active Pra1-green fluorescent protein localizes to the plasma membrane and is constitutively endocytosed. Yup1(ts) mutants that are blocked in the fusion of endocytic transport vesicles with early endosomes are impaired in pheromone perception and conjugation hyphae formation. This is attributable to an accumulation of Pra1-carrying endocytic vesicles in the cytoplasm and the depletion of the receptor from the membrane. Consistently, strong Pra1 expression rescues the signaling defects in endocytosis mutants, but subsequent cell fusion is still impaired. Thus, we conclude that endocytosis is essential for recognition of the partner at the beginning of the pathogenic program but has additional roles in mating as well as spore formation and germination.     

On the move: endosomes in fungal growth and pathogenicity

Fungi invade substrates, such as host tissues, through hyphal tip growth. This article focuses on the corn smut fungus Ustilago maydis, in which tip growth and pathogenicity involve apical endocytic recycling by early endosomes. These organelles rapidly move bi-directionally along microtubules and this movement is mediated by opposing molecular motors. This motility seems to be essential for extended hyphal growth, possibly because it focuses the endocytic machinery at the hyphal tip and mediates communication between the tip and the sub-apical nucleus.     

Apical growth and mitosis are independent processes in Aspergillus nidulans

Summary. It is well established that cytoplasmic microtubules are depolymerized during nuclear division and reassembled as mitotic microtubules. Mounting evidence showing that cytoplasmic microtubules were also involved in apical growth of fungal hyphae posed the question of whether apical growth became disrupted during nuclear division. We conducted simultaneous observations of mitosis (fluorescence microscopy) and apical growth (phase-contrast microscopy) in single hyphae of Aspergillus nidulans to determine if the key parameters of apical growth (elongation rate and Spitzenkörper behavior) were affected during mitosis. To visualize nuclei during mitosis, we used a strain of A. nidulans, SRS27, in which nuclei are labeled with the green-fluorescent protein. To reveal the Spitzenkörper and measure growth with utmost precision, we used computer-enhanced videomicroscopy. Our analysis showed that there is no disruption of apical growth during mitosis. There was no decrease in the rate of hyphal elongation or any alteration in Spitzenkörper presence before, during, or after mitosis. Our findings suggest that apical growth and mitosis do not compete for internal cellular resources. Presumably, the population of cytoplasmic microtubules involved in apical growth operates independently of that involved in mitosis.Present address: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.     

cAMP promotes hyphal branching in <Emphasis Type="Italic">Mucor globosus</Emphasis>

The growth pattern of Mucor globosus cultured on a medium with or without cyclic adenosine monophosphate (cAMP) was examined. Branching remarkably increased in a mycelium grown on an agar medium containing cAMP. In submerged culture containing cAMP, some sporangiospores grew spherically and formed yeast-like cells, and others showed hyphal growth. These hyphae showed septation and swelling and formed spore-like structures. When these hyphae were transferred to cAMP-free medium, a germ tube emerged from each compartment. These results show that cAMP has two different effects on the development of hyphae: one is the promotion of branching, and the other is the suppression of polarized growth.     

A Highlights from MBoC Selection: Dynamics of Multiple Nuclei in Ashbya gossypii Hyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism

Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.     

The multifunctional beta-oxidation enzyme is required for full symptom development by the biotrophic maize pathogen Ustilago maydis

The transition from yeast-like to filamentous growth in the biotrophic fungal phytopathogen Ustilago maydis is a crucial event for pathogenesis. Previously, we showed that fatty acids induce filamentation in U. maydis and that the resulting hyphal cells resemble the infectious filaments observed in planta. To explore the potential metabolic role of lipids in the morphological transition and in pathogenic development in host tissue, we deleted the mfe2 gene encoding the multifunctional enzyme that catalyzes the second and third reactions in beta-oxidation of fatty acids in peroxisomes. The growth of the strains defective in mfe2 was attenuated on long-chain fatty acids and abolished on very-long-chain fatty acids. The mfe2 gene was not generally required for the production of filaments during mating in vitro, but loss of the gene blocked extensive proliferation of fungal filaments in planta. Consistent with this observation, mfe2 mutants exhibited significantly reduced virulence in that only 27% of infected seedlings produced tumors compared to 88% tumor production upon infection by wild-type strains. Similarly, a defect in virulence was observed in developing ears upon infection of mature maize plants. Specifically, the absence of the mfe2 gene delayed the development of teliospores within mature tumor tissue. Overall, these results indicate that the ability to utilize host lipids contributes to the pathogenic development of U. maydis.