Polarized Growth of Fungal Hyphae Is Defined by an Alkaline pH Gradient

Robson, G. D., Prebble, E., Rickers, A., Hosking, S., Denning, D. W., Trinci, A. P. J., and Robertson, W. 1996. Polarized growth of fungal hyphae is defined by an alkaline pH gradient.Fungal Genetics and Biology20,289–298. Polarized cell growth is exhibited by a diverse range of eukaryotic and prokaryotic cells. The events which are responsible for this growth are poorly understood. However, the existence of ion gradients may play an important role in establishing and driving cell polarity. Using a pH-sensitive, ratiometric fluorescent dye to monitor intracellular pH in growing fungal hyphae, we report a gradient at the extending hyphal tip that is up to 1.4 pH units more alkaline than more distal regions. Both the magnitude and the length of the pH gradient were strongly correlated with the rate of hyphal extension and eradication of the gradient-arrested growth. These results suggest that alkaline pH gradients may be integral to hyphal extension in fungi.     

Apical assemblies of intermediate filament‐like protein FilP are highly dynamic and affect polar growth determinant DivIVA in Streptomyces venezuelae

Streptomyces spp. grow as branching hyphae, building the cell wall in restricted zones at hyphal tips. The organization of this mode of polar growth involves three coiled‐coil proteins: DivIVA and Scy, which form apical protein complexes referred to as polarisomes; and the intermediate filament‐like protein FilP, which influences cell shape and interacts with both Scy and DivIVA. Here, we use live cell imaging of Streptomyces venezuelae to clarify the subcellular localization and dynamics of FilP and its effect on hyphal morphology. By monitoring a FilP‐mCherry fusion protein, we show that FilP accumulates in gradient‐like zones behind the hyphal tips. The apical gradient pattern of FilP localization is dependent on hyphal tip extension and immediately dissipates upon growth arrest. Fluorescence recovery after photobleaching experiments show that FilP gradients are dynamic and subject to subunit exchange during vegetative growth. Further, the localization of FilP at hyphal tips is not directly dependent on scy, even though the strongly perturbed morphology of most scy mutant hyphae is associated with mislocalization of FilP. Finally, we find that filP has an effect on the size and position of the foci of key polar growth determinant DivIVA. This effect likely contributes to the phenotype of filP mutants.     

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     

Epichlo? endophytes grow by intercalary hyphal extension in elongating grass leaves.

A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichlo? species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichlo? species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.     

Epichloë endophytes grow by intercalary hyphal extension in elongating grass leaves

A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichlo? species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichlo? species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.     

Evidence That Spitzenkörper Behavior Determines the Shape of a Fungal Hypha: A Test of the Hyphoid Model

Bartnicki-Garcia, S. Bartnicki, D. D., Gierz, G., López-Franco, R., and Bracker, C. E. 1995. Evidence that Spitzenkörper behavior determines the shape of a fungal hypha; A test of the hyphoid model. Experimental Mycology 19, 153-159. Hyphae of the fungus Rhizoctonia solani have a characteristic Spitzenkörper in their growing tips and a cell shape described by the mathematical hyphoid equation. A mild disturbance of hyphae growing in a slide culture chamber on a microscope stage caused the Spitzenkörper to move away from its usual position next to the apical pole and wander briefly inside the apical dome. Hyphal elongation rate declined abruptly, and the apex became rounded and increased in diameter. As the Spitzenkörper migrated back to its polar position, rapid cell elongation resumed, and the contour of the growing hyphal tip returned to the typical hyphoid shape. The brief dislocation of the Spitzenkörper left a permanent bulge in the hyphal profile. This morphogenetic sequence was mimicked by computer simulation, based on the hyphoid equation which relates the generation of hyphal shape to the linear displacement of a vesicle supply center (VSC). The VSC was programmed to retrace the observed movements of the Spitzenkörper during the above sequence. The resulting similarity of shape between real and computer-simulated cells reinforces the mathematical prediction that the Spitzenkörper acts as a VSC and that its continuous linear advancement generates a typical hyphal tube with the characteristic hyphoid shape. Accordingly, the hyphoid model and its VSC concept provide a plausible hypothesis to explain the cellular basis of polarized growth of fungal hyphae.     

A Model for Growth of a Single Fungal Hypha Based on Well-Mixed Tanks in Series: Simulation of Nutrient and Vesicle Transport in Aerial Reproductive Hyphae

Current models that describe the extension of fungal hyphae and development of a mycelium either do not describe the role of vesicles in hyphal extension or do not correctly describe the experimentally observed profile for distribution of vesicles along the hypha. The present work uses the n-tanks-in-series approach to develop a model for hyphal extension that describes the intracellular transport of nutrient to a sub-apical zone where vesicles are formed and then transported to the tip, where tip extension occurs. The model was calibrated using experimental data from the literature for the extension of reproductive aerial hyphae of three different fungi, and was able to describe different profiles involving acceleration and deceleration of the extension rate. A sensitivity analysis showed that the supply of nutrient to the sub-apical vesicle-producing zone is a key factor influencing the rate of extension of the hypha. Although this model was used to describe the extension of a single reproductive aerial hypha, the use of the n-tanks-in-series approach to representing the hypha means that the model has the flexibility to be extended to describe the growth of other types of hyphae and the branching of hyphae to form a complete mycelium.     

Morphogenesis and cell cycle progression in Candida albicans

Candida albicans, an opportunistic human pathogen, displays three modes of growth: yeast, pseudohyphae and true hyphae, all of which differ both in morphology and in aspects of cell cycle progression. In particular, in hyphal cells, polarized growth becomes uncoupled from other cell cycle events. Yeast or pseudohyphae that undergo a cell cycle delay also exhibit polarized growth, independent of cell cycle progression. The Spitzenk?rper, an organelle composed of vesicles associated with hyphal tips, directs continuous hyphal elongation in filamentous fungal species and also in C. albicans hyphae. A polarisome mediates cell cycle dependent growth in yeast and pseudohyphae. Regulation of morphogenesis and cell cycle progression is dependent upon specific cyclins, all of which affect morphogenesis and some of which function specifically in yeast or hyphal cells. Future work will probably focus on the cell cycle checkpoints involved in connecting morphogenesis to cell cycle progression.     

Transcellular ion currents and extension ofNeurospora crassa hyphae

Summary Hyphae ofNeurospora crassa, like many other tipgrowing organisms, drive endogenous electric currents through themselves such that positive charges flow into the apical region and exit from the trunk. In order to identify the ions that carry the current, the complete growth medium was replaced by media lacking various constituents. Omission of K⁺ or of phosphate diminished the zone of inward current, effectively shifting the current pattern towards the apex. Omission of glucose markedly reduced both inward and outward currents; addition of sodium azide virtually abolished the flow of electric current. Growing hyphae also generate a longitudinal pH gradient: the medium surrounding the apex is slightly more alkaline than the bulk phase, while medium adjacent to the trunk turns acid. The results suggest thatNeurospora hyphae generate a proton current; protons are expelled distally by the H⁺-ATPase and return into the apical region by a number of pathways, including the symport of protons with phosphate and potassium ions. Calcium influx may also contribute to the electric current that enters the apical region. There seems to be no simple obligatory linkage between the intensity of the transcellular electric current and the rate of hyphal extension. Calcium ions, however, are required in micromolar concentrations for extensions and morphogenesis of hyphal tips.     

How does a hypha grow? The biophysics of pressurized growth in fungi

The mechanisms underlying the growth of fungal hyphae are rooted in the physical property of cell pressure. Internal hydrostatic pressure (turgor) is one of the major forces driving the localized expansion at the hyphal tip which causes the characteristic filamentous shape of the hypha. Calcium gradients regulate tip growth, and secretory vesicles that contribute to this process are actively transported to the growing tip by molecular motors that move along cytoskeletal structures. Turgor is controlled by an osmotic mitogen-activated protein kinase cascade that causes de novo synthesis of osmolytes and uptake of ions from the external medium. However, as discussed in this Review, turgor and pressure have additional roles in hyphal growth, such as causing the mass flow of cytoplasm from the basal mycelial network towards the expanding hyphal tips at the colony edge.     

High spatial resolution analysis of fungal cell biochemistry – bridging the analytical gap using synchrotron FTIR spectromicroscopy

Fungi impact humans and the environment in many ways, for good and ill. Some fungi support the growth of terrestrial plants or are used in biotechnology, and yet others are established or emerging pathogens. In some cases, the same organism may play different roles depending on the context or the circumstance. A better understanding of the relationship between fungal biochemical composition as related to the fungal growth environment is essential if we are to support or control their activities. Synchrotron FTIR (sFTIR) spectromicroscopy of fungal hyphae is a major new tool for exploring cell composition at a high spatial resolution. Brilliant synchrotron light is essential for this analysis due to the small size of fungal hyphae. sFTIR biochemical characterization of subcellular variation in hyphal composition will allow detailed exploration of fungal responses to experimental treatments and to environmental factors.     

High spatial resolution analysis of fungal cell biochemistry--bridging the analytical gap using synchrotron FTIR spectromicroscopy.

Fungi impact humans and the environment in many ways, for good and ill. Some fungi support the growth of terrestrial plants or are used in biotechnology, and yet others are established or emerging pathogens. In some cases, the same organism may play different roles depending on the context or the circumstance. A better understanding of the relationship between fungal biochemical composition as related to the fungal growth environment is essential if we are to support or control their activities. Synchrotron FTIR (sFTIR) spectromicroscopy of fungal hyphae is a major new tool for exploring cell composition at a high spatial resolution. Brilliant synchrotron light is essential for this analysis due to the small size of fungal hyphae. sFTIR biochemical characterization of subcellular variation in hyphal composition will allow detailed exploration of fungal responses to experimental treatments and to environmental factors.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

On-line study of growth kinetics of single hyphae of Aspergillus oryzae in a flow-through cell

Using image analysis the growth kinetics of the single hyphae of the filamentous fungus Aspergillus oryzae has been determined on-line in a flow-through cell at different glucose concentrations in the range from 26 mg L-1 to 20 g L-1. The tip extension rate of the individual hyphae can be described with saturation type kinetics with respect to the length of the hyphae. The maximum tip extension rate is constant for all hyphae measured at the same glucose concentration, whereas the saturation constant for the hyphae varies significantly between the hyphae even within the same hyphal element. When apical branching occurs, it is observed that the tip extension rate decreases temporarily. The number of branches formed on a hypha is proportional to the length of the hypha that exceeds a certain minimum length required to support the growth of a new branch. The observed kinetics has been used to simulate the outgrowth of a hyphal element from a single spore using a Monte Carlo simulation technique. The simulations shows that the observed kinetics for the individual hyphae result in an experimentally verified growth pattern with exponential growth in both total hyphal length and number of tips.     

Aspergillus fumigatus RasA regulates asexual development and cell wall integrity

The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The DeltarasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The DeltarasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The DeltarasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.     

Mapping the growth of fungal hyphae: orthogonal cell wall expansion during tip growth and the role of turgor

By computer-enhanced videomicroscopy, we mapped the trajectory of external and internal cell surface markers in growing fungal hyphae to determine the pattern of cell wall expansion during apical growth. Carbon particles (India ink) were chosen as external markers for tip expansion of Rhizoctonia solani hyphae. Irregularities in the growing apical walls of R. solani served as internal markers. Marker movement was traced in captured frames from the videotaped sequences. External and internal markers both followed orthogonal trajectories; i.e., they moved perpendicular to the cell surface regardless of their initial position in the hyphal apex. We found no evidence that the tip rotates during elongation. The discovery that the cell wall of a growing hypha expands orthogonally has major repercussions on two fronts: 1) It supports the long-held view that turgor pressure is the main force driving cell wall expansion. 2) It provides crucial information to complete the mathematical derivation of a three-dimensional model of hyphal morphogenesis based on the vesicle supply center concept. In three dimensions, the vesicle gradient generated by the vesicle supply center is insufficient to explain shape; it is also necessary to know the manner in which the existing surface is displaced during wall expansion.     

Structural changes to a mycothallus along a latitudinal transect through the maritime and sub-Antarctic

Previous studies have shown the leafy liverwort Cephaloziella varians to associate consistently with fungi, typically the ericoid mycorrhizal symbiont Rhizoscyphus ericae, across a wide latitudinal gradient in the maritime and sub-Antarctic. Hitherto, however, there are no quantitative data on the intensity of colonisation of C. varians by fungal structures in the natural environment and how colonisation might vary with changing environmental conditions. A study is hence reported showing that the frequency of colonisation by fungal structures of C. varians alters along a latitudinal transect from South Georgia (54° S, 38° W) to Moutonnée Valley on Alexander Island (71° S, 68° W). The percentage of stem length colonised by dark septate (DS) hyphae increased significantly along the transect, from 30% at South Georgia to 97% at Moutonnée Valley. In contrast, the percentage of stem length colonised by hyaline hyphae decreased significantly, from 85% at South Georgia to 13% at Moutonnée Valley, and that colonised by hyphal coils similarly decreased from 71% at the former location to 15% at the latter. The frequencies of DS hyphae were negatively associated with mean annual and seasonal air temperatures, whereas those of hyaline septate hyphae and hyphal coils were positively associated with air temperatures. Coils at northerly locations were more convoluted than those at southerly locations. The data indicate that hyphal coils, usually associated with nutrient exchange between partners in ericoid mycorrhizas, do form in C. varians tissues in the maritime and sub-Antarctic, but that the frequency of these structures diminishes in colder habitats.     

Zinc ions alter morphology and chitin deposition in an ericoid fungus

A sterile mycelium PS IV, an ascomycete capable of establishing ericoid mycorrhizas, was used to investigate how zinc ions affect the cellular mechanisms of fungal growth. A significant reduction of the fungal biomass was observed in the presence of millimolar zinc concentrations; this mirrored conspicuous changes in hyphal morphology which led to apical swellings and increased branching in the subapical parts. Specific probes for fluorescence and electron microscopy localised chitin, the main cell wall polysaccharide, on the inner part of the fungal wall and on septa in control specimens. In Zn-treated mycelium, hyphal walls were thicker and a more intense chitin labelling was detected on the transverse walls. A quantitative assay showed a significant increase in the amount of chitin in metal-treated hyphae.     

Control of growth and differentiation by cyclic AMP in fungi

InSaccharomyces cerivisiae intracellular cAMP mediates environmental signals that regulate cellular metabolism and growth. The studies on the cAMP-requiring mutants and their suppressors in the yeast revealed that cAMP-dependent protein phosphorylation is involved in the G1 phase of the cell cycle, stimulation of the phosphoinositide pathway and the post-meiotic stage of spourlation, and that inhibition of cAMP-dependent protein phosphorylation is required to go into the GO stage of and to induce meiotic division. Growth of some filamentous fungi was observed with significantly reduced levels of cAMP, suggesting that cAMP may not be essential for growth in some species of fungi. Germination of fungal spores, yeast-mycelium dimorphism and hyphal morphogenesis of several species of fungi were affected by cAMP. cAMP was involved in extension of hyphae, formation of hyphal aggregates and fruit body formation. Phosphorylation of cellular proteins is required in these processes, and the nature of these proteins phosphorylated by cAMP-dependent protein kinase is important to the understanding of the role of cAMP for growth and differentistion in fungal cells.