Hex-1, a gene unique to filamentous fungi, encodes the major protein of the Woronin body and functions as a plug for septal pores

We have identified a gene, named hex-1, that encodes the major protein in the hexagonal crystals, or Woronin bodies, of Neurospora crassa. Analysis of a strain with a null mutation in the hex-1 gene showed that the septal pores in this organism were not plugged when hyphae were damaged, leading to extensive loss of cytoplasm. When grown on agar plates containing sorbose, the hex-1(-) strain showed extensive lysis of hyphal tips. The HEX-1 protein was predicted to be 19,125 Da. Analysis of the N-terminus of the purified protein indicated that 16 residues are cleaved, yielding a protein of 17,377 Da. A polyclonal antibody raised to the HEX-1 protein recognized multiple forms of the protein, apparently dimers and tetramers that were resistant to solubilization by sodium dodecyl sulfate and reducing reagents. Treatment of the protein with phosphatase caused dissociation of these oligomers. Preparations enriched in Woronin bodies contained catalase activity, which was not detected in comparable fractions from the hex-1(-) mutant strain. These results support the hypothesis that the Woronin body is a specialized peroxisome that functions as a plug for septal pores.     

A HEX-1 crystal lattice required for Woronin body function in Neurospora crassa

The Woronin body is a dense-core vesicle specific to filamentous ascomycetes (Euascomycetes), where it functions to seal the septal pore in response to cellular damage. The HEX-1 protein self-assembles to form this solid core of the vesicle. Here, we solve the crystal structure of HEX-1 at 1.8 A, which provides the structural basis of its self-assembly. The structure reveals the existence of three intermolecular interfaces that promote the formation of a three-dimensional protein lattice. Consistent with these data, self-assembly is disrupted by mutations in intermolecular contact residues and expression of an assembly-defective HEX-1 mutant results in the production of aberrant Woronin bodies, which possess a soluble noncrystalline core. This mutant also fails to complement a hex-1 deletion in Neurospora crassa, demonstrating that the HEX-1 protein lattice is required for Woronin body function. Although both the sequence and the tertiary structure of HEX-1 are similar to those of eukaryotic initiation factor 5A (eIF-5A), the amino acids required for HEX-1 self-assembly and peroxisomal targeting are absent in eIF-5A. Thus, we propose that a new function has evolved following duplication of an ancestral eIF-5A gene and that this may define an important step in fungal evolution.     

The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenk?rper in Neurospora crassa

Most models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H⁺-translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H⁺ out of the cell, generating a large electrochemical gradient that supplies energy to H⁺-coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 μm) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex.     

Coronin is a component of the endocytic collar of hyphae of Neurospora crassa and is necessary for normal growth and morphogenesis

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenk?rper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenk?rper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis.     

Traffic of chitin synthase 1 (CHS-1) to the Spitzenkörper and developing septa in hyphae of Neurospora crassa: actin dependence and evidence of distinct microvesicle populations

We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1–GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1–GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1–GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.     

Aggregation and collapse in a mechanical model of fungal tip growth

 Interconnected hyphal tubes form the mycelia of a fungal colony. The growth of the colony results from the elongation and branching of these single hyphae. The material being incorporated into the extending hyphal wall is supplied by vesicles which are formed further back in the hyphal tip. Such wall-destined vesicles appear conspicuously concentrated in the interior of the hypha, just before the hyphal apex, in the form of an apical body or Spitzenk?rper. The cytoskeleton of the hyphal tube has been implicated in the organisation of the Spitzenk?rper and the transport of vesicles, but as yet there is no postulated mechanism for this. We propose a mechanism by which forces generated by the cytoskeleton are responsible for biasing the movement of vesicles. A mathematical model is derived where the cytoskeleton is described as a viscoelastic fluid. Viscoelastic forces are coupled to the conservation equation governing the vesicle dynamics, by weighting the diffusion of vesicles via the strain tensor. The model displays collapse and aggregation patterns in one and two dimensions. These are interpreted in terms of the formation of the Spitzenk?rper and the initiation of apical branching. Received: 16 September 1996 / Revised version: 20 July 1998     

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

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

Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2)

Streptomycetes grow by cell wall extension at hyphal tips. The molecular basis for such polar growth in prokaryotes is largely unknown. It is reported here that DivIVASC, the Streptomyces coelicolor homologue of the Bacillus subtilis protein DivIVA, is essential and directly involved in hyphal tip growth and morphogenesis. A DivIVASC-EGFP hybrid was distinctively localized to hyphal tips and lateral branches. Reduction of divIVASC expression to about 10% of the normal level produced a phenotype strikingly similar to that of many tip growth mutants in fungi, including irregular curly hyphae and apical branching. Overexpression of the gene dramatically perturbed determination of cell shape at the growing tips. Furthermore, staining of nascent peptidoglycan with a fluorescent vancomycin conjugate revealed that induction of overexpression in normal hyphae disturbed tip growth, and gave rise to several new sites of cell wall assembly, effectively causing hyperbranching. The results show that DivIVASC is a novel bacterial morphogene, and it is localized at or very close to the apical sites of peptidoglycan assembly in Streptomyces hyphae.     

In vivo imaging of endoplasmic reticulum and distribution of mutant α-amylase in Aspergillus oryzae

Properly folded proteins destined for secretion exit through a specific subdomain of the endoplasmic reticulum (ER) known as transitional ER (tER) sites or ER exit sites (ERES). While such proteins in filamentous fungi localize at the hyphal tips overlapping the Spitzenk?rper, the distribution of misfolded proteins remains unknown. In the present study, we analyzed the distribution of mutant protein as well as ER and tER sites visualized by expression of AoClxA and AoSec13 fused with fluorescent protein, respectively, in the filamentous fungus Aspergillus oryzae. Discrete tER subdomains were visualized as the punctate dots of AoSec13 overlapping or associated with AoClxA distribution. Both ER and tER sites were concentrated near hyphal tips and formed apical gradients. Interestingly, while the expression of wild-type α-amylase fusion protein (AmyB-mDsRed) showed its localization coinciding with the Spitzenk?rper, a disulfide bond-deletion in AmyB causing its misfolding resulted in its accumulation in the subapical and basal ER, creating a reciprocal gradient to the tER sites. Furthermore, the reciprocal gradient enabled a clear distinction between the tER sites and the mutant AmyB accumulation sites near the apex. Based on these findings, we conclude that A. oryzae accumulates aberrant proteins toward basal hyphae while maintaining polarized tER sites for secretion of properly folded proteins at the hyphal tip.     

Aspergillus nidulans ChiA is a glycosylphosphatidylinositol (GPI)-anchored chitinase specifically localized at polarized growth sites

It is believed that chitinases play important physiological roles in filamentous fungi since chitin is one of the major cell wall components in these organisms. In this paper we investigated a chitinase gene, chiA, of Aspergillus nidulans and found that the gene product of chiA consists of a signal sequence, a region including chitinase consensus motifs, a Ser/Thr/Pro-rich region and a glycosylphosphatidylinositol (GPI)-anchor attachment motif. Phosphatidylinositol-specific phospholipase C treatment of the fusion protein of ChiA and enhanced green fluorescent protein (EGFP)-ChiA-EGFP-caused a change in its hydrophobicity, indicating that ChiA is a GPI-anchored protein. ChiA-EGFP localized at the germ tubes of conidia, at hyphal branching sites and hyphal tips. chiA expression was specifically high during conidia germination and in the marginal growth regions of colonies. These results suggest that ChiA functions as a GPI-anchored chitinase at the sites where cell wall remodeling and/or cell wall maturation actively take place.     

CYTOLOGY AND MORPHOGENESIS IN THE RHIZOMORPH OF ARMILLARIA MELLEA

Aided by the techniques of thin sectioning and electron microscopy, the apical region of the rhizomorph of Armillaria mellea has been examined. This region is composed of concentric zones of morphologically distinct tissues derived from a subapical meristematic zone designated the apical center. Meristematic activity is of two types: (1) primary, localized in the apical center, in which new hyphal elements are formed from apical initials, and (2) secondary, localized in the lateral regions of the apex, in which elaboration of the hyphal elements by means of elongation and secondary crosswall formation takes place. From these meristematic zones the tissues of the mature rhizomorph are derived and include: (a) peripheral hyphae, (b) cortex, (c) subcortex, and (d) primary and secondary medulla. The manner of differentiation of an apical initial appears unique and involves synchronous nuclear divisions accompanied by segmentation in many planes. The result of this activity is the formation of multinucleate hyphae. Apical initials are usually highly cytoplasmic and possess peculiar non-membrane-bound fibrous bundles, but in all other respects they resemble the hyphae of most Basidiomycetes thus far examined with the electron microscope.