The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa.

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.

The actin motor MYO-5 effect in the intracellular organization of Neurospora crassa.

In filamentous fungi, polarized growth is the result of vesicle secretion at the hyphal apex. Motor proteins mediate vesicle transport to target destinations on the plasma membrane via actin and microtubule cytoskeletons. Myosins are motor proteins associated with actin filaments. Specifically, class V myosins are responsible for cargo transport in eukaryotes. We studied the dynamics and localization of myosin V in wild type hyphae of Neurospora crassa and in hyphae that lacked MYO-5. In wild type hyphae, MYO-5-GFP was localized concentrated in the hyphal apex and colocalized with Spitzenkörper. Photobleaching studies showed that MYO-5-GFP was transported to the apex from subapical hyphal regions. The deletion of the class V myosin resulted in a reduced rate of hyphal growth, apical hyperbranching, and intermittent loss of hyphal polarity. MYO-5 did not participate in breaking the symmetrical growth during germination but contributed in the apical organization upon establishment of polarized growth. In the Δmyo-5 mutant, actin was organized into thick cables in the apical and subapical hyphal regions, and the number of endocytic patches was reduced. The microvesicles-chitosomes observed with CHS-1-GFP were distributed as a cloud occupying the apical dome and not in the Spitzenkörper as the WT strain. The mitochondrial movement was not associated with MYO-5, but tubular vacuole position is MYO-5-dependent. These results suggest that MYO-5 plays a role in maintaining apical organization and the integrity of the Spitzenkörper and is required for normal hyphal growth, polarity, septation, conidiation, and proper conidial germination.

Imaging the secretory compartments involved in the intracellular traffic of CHS-4, a class IV chitin synthase, in Neurospora crassa.

In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenkörper (SPK) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue, in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the SPK and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the SPK and septa was restored, providing evidence that CSE-7 is necessary for the localization of CHS-4 at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the SPK. The organization of the NEC was dependent on the cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi.

Hyphal tip cytoplasmic organization in four zygomycetous fungi.

We have examined the hyphal tip structure in four zygomycetous fungi: Mortierella verticillata (Mortierellales), Coemansia reversa (Kickxellales), Mucor indicus and Gilbertella persicaria (Mucorales) using both light and transmission electron microscopy. We have used cryofixation and freeze-substitution methods to preserve fungal hyphae for transmission electron microscopy, which yielded improved preservation of ultrastructural details. Our research has confirmed studies that described the accumulation of secretory vesicles as a crescent at the hyphal apex (i.e. the apical vesicle crescent [AVC]) and provided a more detailed understanding of the vesicle populations. In addition, we have been able to observe the behavior of the AVC during hyphal growth in M. indicus and G. persicaria.

A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data.

Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed.

Two microtubule-plus-end binding proteins LIS1-1 and LIS1-2, homologues of human LIS1 in Neurospora crassa.

LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1.

Deletion of the α-(1,3)-glucan synthase genes induces a restructuring of the conidial cell wall responsible for the avirulence of Aspergillus fumigatus.

α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.

Evolution of zygomycetous spindle pole bodies: Evidence from Coemansia reversa mitosis.

PREMISE OF THE STUDY: The earliest eukaryotes were likely flagellates with a centriole that nucleates the centrosome, the microtubule-organizing center (MTOC) for nuclear division. The MTOC in higher fungi, which lack flagella, is the spindle pole body (SPB). Can we detect stages in centrosome evolution leading to the diversity of SPB forms observed in terrestrial fungi? Zygomycetous fungi, which consist of saprobes, symbionts, and parasites of animals and plants, are critical in answering the question, but nuclear division has been studied in only two of six clades. METHODS: Ultrastructure of mitosis was studied in Coemansia reversa (Kickxellomycotina) germlings using cryofixation or chemical fixation. Character evolution was assessed by parsimony analysis, using a phylogenetic tree assembled from multigene analyses. KEY RESULTS: At interphase the SPB consisted of two components: a cytoplasmic, electron-dense sphere containing a cylindrical structure with microtubules oriented nearly perpendicular to the nucleus and an intranuclear component appressed to the nuclear envelope. Markham's rotation was used to reinforce the image of the cylindrical structure and determine the probable number of microtubules as nine. The SPB duplicated early in mitosis and separated on the intact nuclear envelope. Nuclear division appears to be intranuclear with spindle and kinetochore microtubules interspersed with condensed chromatin. CONCLUSIONS: This is the sixth type of zygomycetous SPB, and the third type that suggests a modified centriolar component. Coemansia reversa retains SPB character states from an ancestral centriole intermediate between those of fungi with motile cells and other zygomycetous fungi and Dikarya.

Cytology and molecular phylogenetics of Monoblepharidomycetes provide evidence for multiple independent origins of the hyphal habit in the Fungi.

The evolution of filamentous hyphae underlies an astounding diversity of fungal form and function. We studied the cellular structure and evolutionary origins of the filamentous form in the Monoblepharidomycetes (Chytridiomycota), an early-diverging fungal lineage that displays an exceptional range of body types, from crescent-shaped single cells to sprawling hyphae. To do so, we combined light and transmission electron microscopic analyses of hyphal cytoplasm with molecular phylogenetic reconstructions. Hyphae of Monoblepharidomycetes lack a complex aggregation of secretory vesicles at the hyphal apex (i.e. Spitzenkörper), have centrosomes as primary microtubule organizing centers and have stacked Golgi cisternae instead of tubular/fenestrated Golgi equivalents. The cytoplasmic distribution of actin in Monoblepharidomycetes is comparable to the arrangement observed previously in other filamentous fungi. To discern the origins of Monoblepharidomycetes hyphae, we inferred a phylogeny of the fungi based on 18S and 28S ribosomal DNA sequence data with maximum likelihood and Bayesian inference methods. We focused sampling on Monoblepharidomycetes to infer intergeneric relationships within the class and determined 78 new sequences. Analyses showed class Monoblepharidomycetes to be monophyletic and nested within Chytridiomycota. Hyphal Monoblepharidomycetes formed a clade sister to the genera without hyphae, Harpochytrium and Oedogoniomyces. A likelihood ancestral state reconstruction indicated that hyphae arose independently within the Monoblepharidomycetes lineage and in at least two other lineages. Cytological differences among monoblepharidalean and other fungal hyphae are consistent with these convergent origins.

Interaction of antiparallel microtubules in the phragmoplast is mediated by the microtubule-associated protein MAP65-3 in Arabidopsis.

In plant cells, microtubules (MTs) in the cytokinetic apparatus phragmoplast exhibit an antiparallel array and transport Golgi-derived vesicles toward MT plus ends located at or near the division site. By transmission electron microscopy, we observed that certain antiparallel phragmoplast MTs overlapped and were bridged by electron-dense materials in Arabidopsis thaliana. Robust MT polymerization, reported by fluorescently tagged End Binding1c (EB1c), took place in the phragmoplast midline. The engagement of antiparallel MTs in the central spindle and phragmoplast was largely abolished in mutant cells lacking the MT-associated protein, MAP65-3. We found that endogenous MAP65-3 was selectively detected on the middle segments of the central spindle MTs at late anaphase. When MTs exhibited a bipolar appearance with their plus ends placed in the middle, MAP65-3 exclusively decorated the phragmoplast midline. A bacterially expressed MAP65-3 protein was able to establish the interdigitation of MTs in vitro. MAP65-3 interacted with antiparallel microtubules before motor Kinesin-12 did during the establishment of the phragmoplast MT array. Thus, MAP65-3 selectively cross-linked interdigitating MTs (IMTs) to allow antiparallel MTs to be closely engaged in the phragmoplast. Although the presence of IMTs was not essential for vesicle trafficking, they were required for the phragmoplast-specific motors Kinesin-12 and Phragmoplast-Associated Kinesin-Related Protein2 to interact with MT plus ends. In conclusion, we suggest that the phragmoplast contains IMTs and highly dynamic noninterdigitating MTs, which work in concert to bring about cytokinesis in plant cells.

A bird's-eye view of the endoplasmic reticulum in filamentous fungi.

SUMMARYThe endoplasmic reticulum (ER) is one of the most extensive organelles in eukaryotic cells. It performs crucial roles in protein and lipid synthesis and Ca2+ homeostasis. Most information on ER types, functions, organization, and domains comes from studies in uninucleate animal, plant, and yeast cells. In contrast, there is limited information on the multinucleate cells of filamentous fungi, i.e., hyphae. We provide an analytical review of existing literature to categorize different types of ER described in filamentous fungi while emphasizing the research techniques and markers used. Additionally, we identify the knowledge gaps that need to be resolved better to understand the structure-function correlation of ER in filamentous fungi. Finally, advanced technologies that can provide breakthroughs in understanding the ER in filamentous fungi are discussed.

Gross morphological changes in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp. PCC 6803.

Cells of Synechocystis sp. PCC 6803 lacking photosystem I (PSI-less) and containing only photosystem II (PSII) or lacking both photosystems I and II (PSI/PSII-less) were compared to wild type (WT) cells to investigate the role of the photosystems in the architecture, structure, and number of thylakoid membranes. All cells were grown at 0.5µmol photons m(-2)s(-1). The lumen of the thylakoid membranes of the WT cells grown at this low light intensity were inflated compared to cells grown at higher light intensity. Tubular as well as sheet-like thylakoid membranes were found in the PSI-less strain at all stages of development with organized regular arrays of phycobilisomes on the surface of the thylakoid membranes. Tubular structures were also found in the PSI/PSII-less strain, but these were smaller in diameter to those found in the PSI-less strain with what appeared to be a different internal structure and were less common. There were fewer and smaller thylakoid membrane sheets in the double mutant and the phycobilisomes were found on the surface in more disordered arrays. These differences in thylakoid membrane structure most likely reflect the altered composition of photosynthetic particles and distribution of other integral membrane proteins and their interaction with the lipid bilayer. These results suggest an important role for the presence of PSII in the formation of the highly ordered tubular structures.

Polyhydroxybutyrate particles in Synechocystis sp. PCC 6803: facts and fiction.

Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Spherical inclusions inside the cell that are electron-transparent and/or slightly electron-dense and that are found in transmission electron micrographs of cyanobacteria are generally assumed to be PHB granules. The aim of this study was to test this assumption in different strains of the cyanobacterium Synechocystis sp. PCC 6803. Inclusions that resemble PHB granules were present in strains lacking a pair of genes essential for PHB synthesis and in wild-type cells under conditions that no PHB granules could be detected by fluorescence staining of PHB. Indeed, in these cells PHB could not be demonstrated chemically by GC/MS either. Based on the results gathered, it is concluded that not all the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria. Alternate assignments for these inclusions are discussed.

Meredithblackwellia eburnea gen. et sp. nov., Kriegeriaceae fam. nov. and Kriegeriales ord. nov.--toward resolving higher-level classification in Microbotryomycetes.

A field survey of ballistosporic yeasts in a Neotropical forest yielded a new species isolated from a fern leaf. The isolate is a cream-colored butyrous yeast that reproduces by budding. Budding occurs at both the apical and basal cell poles; occasionally multiple budding events co-occur, giving rise to rosette-like clusters of cells at both poles of the yeast mother cell. DNA sequences of large and small subunit and the internal transcribed spacer regions of the nuclear ribosomal DNA cistron indicated an affinity to Microbotryomycetes, Pucciniomycotina. A new genus, Meredithblackwellia, is proposed to accommodate the new species, M. eburnea (type strain MCA4105). Based on phylogenetic analyses, Meredithblackwellia is related to Kriegeria eriophori, a sedge parasite, to an aquatic fungus Camptobasidium hydrophilum and to several recently described anamorphic yeasts that have been isolated from plant material or psychrophilic environments. Morphological and ultrastructural studies confirm the relatedness of M. eburnea to these taxa and prompted the re-evaluation of higher-level classification within Microbotryomycetes. We propose here a new order, Kriegeriales, and place two families, Kriegeriaceae fam. nov. and Camptobasidiaceae R.T. Moore, within it. Our study re-emphasizes the need for systematic revision of species described in Rhodotorula.

Cellular organization in germ tube tips of Gigaspora and its phylogenetic implications.

Comparative morphology of the fine structure of fungal hyphal tips often is phylogenetically informative. In particular, morphology of the Spitzenkörper varies among higher taxa. To date no one has thoroughly characterized the hyphal tips of members of the phylum Glomeromycota to compare them with other fungi. This is partly due to difficulty growing and manipulating living hyphae of these obligate symbionts. We observed growing germ tubes of Gigaspora gigantea, G. margarita and G. rosea with a combination of light microscopy (LM) and transmission electron microscopy (TEM). For TEM, we used both traditional chemical fixation and cryo-fixation methods. Germ tubes of all species were extremely sensitive to manipulation. Healthy germ tubes often showed rapid bidirectional cytoplasmic streaming, whereas germ tubes that had been disturbed showed reduced or no cytoplasmic movement. Actively growing germ tubes contain a cluster of 10-20 spherical bodies approximately 3-8 µm behind the apex. The bodies, which we hypothesize are lipid bodies, move rapidly in healthy germ tubes. These bodies disappear immediately after any cellular perturbation. Cells prepared with cryo-techniques had superior preservation compared to those that had been processed with traditional chemical protocols. For example, cryo-prepared samples displayed two cell-wall layers, at least three vesicle types near the tip and three distinct cytoplasmic zones were noted. We did not detect a Spitzenkörper with either LM or TEM techniques and the tip organization of Gigaspora germ tubes appeared to be similar to hyphae in zygomycetous fungi. This observation was supported by a phylogenetic analysis of microscopic characters of hyphal tips from members of five fungal phyla. Our work emphasizes the sensitive nature of cellular organization, and the need for as little manipulation as possible to observe germ tube structure accurately.

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.

Hyphal and cytoskeleton polarization in Tuber melanosporum: a genomic and cellular analysis.

Filamentous polarized growth involves a series of events including polarization of the cytoskeleton to selected growth sites, and the transport of secretory vesicles containing the components required for growth. The availability of fungal genome sequences has recently led to the identification of a large number of proteins involved in these processes. We have explored the Tuber melanosporum genome sequence by searching for homologs of genes known to play crucial roles in the morphogenesis and cell polarity of yeasts and filamentous fungi. One hundred and forty-nine genes have been identified and functionally grouped according to the deduced amino acid sequences (44 genes involved in cell polarity/morphogenesis, 39 belonging to the actin cytoskeleton and 66 involved in membrane dynamics, septation and exocytosis). A detailed gene annotation has shown that most components of the cell polarity machinery, morphogenesis and cytoskeleton found in yeasts and filamentous fungi are conserved, although the degree of similarity varies from strong to weak. Microscopic analysis of quick-frozen truffle hyphae detected the characteristic subcellular components of the hyphal tip in septate filamentous fungi, while transcript profiles revealed a moderately variable pattern during the biological cycle.

An ontology of fungal subcellular traits.

UNLABELLED: • PREMISE OF THE STUDY: The Fungal Subcellular Ontology used in the Assembling the Fungal Tree of Life project is a taxon-wide ontology (controlled vocabulary for attributes) designed to clarify and integrate the broad range of subcellular characters and character states used in higher-level fungal systematics. As in the algae, cellular characters are important phylogenetic markers in kingdom Fungi. The Fungal Subcellular Ontology has been developed primarily to help researchers, especially systematists, in their search for information on subcellular characters across the Fungi, and it complements existing biological ontologies, including the Gene Ontology. • METHODS: The character and character state data set used in the Assembling the Fungal Tree of Life Structural and Biochemical Database (http://aftol.umn.edu) is the source of terms for generating the ontology. After the terms were accessioned and defined, they were combined in OBO-Edit file format, and the ontology was edited using OBO-Edit, an open source Java tool supported by the Gene Ontology project. • KEY RESULTS: The Fungal Subcellular Ontology covers both model and nonmodel fungi in great detail and is downloadable in OBO-Edit format at website http://aftol.umn.edu/ontology/fungal_subcellular.obo. • CONCLUSIONS: The ontology provides a controlled vocabulary of fungal subcellular terms and functions as an operating framework for the Assembling the Fungal Tree of Life Structural and Biochemical Database. An ontology-based design enhances reuse of data deposited in the Structural and Biochemical Database from other independent biological and genetic databases. Data integration approaches that advance access to data from the diversity of biological databases are imperative as interdisciplinary research gains importance. In this sense, the Fungal Subcellular Ontology becomes highly relevant to mycologists as well as nonmycologists because fungi interact actively as symbionts and parasites or passively with many other life forms.

Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa.

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

Cytoplasmic bulk flow propels nuclei in mature hyphae of Neurospora crassa.

We used confocal microscopy to evaluate nuclear dynamics in mature, growing hyphae of Neurospora crassa whose nuclei expressed histone H1-tagged green fluorescent protein (GFP). In addition to the H1-GFP wild-type (WT) strain, we examined nuclear displacement (passive transport) in four mutants deficient in microtubule-related motor proteins (ro-1, ro-3, kin-1, and a ro-1 kin-1 double mutant). We also treated the WT strain with benomyl and cytochalasin A to disrupt microtubules and actin microfilaments, respectively. We found that the degree of nuclear displacement in the subapical regions of all strains correlated with hyphal elongation rate. The WT strain and that the ro-1 kin-1 double mutant showed the highest correlation between nuclear movement and hyphal elongation. Although most nuclei seemed to move forward passively, presumably carried by the cytoplasmic bulk flow, a small proportion of the movement detected was either retrograde or accelerated anterograde. The absence of a specific microtubule motor in the mutants ro-1, ro-3, or kin-1 did not prevent the anterograde and retrograde migration of nuclei; however, in the ro-1 kin-1 double mutant retrograde migration was absent. In the WT strain, almost all nuclei were elongated, whereas in all other strains a majority of nuclei were nearly spherical. With only one exception, a sizable exclusion zone was maintained between the apex and the leading nucleus. The ro-1 mutant showed the largest nucleus exclusion zone; only the treatment with cytochalasin A abolished the exclusion zone. In conclusion, the movement and distribution of nuclei in mature hyphae appear to be determined by a combination of forces, with cytoplasmic bulk flow being a major determinant. Motor proteins probably play an active role in powering the retrograde or accelerated anterograde migrations of nuclei and may also contribute to passive anterograde displacement by binding nuclei to microtubules.