Robert W. Roberson

Polarisome meets spitzenkörper: microscopy, genetics, and genomics converge.

The impact of filamentous fungi on human welfare has never been greater. Fungi are acknowledged as the most economically devastating plant pathogens ([1][1]) and are attaining increasing notoriety for their ability to cause life-threatening infections in humans ([57][2], [71][3]), and fungal

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.

Architecture and development of the Neurospora crassa hypha -- a model cell for polarized growth.

Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation -- the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.

Growth-speed-correlated localization of exocyst and polarisome components in growth zones of Ashbya gossypii hyphal tips

We use the fungus Ashbya gossypii to investigate how its polar growth machinery is organized to achieve sustained hyphal growth. In slowly elongating hyphae exocyst, cell polarity and polarisome proteins permanently localize as cortical cap at hyphal tips, thus defining the zone of secretory vesicle fusion. In tenfold faster growing hyphae, this zone is only slightly enlarged demonstrating a capacity of hyphal growth zones to increase rates of vesicle processing to reach higher speeds. Concomitant with this increase, vesicles accumulate as spheroid associated with the tip cortex, indicating that a Spitzenkörper forms in fast hyphae. We also found spheroid-like accumulations for the exocyst components AgSec3, AgSec5, AgExo70 and the polarisome components AgSpa2, AgBni1 and AgPea2 (but not AgBud6 or cell polarity factors such as AgCdc42 or AgBem1). The localization of AgSpa2, AgPea2 and AgBni1 depend on each other but only marginally on AgBud6, as concluded from a set of deletions. Our data define three conditions to achieve fast growth at hyphal tips: permanent presence of the polarity machinery in a confined cortical area, organized accumulation of vesicles and a subset of polarity components close to this area, and spatial separation of the zones of exocytosis (tip front) and endocytosis (tip rim).

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.

The effects of ropy-1 mutation on cytoplasmic organization and intracellular motility in mature hyphae of Neurospora crassa

We have used light and electron microscopy to document the cytoplasmic effects of the ropy (ro-1) mutation in mature hyphae of Neurospora crassa and to better understand the role(s) of dynein during hyphal tip growth. Based on video-enhanced DIC light microscopy, the mature, growing hyphae of N. crassa wild type could be divided into four regions according to cytoplasmic organization and behavior: the apical region (I) and three subapical regions (II, III, and IV). A well-defined Spitzenkörper dominated the cytoplasm of region I. In region II, vesicles ( approximately 0.48 micro m diameter) and mitochondria maintained primarily a constant location within the advancing cytoplasm. This region was typically void of nuclei. Vesicles exhibited anterograde and retrograde motility in regions III and IV and followed generally parallel paths along the longitudinal axis of the cell. A small population of mitochondria displayed rapid anterograde and retrograde movements, while most maintained a constant position in the advancing cytoplasm in regions III and IV. Many nuclei occupied the cytoplasm of regions III and IV. In ro-1 hyphae, discrete cytoplasmic regions were not recognized and the motility and/or positioning of vesicles, mitochondria, and nuclei were altered to varying degrees, relative to the wild type cells. Immunofluorescence microscopy revealed that the microtubule cytoskeleton was severely disrupted in ro-1 cells. Transmission electron microscopy of cryofixed cells confirmed that region I of wild-type hyphae contained a Spitzenkörper composed of an aggregation of small apical vesicles that surrounded entirely or partially a central core composed, in part, of microvesicles embedded in a dense granular to fibrillar matrix. The apex of ro-1 the hypha contained a Spitzenkörper with reduced numbers of apical vesicles but maintained a defined central core. Clearly, dynein deficiency in the mutant caused profound perturbation in microtubule organization and function and, consequently, organelle dynamics and positioning. These perturbations impact negatively on the organization and stability of the Spitzenkörper, which, in turn, led to severe reduction in growth rate and altered hyphal morphology.

THE ACTIN CYTOSKELETON IN HYPHAL CELLS OF SCLEROTIUM ROLFSII

The distribution of the actin cytoskeleton was examined in hyphal tips of the plant pathogenic fungus Sclerotium rolfsii. Actin was visualized with light microscopy using immunofluorescence techniques and with ultrastructural immunocytochemistry. Computer-aided, three-dimensional reconstruction analysis of ultrathin serial sections and immunoblot analysis techniques were also employed in this study. Immunofluorescence data indicated that the majority of actin was localized in brightly fluorescent plaques that were principally localized in the first 10 to 12 gm of the hyphal tip. Focusing through the hyphal cell revealed that most plaques were positioned in the peripheral cytoplasmic regions. Actin microfilaments were not resolved at the light microscopic level; however, coarse actin fibers oriented parallel to the long hyphal axis were observed in subapical regions and in association with septa. A diffuse, amorphous cytoplasmic staining was present in the apical regions of the hypha except for that area occupied by the Spitzenk6rper. Ultrastructural immunocytochemistry and three-dimensional reconstruction analysis suggested that the fibrillar coating of filasomes contained actin and that filasomes represented the ultrastructural equivalent of actin plaques. Immunoblot analysis of total protein extracts separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis identified two actin related proteins, one with a relative molecular mass of 43,000 and the other migrating with a slightly higher molecular mass of 47,500.

Interaction of Antiparallel Microtubules in the Phragmoplast Is Mediated by the Microtubule-Associated Protein MAP65-3 in Arabidopsis

This study identifies Arabidopsis MAP65-3 as a protein that links antiparallel microtubules together in the midline of the phragmoplast and presents a modular model for microtubule organization during cytokinesis. Furthermore, MAP65-3 is found to be essential for the binding of the motor proteins Kinesin-12 and PAKRP2 to MT plus ends in the phragmoplast. 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.

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

ABSTRACT 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.

Myxoxanthophyll Is Required for Normal Cell Wall Structure and Thylakoid Organization in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Myxoxanthophyll is a carotenoid glycoside in cyanobacteria that is of unknown biological significance. The sugar moiety of myxoxanthophyll in Synechocystis sp. strain PCC 6803 was identified as dimethyl fucose. The open reading frame sll1213 encoding a fucose synthetase orthologue was deleted to probe the role of fucose and to determine the biological significance of myxoxanthophyll in Synechocystis sp. strain PCC 6803. Upon deletion of sll1213, a pleiotropic phenotype was obtained: when propagated at 0.5 micromol photons m(-2) s(-1), photomixotrophic growth of cells lacking sll1213 was poor. When grown at 40 micromol photons m(-2) s(-1), growth was comparable to that of the wild type, but cells showed a severe reduction in or loss of the glycocalyx (S-layer). As a consequence, cells aggregated in liquid as well as on plates. At both light intensities, new carotenoid glycosides accumulated, but myxoxanthophyll was absent. New carotenoid glycosides may be a consequence of less-specific glycosylation reactions that gained prominence upon the disappearance of the native sugar moiety (fucose) of myxoxanthophyll. In the mutant, the N-storage compound cyanophycin accumulated, and the organization of thylakoid membranes was altered. Altered cell wall structure and thylakoid membrane organization and increased cyanophycin accumulation were also observed for deltaslr0940K, a strain lacking zeta-carotene desaturase and thereby all carotenoids but retaining fucose. Therefore, lack of myxoxanthophyll and not simply of fucose results in most of the phenotypic effects described here. It is concluded that myxoxanthophyll contributes significantly to the vigor of cyanobacteria, as it stabilizes thylakoid membranes and is critical for S-layer formation.