Eremothecium coryli bloodstream infection in a patient with acute myeloid leukemia: first case report of human infection.

Eremothecium coryli is a dimorphic fungus of the Saccharomycetes class. While species within this class are known to cause human infection, Eremothecium species have previously only been known as phytopathogens and never been isolated from a human sample. Here, we report the first known case of human E. coryli infection.

Mechanism of nuclear movements in a multinucleated cell.

Multinucleated cells are important in many organisms, but the mechanisms governing the movements of nuclei sharing a common cytoplasm are not understood. In the hyphae of the plant pathogenic fungus Ashbya gossypii, nuclei move back and forth, occasionally bypassing each other, preventing the formation of nuclear clusters. This is essential for genetic stability. These movements depend on cytoplasmic microtubules emanating from the nuclei that are pulled by dynein motors anchored at the cortex. Using three-dimensional stochastic simulations with parameters constrained by the literature, we predict the cortical anchor density from the characteristics of nuclear movements. The model accounts for the complex nuclear movements seen in vivo, using a minimal set of experimentally determined ingredients. Of interest, these ingredients power the oscillations of the anaphase spindle in budding yeast, but in A. gossypii, this system is not restricted to a specific nuclear cycle stage, possibly as a result of adaptation to hyphal growth and multinuclearity.

Oxidative stress protection and glutathione metabolism in response to hydrogen peroxide and menadione in riboflavinogenic fungus Ashbya gossypii.

Ashbya gossypii is a plant pathogen and a natural overproducer of riboflavin and is used for industrial riboflavin production. A few literature reports depict a link between riboflavin overproduction and stress in this fungus. However, the stress protection mechanisms and glutathione metabolism are not much explored in A. gossypii. In the present study, an increase in the activity of catalase and superoxide dismutase was observed in response to hydrogen peroxide and menadione. The lipid peroxide and membrane lipid peroxide levels were increased by H2O2 and menadione, indicating oxidative damage. The glutathione metabolism was altered with a significant increase in oxidized glutathione (GSSG), glutathione peroxidase (GPX), glutathione S transferase (GST), and glutathione reductase (GR) and a decrease in reduced glutathione (GSH) levels in the presence of H2O2 and menadione. Expression of the genes involved in stress mechanism was analyzed in response to the stressors by semiquantitative RT-PCR. The messenger RNA (mRNA) levels of CTT1, SOD1, GSH1, YAP1, and RIB3 were increased by H2O2 and menadione, indicating the effect of stress at the transcriptional level. A preliminary bioinformatics study for the presence of stress response elements (STRE)/Yap response elements (YRE) depicted that the glutathione metabolic genes, stress genes, and the RIB genes hosted either STRE/YRE, which may enable induction of these genes during stress.

Nuclear repulsion enables division autonomy in a single cytoplasm.

BACKGROUND: Current models of cell-cycle control, based on classic studies of fused cells, predict that nuclei in a shared cytoplasm respond to the same CDK activities to undergo synchronous cycling. However, synchrony is rarely observed in naturally occurring syncytia, such as the multinucleate fungus Ashbya gossypii. In this system, nuclei divide asynchronously, raising the question of how nuclear timing differences are maintained despite sharing a common milieu. RESULTS: We observe that neighboring nuclei are highly variable in division-cycle duration and that neighbors repel one another to space apart and demarcate their own cytoplasmic territories. The size of these territories increases as a nucleus approaches mitosis and can influence cycling rates. This nonrandom nuclear spacing is regulated by microtubules and is required for nuclear asynchrony, as nuclei that transiently come in very close proximity will partially synchronize. Sister nuclei born of the same mitosis are generally not persistent neighbors over their lifetimes yet remarkably retain similar division cycle times. This indicates that nuclei carry a memory of their birth state that influences their division timing and supports that nuclei subdivide a common cytosol into functionally distinct yet mobile compartments. CONCLUSIONS: These findings support that nuclei use cytoplasmic microtubules to establish "cells within cells." Individual compartments appear to push against one another to compete for cytoplasmic territory and insulate the division cycle. This provides a mechanism by which syncytial nuclei can spatially organize cell-cycle signaling and suggests size control can act in a system without physical boundaries.

Protein aggregation behavior regulates cyclin transcript localization and cell-cycle control.

Little is known about the active positioning of transcripts outside of embryogenesis or highly polarized cells. We show here that a specific G1 cyclin transcript is highly clustered in the cytoplasm of large multinucleate cells. This heterogeneous cyclin transcript localization results from aggregation of an RNA-binding protein, and deletion of a polyglutamine stretch in this protein results in random transcript localization. These multinucleate cells are remarkable in that nuclei cycle asynchronously despite sharing a common cytoplasm. Notably, randomization of cyclin transcript localization significantly diminishes nucleus-to-nucleus differences in the number of mRNAs and synchronizes cell-cycle timing. Thus, nonrandom cyclin transcript localization is important for cell-cycle timing control and arises due to polyQ-dependent behavior of an RNA-binding protein. There is a widespread association between polyQ expansions and RNA-binding motifs, suggesting that this is a broadly exploited mechanism to produce spatially variable transcripts and heterogeneous cell behaviors. PAPERCLIP:

A network involving Rho-type GTPases, a paxillin and a formin homologue regulates spore length and spore wall integrity in the filamentous fungus Ashbya gossypii.

Fungi produce spores that allow for their dispersal and survival under harsh environmental conditions. These spores can have an astonishing variety of shapes and sizes. Using the highly polar, needle-shaped spores of the ascomycete Ashbya gossypii as a model, we demonstrated that spores produced by this organism are not simple continuous structures but rather consist of three different segments that correlate with the accumulation of different materials: a rigid tip segment, a more fragile main spore-compartment and a solid tail segment. Little is currently known about the regulatory mechanisms that control the formation of the characteristic spore morphologies. We tested a variety of mutant strains for their spore phenotypes, including spore size, shape and wall defects. The mutants that we identified as displaying such phenotypes are all known for their roles in the regulation of hyphal tip growth, including the formin protein AgBni1, the homologous Rho-type GTPases AgRho1a and AgRho1b and the scaffold protein AgPxl1. Our observations suggest that these proteins form a signalling network controlling spore length by regulating the formation of actin structures.

Axl2 integrates polarity establishment, maintenance, and environmental stress response in the filamentous fungus Ashbya gossypii.

In budding yeast, new sites of polarity are chosen with each cell cycle and polarization is transient. In filamentous fungi, sites of polarity persist for extended periods of growth and new polarity sites can be established while existing sites are maintained. How the polarity establishment machinery functions in these distinct growth forms found in fungi is still not well understood. We have examined the function of Axl2, a transmembrane bud site selection protein discovered in Saccharomyces cerevisiae, in the filamentous fungus Ashbya gossypii. A. gossypii does not divide by budding and instead exhibits persistent highly polarized growth, and multiple axes of polarity coexist in one cell. A. gossypii axl2Δ (Agaxl2Δ) cells have wavy hyphae, bulbous tips, and a high frequency of branch initiations that fail to elongate, indicative of a polarity maintenance defect. Mutant colonies also have significantly lower radial growth and hyphal tip elongation speeds than wild-type colonies, and Agaxl2Δ hyphae have depolarized actin patches. Consistent with a function in polarity, AgAxl2 localizes to hyphal tips, branches, and septin rings. Unlike S. cerevisiae Axl2, AgAxl2 contains a Mid2 homology domain and may function to sense or respond to environmental stress. In support of this idea, hyphae lacking AgAxl2 also display hypersensitivity to heat, osmotic, and cell wall stresses. Axl2 serves to integrate polarity establishment, polarity maintenance, and environmental stress response for optimal polarized growth in A. gossypii.

Regulation of exit from mitosis in multinucleate Ashbya gossypii cells relies on a minimal network of genes.

In Saccharomyces cerevisiae, mitosis is coupled to cell division by the action of the Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN) regulatory networks, which mediate exit from mitosis by activation of the phosphatase Cdc14. The closely related filamentous ascomycete Ashbya gossypii provides a unique cellular setting to study the evolution of these networks. Within its multinucleate hyphae, nuclei are free to divide without the spatial and temporal constraints described for budding yeast. To investigate how this highly conserved system has adapted to these circumstances, we constructed a series of mutants lacking homologues of core components of MEN and FEAR and monitored phenomena such as progression through mitosis and Cdc14 activation. MEN homologues in A. gossypii were shown to have diverged from their anticipated role in Cdc14 release and exit from mitosis. We observed defects in septation, as well as a partial metaphase arrest, in Agtem1Δ, Agcdc15Δ, Agdbf2/dbf20Δ, and Agmob1Δ. A. gossypii homologues of the FEAR network, on the other hand, have a conserved and more pronounced role in regulation of the M/G1 transition. Agcdc55Δ mutants are unable to sequester AgCdc14 throughout interphase. We propose a reduced model of the networks described in yeast, with a low degree of functional redundancy, convenient for further investigations into these networks.

Clustering of nuclei in multinucleated hyphae is prevented by dynein-driven bidirectional nuclear movements and microtubule growth control in Ashbya gossypii.

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.

Mobility, microtubule nucleation and structure of microtubule-organizing centers in multinucleated hyphae of Ashbya gossypii.

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 microm/min), rotations (up to 180 degrees in 30 s), and long-range nuclear bypassing (up to 9 microm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis-oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

Development of a yeast bio-assay to screen anti-mitochondrial drugs.

Previous studies show that acetylsalicylic acid (aspirin) at low concentrations affects yeast sexual structure development in a similar fashion than oxygen depletion. This is ascribed to its anti-mitochondrial action. In this study, we report the same for other anti-inflammatory (i.e. ibuprofen, indomethacin, salicylic acid, benzoic acid) as well as anticancer (Lonidamine) drugs, also known for inhibiting mitochondrial activity in mammalian cells. This is shown by a unique yeast bio-assay, with the mitochondrion-dependent sexual structure, riboflavin production, and hyphal morphology of the yeast Eremothecium ashbyi serving as indicators. These drugs affect this yeast in a similar way as found under oxygen limitation conditions by inhibiting sexual structure development (most sensitive), riboflavin production, and yielding characteristically wrinkled and granular hyphae, presenting a unique "anoxic" morphological pattern for this yeast. Only drugs associated with anti-mitochondrial activity presented such a pattern. This bio-assay may find application in the screening for novel drugs from various sources with anti-mitochondrial actions.

Regulation of distinct septin rings in a single cell by Elm1p and Gin4p kinases.

Septins are conserved, GTP-binding proteins that assemble into higher order structures, including filaments and rings with varied cellular functions. Using four-dimensional quantitative fluorescence microscopy of Ashbya gossypii fungal cells, we show that septins can assemble into morphologically distinct classes of rings that vary in dimensions, intensities, and positions within a single cell. Notably, these different classes coexist and persist for extended times, similar in appearance and behavior to septins in mammalian neurons and cultured cells. We demonstrate that new septin proteins can add through time to assembled rings, indicating that septins may continue to polymerize during ring maturation. Different classes of rings do not arise from the presence or absence of specific septin subunits and ring maintenance does not require the actin and microtubule cytoskeletons. Instead, morphological and behavioral differences in the rings require the Elm1p and Gin4p kinases. This work demonstrates that distinct higher order septin structures form within one cell because of the action of specific kinases.