F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

Relationship between Phylogenetic Distribution and Genomic Features in Neurospora crassa

In the post-genome era, insufficient functional annotation of predicted genes greatly restricts the potential of mining genome data. We demonstrate that an evolutionary approach, which is independent of functional annotation, has great potential as a tool for genome analysis. We chose the genome of a model filamentous fungus Neurospora crassa as an example. Phylogenetic distribution of each predicted protein coding gene (PCG) in the N. crassa genome was used to classify genes into six mutually exclusive lineage specificity (LS) groups, i.e. Eukaryote/Prokaryote-core, Dikarya-core, Ascomycota-core, Pezizomycotina-specific, N. crassa-orphans and Others. Functional category analysis revealed that only ∼23% of PCGs in the two most highly lineage-specific grouping, Pezizomycotina-specific and N. crassa-orphans, have functional annotation. In contrast, ∼76% of PCGs in the remaining four LS groups have functional annotation. Analysis of chromosomal localization of N. crassa-orphan PCGs and genes encoding for secreted proteins showed enrichment in subtelomeric regions. The origin of N. crassa-orphans is not known. We found that 11% of N. crassa-orphans have paralogous N. crassa-orphan genes. Of the paralogous N. crassa-orphan gene pairs, 33% were tandemly located in the genome, implying a duplication origin of N. crassa-orphan PCGs in the past. LS grouping is thus a useful tool to explore and understand genome organization, evolution and gene function in fungi.     

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis,Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P₂. In N. crassa hyphae, PtdIns(4,5)P₂ localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P₂ that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.     

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2^Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication.     

Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.     

DCAF26, an Adaptor Protein of Cul4-Based E3, Is Essential for DNA Methylation in Neurospora crassa

DNA methylation is involved in gene silencing and genome stability in organisms from fungi to mammals. Genetic studies in Neurospora crassa previously showed that the CUL4-DDB1 E3 ubiquitin ligase regulates DNA methylation via histone H3K9 trimethylation. However, the substrate-specific adaptors of this ligase that are involved in the process were not known. Here, we show that, among the 16 DDB1- and Cul4-associated factors (DCAFs) encoded in the N. crassa genome, three interacted strongly with CUL4-DDB1 complexes. DNA methylation analyses of dcaf knockout mutants revealed that dcaf26 was required for all of the DNA methylation that we observed. In addition, histone H3K9 trimethylation was also eliminated in dcaf26^KO mutants. Based on the finding that DCAF26 associates with DDB1 and the histone methyltransferase DIM-5, we propose that DCAF26 protein is the major adaptor subunit of the Cul4-DDB1-DCAF26 complex, which recruits DIM-5 to DNA regions to initiate H3K9 trimethylation and DNA methylation in N. crassa.     

Differences in Gene Expression between Mouse and Human for Dynamically Regulated Genes in Early Embryo

Infertility is a worldwide concern that can be treated with in vitro fertilization (IVF). Improvements in IVF and infertility treatment depend largely on better understanding of the molecular mechanisms for human preimplantation development. Several large-scale studies have been conducted to identify gene expression patterns for the first five days of human development, and many functional studies utilize mouse as a model system. We have identified genes of possible importance for this time period by analyzing human microarray data and available data from online databases. We selected 70 candidate genes for human preimplantation development and investigated their expression in the early mouse development from oocyte to the 8-cell stage. Maternally loaded genes expectedly decreased in expression during development both in human and mouse. We discovered that 25 significantly upregulated genes after fertilization in human included 13 genes whose orthologs in mouse behaved differently and mimicked the expression profile of maternally expressed genes. Our findings highlight many significant differences in gene expression patterns during mouse and human preimplantation development. We also describe four cancer-testis antigen families that are also highly expressed in human embryos: PRAME, SSX, GAGE and MAGEA.     

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.     

Differences in Crossover Frequency and Distribution among Three Sibling Species of Drosophila

Comparisons of the genetic and cytogenetic maps of three sibling species of Drosophila reveal marked differences in the frequency and cumulative distribution of crossovers during meiosis. The maps for two of these species, Drosophila melanogaster and D. simulans, have previously been described, while this report presents new map data for D. mauritiana, obtained using a set of P element markers. A genetic map covering nearly the entire genome was constructed by estimating the recombination fraction for each pair of adjacent inserts. The P-based genetic map of mauritiana is ~1.8 times longer than the standard melanogaster map. It appears that mauritiana has higher recombination along the entire length of each chromosome, but the difference is greatest in centromere-proximal regions of the autosomes. The mauritiana autosomes show little or no centromeric recombinational suppression, a characteristic that is prominent in melanogaster. D. simulans appears to be intermediate both in terms of total map length and intensity of the autosomal centromeric effect. These interspecific differences in recombination have important evolutionary implications for DNA sequence organization and variability. In particular, mauritiana is expected to differ from melanogaster in patterns and amounts of sequence variation and transposon insertions.     

Methods for the Induction of Reproduction in a Tropical Species of Filamentous Ulva

The green seaweed Ulva is a major fouling organism but also an edible aquaculture product in Asia. This study quantified for the first time the effect of key factors on the reproduction of a tropical species of filamentous Ulva (Ulva sp. 3). The controlled timing of release of swarmers (motile reproductive bodies) was achieved when experiments were initiated in the early afternoon by exposing the thalli to a temperature shock (4°C) for 10 min and subsequently placing them into autoclaved filtered seawater under a 12 h light: 12 h dark photoperiod at 25°C. The release of swarmers then peaked two days after initiation. In contrast, segmentation, dehydration, salinity or time of initiation of experiments had no effect of any magnitude on reproduction. The released swarmers were predominantly biflagellate (95%), negatively phototactic and germinated without complementary gametes. This indicates that Ulva sp. 3 has a simple asexual life history dominated by biflagellate zoids.