A versatile set of Lifeact-RFP expression plasmids for live-cell imaging of F-actin in filamentous fungi

Here we report the construction and application of a range of expression plasmids designed to facilitate live-cell imaging of F-actin dynamics in filamentous fungi simultaneously with other, preferably GFP-tagged fusion proteins. Pros and cons of the use of three different red fluorescent proteins (RFPs), two different promoters and three different selection markers are addressed

Cell fusion in Neurospora crassa

The primary research aims of this thesis were the identification of novel cell fusion mutants of Neurospora crassa and the subsequent functional characterization of selected candidate proteins during conidial anastomosis tube (CAT)-mediated cell fusion by means of genetic, molecular, biochemical and live-cell imaging analysis. Chapter 1 provides a general introduction of the model organism and the cell fusion processes studied during different stages of the fungal lifecycle. Chapter 2 summarizes the materials and methods used. Chapter 3 introduces the comparative genomics screen conducted between appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae and hyphal fusion in Neurospora crassa. Novel cell fusion mutants were identified in MAP kinase signalling, redox-signalling and Rho-type GTPase signalling pathways, whereas no functional overlap in the cAMP response pathway between both species could be found. Chapter 4 demonstrates how newly developed fluorescent reporters for F-actin and activated Rho GTPases in filamentous fungi lead to novel insights into the dynamic rearrangement of the F-actin cytoskeleton and cortical activation of Rho GTPases during cell symmetry breaking, polarized tip growth and cell fusion. Chapter 5 focuses on the role of the cell wall integrity (CWI) MAP kinase pathway during cell fusion, and in particular, on the function of the terminal MAP kinase MAK-1 during CAT homing and fusion pore formation. Inhibitor studies indicated that MAK-1 kinase activity is required for its own recruitment to the fusion site already during homing and for cell wall remodelling during fusion pore enlargement between interacting cells. Chapter 6 presents ultrastructural scanning electron microscope (SEM) studies which indicate that defects in hyphal attachment, extracellular matrix deposition and cell wall remodelling prematurely abort morphogenesis of the female fruitbody. These findings are put into context with defects observed in mutants of components acting in related signalling pathways which appear to regulate non-self fusion events at later stages of sexual development leading to fertilization in N. crassa. Chapter 7 provides the first evidence for a role of NADPH-oxidase (NOX)-generated reactive oxygen species (ROS) in the regulation of morphogenetic changes required for CAT-mediated cell fusion. Redox-modification of signalling proteins might be involved in cell-cell chemoattraction. Chapter 8 provides a summary of the key findings and discusses future directions

Parameters of Stromal Activation and Epithelial to Mesenchymal Transition as Predictive Biomarkers for Induction Chemotherapy in Patients With Locally Advanced Oral Cavity and Oropharyngeal Squamous Cell Cancer

Objectives Induction chemotherapy (IC) is likely to be effective for biologically distinct subgroups of oral cancer and biomarker development may lead to identification of those patients. Methods We evaluated immune cell infiltration, stroma formation and structure of the invasive front as well as the immunohistochemical expression of alpha smooth muscle actin (ASMA), CD163, E-cadherin, N-cadherin, and the laminin gamma 2 chain in pretreatment biopsy specimens and surgical resections after IC in 20 patients with locally advanced oral cancer who were treated in a prospective, ongoing, phase II trial on IC using docetaxel, cisplatin, and 5-fluorouracil (TPF). Results Significant negative prognostic factors for incomplete pathological tumor response to IC were alcohol abuse (P=0.032), cN+ (P=0.042), and <30% tumor reduction after first cycle of IC (P=0.034). Of the investigated histological parameters and biomarkers only a low membrane-bound expression of E-cadherin showed a trend to be associated with incomplete response to IC (P=0.061). Low expression of ASMA in stromal vessels and a strong tumor invasion front were significantly associated to tumor recurrence (P=0.024 and P=0.004, respectively). The median follow-up of all patients was 35 months. Alcohol abuse (P<0.001), <30% tumor reduction after first cycle of IC (P=0.005), and a strong tumor invasion front (P=0.019) were negative prognostic factors for overall survival. Conclusion A strong predictive biomarker among the investigated parameters for benefitting from TPF IC could not be found. The extent of the tumor invasion front was a negative prognostic marker for recurrence and survival in oral cancer treated by TPF IC followed by surgery and postoperative radiochemotherapy

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

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion

Comparative proteomic analysis of normal and tumor stromal cells by tissue on chip based mass spectrometry (toc-MS)

In carcinoma tissues, genetic and metabolic changes not only occur at the tumor cell level, but also in the surrounding stroma. This carcinoma-reactive stromal tissue is heterogeneous and consists e.g. of non-epithelial cells such as fibroblasts or fibrocytes, inflammatory cells and vasculature-related cells, which promote carcinoma growth and progression of carcinomas. Nevertheless, there is just little knowledge about the proteomic changes from normal connective tissue to tumor stroma. In the present study, we acquired and analysed specific protein patterns of small stromal sections surrounding head and neck cell complexes in comparison to normal subepithelial connective tissue. To gain defined stromal areas we used laser-based tissue microdissection. Because these stromal areas are limited in size we established the highly sensitive 'tissue on chip based mass spectrometry' (toc-MS). Therefore, the dissected areas were directly transferred to chromatographic arrays and the proteomic profiles were subsequently analysed with mass spectrometry. At least 100 cells were needed for an adequate spectrum. The locating of differentially expressed proteins enables a precise separation of normal and tumor stroma. The newly described toc-MS technology allows an initial insight into proteomic differences between small numbers of exactly defined cells from normal and tumor stroma

Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome

A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture

Erratum to: Genome sequencing of the Trichoderma reesei QM9136 mutant identifies a truncation of the transcriptional regulator XYR1 as the cause for its cellulase-negative phenotype

International audienceBackground : Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a.Results : In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn2Cys6 transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be expressed over the basal constitutive level.Conclusion : The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype

Functional Analyses of Trichoderma reesei LAE1 Reveal Conserved and Contrasting Roles of This Regulator

The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in Aspergillus nidulans. We have recently shown that the LaeA ortholog of Trichoderma reesei (LAE1), a fungus that is an industrial producer of cellulase and hemicellulase enzymes, regulates the expression of cellulases and polysaccharide hydrolases. To learn more about the function of LAE1 in T. reesei, we assessed the effect of deletion and overexpression of lae1 on genome-wide gene expression. We found that in addition to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrin-proteins, iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases are major gene categories also regulated by LAE1. chromatin immunoprecipitation sequencing with antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and -me3) or silent (H3K9me3) chromatin detected 4089 genes bearing one or more of these methylation marks, of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1. Transformation of a laeA-null mutant of A. nidulans with the T. reesei lae1 gene did not rescue sterigmatocystin formation and further impaired sexual development. LAE1 did not interact with A. nidulans VeA in yeast two-hybrid assays, whereas it interacted with the T. reesei VeA ortholog, VEL1. LAE1 was shown to be required for the expression of vel1, whereas the orthologs of velB and VosA are unaffected by lae1 deletion. Our data show that the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. In T. reesei, LAE1 appears predominantly to regulate genes increasing relative fitness in its environment.Keywords: Flavus, Secondary metabolism, Aspergillus nidulans, Gene expression, Hypocrea jecorina, DNA methylation, Histone H3, Protein kinase, Neurospora crassa, Penicillin biosynthesi