A. Pedro Gonçalves

MODULATION OF FUNGAL SENSITIVITY TO STAUROSPORINE BY TARGETING PROTEINS IDENTIFIED BY TRANSCRIPTIONAL PROFILING

An analysis of the time-dependent genetic response to the death-inducer staurosporine was performed in Neurospora crassa by transcriptional profiling. Staurosporine induced two major genes encoding an ABC transporter and a protein with similarity to regulatory subunits of potassium channels. The transcriptional response is dependent on the activity of a novel transcription factor. Deletion mutants in differentially expressed genes displayed altered sensitivity to staurosporine, underscoring significant proteins involved in the response to the drug. A null-mutant of the ABC transporter (abc3) is extremely sensitive to staurosporine, accumulates more staurosporine than the wild type strain and is defective in energy-dependent export of the drug, indicating that the ABC3 protein is the first described staurosporine transporter. It was located in the plasma membrane by immunofluorescence microscopy. The combination of inhibitors of ABC transporters or of potassium channels with staurosporine leads to an enhanced activity against N. crassa and pathogenic fungi paving the way to the development of more potent and specific antifungals. Our results highlight the general use of transcriptional profiling for the identification of novel proteins involved in cell death and their potential use as drug targets.

CZT-1 Is a Novel Transcription Factor Controlling Cell Death and Natural Drug Resistance in Neurospora crassa

We pinpoint CZT-1 (cell death–activated zinc cluster transcription factor) as a novel transcription factor involved in tolerance to cell death induced by the protein kinase inhibitor staurosporine in Neurospora crassa. Transcriptional profiling of staurosporine-treated wild-type cells by RNA-sequencing showed that genes encoding the machinery for protein synthesis are enriched among the genes repressed by the drug. Functional category enrichment analyses also show that genes encoding components of the mitochondrial respiratory chain are downregulated by staurosporine, whereas genes involved in endoplasmic reticulum activities are upregulated. In contrast, a staurosporine-treated Δczt-1 deletion strain is unable to repress the genes for the respiratory chain and to induce the genes related to the endoplasmic reticulum, indicating a role for CZT-1 in the regulation of activity of these organelles. The Δczt-1 mutant strain displays increased reactive oxygen species accumulation on insult with staurosporine. A genome-wide association study of a wild population of N. crassa isolates pointed out genes associated with a cell death role of CZT-1, including catalase-1 (cat-1) and apoptosis-inducing factor–homologous mitochondrion-associated inducer of death 2 (amid-2). Importantly, differences in the expression of czt-1 correlates with resistance to staurosporine among wild isolate strains. Our results reveal a novel transcription factor that regulates drug resistance and cell death in response to staurosporine in laboratory strains as well as in wild isolates of N. crassa.

Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death

ABSTRACT The model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca2+ ([Ca2+]c) dynamics and a distinct Ca2+ signature that involves Ca2+ influx from the external medium and internal Ca2+ stores. We investigated the molecular basis of this Ca2+ response by using [Ca2+]c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca2+ signature. Using &Dgr;cch-1, &Dgr;fig-1 and &Dgr;yvc-1 mutants and a range of inhibitors, we show that extracellular Ca2+ entry does not occur through the hitherto described high- and low-affinity Ca2+ uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca2+ release from internal stores following IP3 formation combines with the extracellular Ca2+ influx.

Regulated Forms of Cell Death in Fungi

Cell death occurs in all domains of life. While some cells die in an uncontrolled way due to exposure to external cues, other cells die in a regulated manner as part of a genetically encoded developmental program. Like other eukaryotic species, fungi undergo programmed cell death (PCD) in response to various triggers. For example, exposure to external stress conditions can activate PCD pathways in fungi. Calcium redistribution between the extracellular space, the cytoplasm and intracellular storage organelles appears to be pivotal for this kind of cell death. PCD is also part of the fungal life cycle, in which it occurs during sexual and asexual reproduction, aging, and as part of development associated with infection in phytopathogenic fungi. Additionally, a fungal non-self-recognition mechanism termed heterokaryon incompatibility (HI) also involves PCD. Some of the molecular players mediating PCD during HI show remarkable similarities to major constituents involved in innate immunity in metazoans and plants. In this review we discuss recent research on fungal PCD mechanisms in comparison to more characterized mechanisms in metazoans. We highlight the role of PCD in fungi in response to exogenic compounds, fungal development and non-self-recognition processes and discuss identified intracellular signaling pathways and molecules that regulate fungal PCD.

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics.

Programmed Cell Death in Neurospora crassa

Programmed cell death has been studied for decades in mammalian cells, but simpler organisms, including prokaryotes, plants, and fungi, also undergo regulated forms of cell death. We highlight the usefulness of the filamentous fungus Neurospora crassa as a model organism for the study of programmed cell death. In N. crassa, cell death can be triggered genetically due to hyphal fusion between individuals with different allelic specificities at het loci, in a process called “heterokaryon incompatibility.” Chemical induction of cell death can also be achieved upon exposure to death-inducing agents like staurosporine, phytosphingosine, or hydrogen peroxide. A summary of the recent advances made by our and other groups on the discovery of the mechanisms and mediators underlying the process of cell death in N. crassa is presented.

Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa

Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS). Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology.

Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

ABSTRACT The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion.

The Major Cellulases CBH-1 and CBH-2 of Neurospora crassa Rely on Distinct ER Cargo Adaptors for Efficient ER-Exit

Filamentous fungi are native secretors of lignocellulolytic enzymes and are used as protein‐producing factories in the industrial biotechnology sector. Despite the importance of these organisms in industry, relatively little is known about the filamentous fungal secretory pathway or how it might be manipulated for improved protein production. Here, we use Neurospora crassa as a model filamentous fungus to interrogate the requirements for trafficking of cellulase enzymes from the endoplasmic reticulum to the Golgi. We characterized the localization and interaction properties of the p24 and ERV‐29 cargo adaptors, as well as their role in cellulase enzyme trafficking. We find that the two most abundantly secreted cellulases, CBH‐1 and CBH‐2, depend on distinct ER cargo adaptors for efficient exit from the ER. CBH‐1 depends on the p24 proteins, whereas CBH‐2 depends on the N. crassa homolog of yeast Erv29p. This study provides a first step in characterizing distinct trafficking pathways of lignocellulolytic enzymes in filamentous fungi.

Transcription profiling of the Neurospora crassa response to a group of synthetic (thio)xanthones and a natural acetophenone.

Xanthones are a class of heterocyclic compounds characterized by a dibenzo-γ-pyrone nucleus. Analysis of their mode of action in cells, namely uncovering alterations in gene expression, is important because these compounds have potential therapeutic applications. Thus, we studied the transcriptional response of the filamentous fungus Neurospora crassa to a group of synthetic (thio)xanthone derivatives with antitumor activity using high throughput RNA sequencing. The induction of ABC transporters in N. crassa, particularly atrb and cdr4, is a common consequence of the treatment with xanthones. In addition, we found a group of genes repressed by all of the tested (thio)xanthone derivatives, that are evocative of genes downregulated during oxidative stress. The transcriptional response of N. crassa treated with an acetophenone isolated from the soil fungus Neosartorya siamensis shares some features with the (thio)xanthone-elicited gene expression profiles. Two of the (thio)xanthone derivatives and the N. siamensis-derived acetophenone inhibited the growth of N. crassa. Our work also provides framework datasets that may orientate future studies on the mechanisms of action of some groups of xanthones.