A member of the OSCA/TMEM63 family of mechanosensitive calcium channels participates in cell wall integrity maintenance in Aspergillus nidulans.

The calF7 mutation in Aspergillus nidulans causes hypersensitivity to the cell wall compromising agents Calcofluor White (CFW) and Congo Red. In this research we demonstrate that the calF7 mutation resides in gene AN2880, encoding a predicted member of the OSCA/TMEM63 family of transmembrane glycoproteins. Those members of the family whose physiological functions have been investigated have been shown to act as mechanosensitive calcium transport channels. Deletion of AN2880 replicates the CFW hypersensitivity phenotype. Separately, we show that CFW hypersensitivity of calF deletion strains can be overcome by inclusion of elevated levels of extracellular calcium ions in the growth medium, and, correspondingly, wild type strains grown in media deficient in calcium ions are no longer resistant to CFW. These observations support a model in which accommodation to at least some forms of cell wall stress is mediated by a calcium ion signaling system in which the AN2880 gene product plays a role. The genetic lesion in calF7 is predicted to result in a glycine-to-arginine substitution at position 638 of the 945-residue CalF protein in a region of the RSN1_7TM domain that is highly conserved amongst filamentous fungi. Homology modeling predicts that the consequence of a G638R substitution is to structurally occlude the principal conductance pore in the protein. GFP-tagged wild type CalF localizes principally to the Spitzenkörper and the plasma membrane at growing tips and forming septa. However, both septation and hyphal morphology appear to be normal in calF7 and AN2880 deletion strains, indicating that any role played by CalF in normal hyphal growth and cytokinesis is dispensable.

The Aspergillus nidulans IQGAP orthologue SepG is required for constriction of the contractile actomyosin ring.

In this research we report that the sepG1 mutation in Aspergillus nidulans resides in gene AN9463, which is predicted to encode an IQGAP orthologue. The genetic lesion is predicted to result in a G-to-R substitution at residue 1637 of the 1737-residue protein in a highly conserved region of the RasGAP-C-terminal (RGCT) domain. When grown at restrictive temperature, strains expressing the sepGG1637R (sepG1) allele are aseptate, with reduced colony growth and aberrantly formed conidiophores. The aseptate condition can be replicated by deletion of AN9463 or by downregulating its expression via introduced promoters. The mutation does not prevent assembly of a cortical contractile actomyosin ring (CAR) at putative septation sites, but tight compaction of the rings is impaired and the rings fail to constrict. Both GFP::SepG wild type and the GFP-tagged product of the sepG1 allele localize to the CAR at both permissive and restrictive temperatures. Downregulation of myoB (encoding the A. nidulans type-II myosin heavy chain) does not prevent formation of SepG rings at septation sites, but filamentous actin is required for CAR localization of SepG and MyoB. We identify fourteen probable IQ-motifs (EF-hand protein binding sites) in the predicted SepG sequence. Two of the A. nidulans EF-hand proteins, myosin essential light chain (AnCdc4) and myosin regulatory light chain (MrlC), colocalize with SepG and MyoB at all stages of CAR formation and constriction. However, calmodulin (CamA) appears at septation sites only after the CAR has become fully compacted. When expression of sepG is downregulated, leaving MyoB as the sole IQ-motif protein in the pre-compaction CAR, both MrlC and AnCdc4 continue to associate with the forming CAR. When myoB expression is downregulated, leaving SepG as the sole IQ-motif protein in the CAR, AnCdc4 association with the forming CAR continues but MrlC fails to associate. This supports a model in which the IQ motifs of MyoB bind both MrlC and AnCdc4, while the IQ motifs of SepG bind only AnCdc4. Downregulation of either mrlC or Ancdc4 results in an aseptate phenotype, but has no effect on association of either SepG or MyoB with the CAR.

Aspergillus nidulans protein kinase C forms a complex with the formin SepA that is involved in apical growth and septation.

The Aspergillus nidulans orthologue of Protein kinase C (PkcA) and the A. nidulans formin SepA participate in polarized growth. PkcA localizes to growing hyphal apices and septation sites, and amino acid sequences within PkcA that are required for PkcA to localize to these sites of cell wall synthesis have been identified. SepA is associated with the contractile actomyosin ring (CAR), and it localizes at hyphal tips in association with the Spitzenkörper (SPK) and as an apical dome. A mutation in the sepA gene (sepA1) renders A. nidulans aseptate at elevated temperature. Progress towards understanding the spatiotemporal relationship between PkcA and SepA during polarized growth is presented here. Fluorescent chimeras of PkcA and SepA strongly overlapped in some hyphal tips in a dome pattern, while other tips displayed SepA SPK and PkcA dome localization within the same tip. At septation sites PkcA and SepA consistently colocalized through late stages of CAR constriction. Bimolecular fluorescence complementation experimental results provide evidence that SepA and PkcA are both present in complexes at both hyphal tip domes and at cortical rings. A Gal4-based yeast two-hybrid analysis confirmed the physical interaction between SepA and PkcA, and indicted that the FH2 domain of SepA is involved in its physical interaction with PkcA. A functional interaction between PkcA and SepA was shown through complementation of the pkcA calC2 mutant's hypersensitivity to cell wall perturbing agents by overexpressed sepA and by the ability of the sepA1 mutation to block PkcA's ability to form cortical rings. Taken together these results suggest that a PkcA/SepA complex is involved in polarized growth. Through experiments using the actin disrupter latrunculin B, evidence is presented suggesting that actin plays a role in the PkcA/SepA complex.

Two amino acid sequences direct Aspergillus nidulans protein kinase C (PkcA) localization to hyphal apices and septation sites.

The Aspergillus nidulans ortholog of protein kinase C (pkcA) is involved in the organism's putative cell wall integrity (CWI) pathway, and PkcA also is highly localized at growing tips and forming septa. In the present work we identify the regions within PkcA that are responsible for its localization to hyphal tips and septation sites. To this end, we used serially truncated pkcA constructs and expressed them as green fluorescent protein (GFP) chimeras and identified two regions that direct PkcA localization. The first region is a 10 amino-acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips. Proteins containing this sequence also localize to septation sites. A second region between C2 and C1B (encompassing C1A) is sufficient for localization to septation sites but not to hyphal tips. We also report that localization to hyphal tips and septation sites alone is not sufficient for truncated constructs to complement hypersensitivity to the cell wall compromising agent calcofluor white in a strain bearing a mutation in the pkcA gene. Taken together, these results suggest that localization and stress response might be independent.

A mutation in the converter subdomain of Aspergillus nidulans MyoB blocks constriction of the actomyosin ring in cytokinesis.

We have identified a mutant allele of the Aspergillus nidulans homologue of myosin II (myoB; AN4706), which prevents normal septum formation. This is the first reported myosin II mutation in a filamentous fungus. Strains expressing the myoB(G843D) allele produce mainly aberrant septa at 30 °C and are completely aseptate at temperatures above 37 °C. Conidium formation is greatly reduced at 30 °C and progressively impaired with increasing temperature. Sequencing of the myoB(G843D) allele identified a point mutation predicted to result in a glycine-to-aspartate amino acid substitution at residue 843 in the myosin II converter domain. This residue is conserved in all fungal, plant, and animal myosin sequences that we have examined. The mutation does not prevent localization of the myoB(G843D) gene product to contractile rings, but it does block ring constriction. MyoB(G843D) rings at sites of abortive septation disassemble after an extended period and dissipate into the cytoplasm. During contractile ring formation, both wild type and mutant MyoB::GFP colocalize with actin--an association that begins at the pre-ring "string" stage. Down-regulation of wild-type myoB expression under control of the alcA promoter blocks septation but does not prevent actin from aggregating at putative septation sites--the actin rings, however, do not fully coalesce. Both septation and targeting of MyoB are blocked by disruption of filamentous actin using latrunculin B. We propose a model in which myosin assembly at septation sites depends upon the presence of F-actin, but assembly of the actin component of contractile rings depends upon normal levels of myosin only for the final stages of ring compaction.

GDP-mannose transporter paralogues play distinct roles in polarized growth of Aspergillus nidulans.

GDP-mannose transporters (GMT) carry GDP-mannose nucleotide sugars from the cytosol across the Golgi apparatus membrane for use as substrates in protein glycosylation in plants, animals and fungi. Genomes of some fungal species, such as the yeast Saccharomyces cerevisiae, contain only one gene encoding a GMT, while others, including Aspergillus nidulans, contain two (gmtA and gmtB). We previously showed that cell wall integrity and normal hyphal morphogenesis in A. nidulans depend upon the function of GmtA and that GmtA localizes to a Golgi-like compartment. Cells bearing the calI11 mutation in gmtA also have reduced cell surface mannosylation. Here we show that GmtB colocalizes with GmtA, suggesting that the role of GmtB is similar to that of GmtA, although the respective transcript levels differ during spore germination and early development. Transcript levels of gmtB are high in ungerminated spores and remain so throughout the first 16 h of germination. In contrast, transcript levels of gmrtA are negligible in ungerminated spores but increase to levels comparable to those of gmtB during germination. These observations suggest that although GmtA and GmtB reside within the same subcellular compartments, they nevertheless perform distinct functions at different stages of development.

Two GDP-mannose transporters contribute to hyphal form and cell wall integrity in Aspergillus nidulans.

In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans that show hypersensitivity to the chitin-binding agent Calcofluor White (CFW). Affected loci are designated cal loci. The phenotype of one of these alleles, calI11, also includes shortened hyphal compartments and increased density of branching in the absence of CFW, as well as reduced staining of cell walls by the lectin FITC-Concanavalin A (ConA), which has strong binding affinity for mannosyl residues. We have identified two A. nidulans genes (AN8848.3 and AN9298.3, designated gmtA and gmtB, respectively) that complement all aspects of the phenotype. Both genes show strong sequence similarity to GDP-mannose transporters (GMTs) of Saccharomyces and other yeasts. Sequencing of gmtA from the calI11 mutant strain reveals a G to C mutation at position 943, resulting in a predicted alanine to proline substitution at amino acid position 315 within a region that is highly conserved among other fungi. No mutations were observed in the mutant strain's allele of gmtB. Meiotic mapping demonstrated a recombination frequency of under 1 % between the calI locus and the phenA locus (located approximately 9.5 kb from AN8848.3), confirming that gmtA and calI are identical. A GmtA-GFP chimera exhibits a punctate distribution pattern, consistent with that shown by putative Golgi markers in A. nidulans. However, this distribution did not overlap with that of the putative Golgi equivalent marker CopA-monomeric red fluorescent protein (mRFP), which may indicate that the physically separated Golgi-equivalent organelles of A. nidulans represent physiologically distinct counterparts of the stacked cisternae of plants and animals. These findings demonstrate that gmtA and gmtB play roles in cell wall metabolism in A. nidulans similar to those previously reported for GMTs in yeasts.

Peroxisomal proliferator activated receptor gamma coactivator (PGC-1alpha) stimulates carnitine palmitoyltransferase I (CPT-Ialpha) through the first intron.

Peroxisomal proliferator activated receptor gamma coactivator-1 (PGC-1alpha) is a transcriptional coactivator that promotes mitochondrial biogenesis and energy metabolism in brown fat, skeletal muscle and heart. Previous studies demonstrated that PGC-1alpha is present at low levels in the liver but that the hepatic abundance of PGC-1alpha is elevated in diabetic and fasted animals. Elevated PGC-1alpha expression is associated with increased fatty acid oxidation and hepatic glucose production. Carnitine palmitoyltransferase-I (CPT-I) is a rate controlling step in the mitochondrial oxidation of long chain fatty acids. CPT-I transfers the acyl moiety from fatty acyl-CoA to carnitine for the translocation of long chain fatty acids across the mitochondrial membrane. There are two isoforms of CPT-I including a liver isoform CPT-Ialpha and a muscle isoform CPT-Ibeta. Here, we characterized the regulation of CPT-Ialpha isoform by PGC-1alpha. PGC-1alpha stimulates CPT-Ialpha primarily through multiple sites in the first intron. We found that PGC-1alpha can induce CPT-Ialpha gene expression in cardiac myocytes and primary hepatocytes. Our results indicate that PGC-1alpha elevates the expression of CPT-Ialpha via a unique mechanism that utilizes elements within the intron.

A thyroid hormone response unit formed between the promoter and first intron of the carnitine palmitoyltransferase-Ialpha gene mediates the liver-specific induction by thyroid hormone.

Carnitine palmitoyltransferase-I (CPT-I) catalyzes the rate-controlling step of fatty acid oxidation. CPT-I converts long-chain fatty acyl-CoAs to acylcarnitines for translocation across the mitochondrial membrane. The mRNA levels and enzyme activity of the liver isoform, CPT-Ialpha, are greatly increased in the liver of hyperthyroid animals. Thyroid hormone (T3) stimulates CPT-Ialpha transcription far more robustly in the liver than in non-hepatic tissues. We have shown that the thyroid hormone receptor (TR) binds to a thyroid hormone response element (TRE) located in the CPT-Ialpha promoter. In addition, elements in the first intron participate in the T3 induction of CPT-Ialpha gene expression, but the CPT-Ialpha intron alone cannot confer a T3 response. We found that deletion of sequences in the first intron between +653 and +744 decreased the T3 induction of CPT-Ialpha. Upstream stimulatory factor (USF) and CCAAT enhancer binding proteins (C/EBPs) bind to elements within this region, and these factors are required for the T3 response. The binding of TR and C/EBP to the CPT-Ialpha gene in vivo was shown by the chromatin immunoprecipitation assay. We determined that TR can physically interact with USF-1, USF-2, and C/EBPalpha. Transgenic mice were created that carry CPT-Ialpha-luciferase transgenes with or without the first intron of the CPT-Ialpha gene. In these mouse lines, the first intron is required for T3 induction as well as high levels of hepatic expression. Our data indicate that the T3 stimulates CPT-Ialpha gene expression in the liver through a T3 response unit consisting of the TRE in the promoter and additional factors, C/EBP and USF, bound in the first intron.