Fibroblast growth factor 22 is not essential for skin development and repair but plays a role in tumorigenesis

Fibroblast Growth Factors play critical roles during development, tissue homeostasis and repair by controlling cell proliferation, survival, migration and differentiation. Of the 22 mammalian FGFs, FGF22, a member of the FGF7/10/22 subfamily, has been shown to have a clear role in synaptogenesis, but its roles in other tissues have not been studied. We have investigated the in vivo functions of FGF22 in mice. Fgf22 null animals were viable, fertile and did not display any obvious abnormalities. Despite the known expression profile of FGF22 in the skin, no differences in either skin or pelage were observed, demonstrating that FGF22 is dispensable during embryogenesis and in unchallenged adult skin. Mice lacking FGF22 were able to heal acute wounds just as efficiently as wild type mice. However, classical two-step skin carcinogenesis challenge revealed that FGF22 null mice developed fewer papillomas than wild type controls, suggesting a potential pro-oncogenic role for FGF22 in the skin.     

The septin AspB in Aspergillus nidulans forms bars and filaments and plays roles in growth emergence and conidiation

In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungus Aspergillus nidulans there are five septin genes, aspA (an ortholog of Saccharomyces cerevisiae CDC11), aspB (an ortholog of S. cerevisiae CDC3), aspC (an ortholog of S. cerevisiae CDC12), aspD (an ortholog of S. cerevisiae CDC10), and aspE (found only in filamentous fungi). The aspB gene was previously reported to be the most highly expressed Aspergillus nidulans septin and to be essential. Using improved gene targeting techniques, we found that deletion of aspB is not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained in ΔaspD and lost in ΔaspA and ΔaspC strains. Interestingly, in the ΔaspE mutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.     

SwoHp, a nucleoside diphosphate kinase, is essential in Aspergillus nidulans

The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had ~20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance.     

Ca(2+)/calmodulin-dependent kinase is essential for both growth and nuclear division in Aspergillus nidulans.

The calmodulin gene has been shown to be essential for cell cycle progression in a number of eukaryotic organisms. In vertebrates and Aspergillus nidulans the calmodulin dependence also requires calcium. We demonstrate that the unique gene encoding a multifunctional calcium/calmodulin-dependent protein kinase (CaMK) is also essential in A. nidulans. This enzyme is required both for the nuclear division cycle and for hyphal growth, because spores containing the disrupted gene arrest with a single nucleus and fail to extend a germ tube. A strain conditional for the expression of CaMK was created. When grown under conditions that resulted in a 90% decrease in the enzyme, both nuclear division and growth were markedly slowed. The CaMK seems to be important for progression from G2 to mitosis.     

Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that Aspergillus nidulans is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that A. nidulans ArfB shares similarity with ARF6 of Homo sapiens and Arf3p of Saccharomyces cerevisiae. An arfB null allele (arfB disrupted by a transposon [arfB::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the arfB::Tn strain revealed that another phenotypic characteristic seen for arfB::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfB^G2A::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension.     

Cytochrome c is not essential for viability of the fungus Aspergillus nidulans

The filamentous fungus Aspergillus nidulans is an obligate aerobe, which is capable of anaerobic survival, but not anaerobic growth. Since cytochrome c forms an essential part of the oxidative respiratory pathway it was expected that mutants lacking this component would be non-viable. Gene replacement of one homologue of the cycA (cytochrome c) gene was carried out in a diploid strain. Benomyl-induced haploidisation of this diploid yielded all cycA+ haploid colonies, initially suggesting that loss of cycA was indeed lethal. However, use of an alternative unbiased method to recover haploids yielded viable, but slow-growing, cycA- mutants. Replacement of the cycA locus in the cycA- mutants was verified by Southern blotting. Spectral analysis confirmed the absence of detectable levels of cytochrome c, and respiratory insensitivity to cyanide suggested the absence of cytochrome c-dependent respiration. Growth parameters were consistent with those expected of a CycA- mutant. Compared to the wild type, the mutants grew slowly on fermentable carbon sources, did not grow on non-fermentable carbon sources, and produced higher levels of ethanol. To our knowledge, this is the first report of a filamentous fungus that remains viable after complete elimination of a functional cytochrome c gene. We propose that the mutants are viable due to their ability to ferment and to use alternative respiratory pathways.     

The protein kinase C orthologue PkcA plays a role in cell wall integrity and polarized growth in Aspergillus nidulans

The calC2 mutation in Aspergillus nidulans causes hypersensitivity to Calcofluor White, along with other drug sensitivities that indicate a defect in cell wall integrity. We have cloned CalC by complementation, isolating the A. nidulans orthologue of protein kinase C (PkcA). The pkcA allele of the calC2 strain contains a mutation predicted to introduce a charged arginine residue in place of neutral glycine at a conserved site located immediately beside the C1B regulatory domain. Both PkcA and calC2 map to the same region of chromosome VIII. A PkcA::GFP chimera localizes to hyphal apices and growing septa, as well as to the conidiogenous apices of phialides, indicating a role for PkcA in polarized cell wall growth. These observations support the hypothesis that the role of PkcA in A. nidulans, is comparable to that played by Pkc1p in the Saccharomyces cerevisiae cell wall integrity pathway.     

Multiple roles of a heterotrimeric G-protein gamma-subunit in governing growth and development of Aspergillus nidulans

Vegetative growth signaling in the filamentous fungus Aspergillus nidulans is primarily mediated by the heterotrimeric G-protein composed of FadA (G alpha), SfaD (G beta), and a presumed G gamma. Analysis of the A. nidulans genome identified a single gene named gpgA encoding a putative G gamma-subunit. The predicted GpgA protein consists of 90 amino acids showing 72% similarity with yeast Ste18p. Deletion (delta) of gpgA resulted in restricted vegetative growth and lowered asexual sporulation. Moreover, similar to the delta sfaD mutant, the delta gpgA mutant was unable to produce sexual fruiting bodies (cleistothecia) in self-fertilization and was severely impaired with cleistothecial development in outcross, indicating that both SfaD and GpgA are required for fruiting body formation. Developmental and morphological defects caused by deletion of flbA encoding an RGS protein negatively controlling FadA-mediated vegetative growth signaling were suppressed by delta gpgA, indicating that GpgA functions in FadA-SfaD-mediated vegetative growth signaling. However, deletion of gpgA could not bypass the need for the early developmental activator FluG in asexual sporulation, suggesting that GpgA functions in a separate signaling pathway. We propose that GpgA is the only A. nidulans G gamma-subunit and is required for normal vegetative growth as well as proper asexual and sexual developmental progression.     

Aspergillus nidulans UDP-galactopyranose mutase, encoded by ugmA plays key roles in colony growth, hyphal morphogenesis, and conidiation

Growing resistance to current anti-fungal drugs is spurring investigation of new targets, including those in fungal wall metabolism. Galactofuranose (Galf) is found in the cell walls of many fungi including Aspergillus fumigatus, which is currently the most prevalent opportunistic fungal pathogen in developed countries, and A. nidulans, a closely-related, tractable model system. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose into UDP-Galf prior to incorporation into the fungal wall. We deleted the single-copy UGM sequence (AN3112.4, which we call ugmA) from an A. nidulans nkuADelta strain, creating ugmADelta. Haploid ugmADelta strains were able to complete their asexual life cycle, showing that ugmA is not essential. However, ugmADelta strains had compact colonial growth, which was associated with substantially delayed and abnormal conidiation. Compared to a wildtype morphology strain, ugmADelta strains had aberrant hyphal morphology, producing wide, uneven, highly-branched hyphae, with thick, relatively electron-dense walls as visualized by transmission electron microscopy. These effects were partially remediated by growth on high osmolarity medium, or on medium containing 10 microg/mL Calcofluor, consistent with Galf being important in cell wall structure and/or function.     

The Aspergillus nidulans swoC1 mutant shows defects in growth and development

Previous work identified swoC1 as a single-gene mutant with defects in polarity establishment. In this study swoC1 was shown to have defects in endocytosis, compartmentation, nuclear distribution, and conidiation. Temperature-shift experiments showed that the swoC1 mutant establishes multiple random sites of germ tube emergence. Surprisingly, these experiments also showed that even a slight delay in polarity establishment causes defects in later vegetative growth and asexual reproduction. The swoC gene was mapped to the centromere of chromosome III and cloned by complementation of the temperature-sensitive phenotype. The predicted SwoCp is homologous to rRNA pseudouridine synthases of yeast (Cbf5p) and humans (Dkc1p). However, neither rRNA pseudouridine synthesis nor rRNA processing appears to be affected in the swoC1 mutant. The swoC1 mutation occurs in the putative RNA-binding domain upstream of the C terminus, leaving the N-terminal TRUB catalytic domain intact. Interestingly, while deletion of the swoC gene was lethal in A. nidulans, the C terminus, including NLS, microtubule-binding, and coiled-coil domains, was dispensable for growth. SwoCp likely plays an important role in polar growth and nuclear distribution in A. nidulans, functions not yet described for its homologs.     

The Aspergillus nidulans putative kinase, KfsA (kinase for septation), plays a role in septation and is required for efficient asexual spore formation

In Aspergillus nidulans nuclear division and cytokinesis are coupled processes during asexual sporulation. Metulae, phialides and conidia contain a single nucleus. Here we describe the role of a putative Saccharomyces cerevisiae Kin4-related kinase, KfsA (kinase for septation) in the control of septum formation in A. nidulans. The kfsA deletion caused an increase in the number of conidiophores with septa in their stalks from 20% in wild type to 60% in the mutant strain. Interestingly, 7% of metulae contained two nuclei and the corresponding phialides remained anucleate, suggesting septum formation before proper segregation of nuclei. This points to a checkpoint control of KfsA, which prevents septum formation before nuclear separation. KfsA localized to the cortex and septa in hyphae and in conidiophores but not to the spindle-pole bodies, as it was shown for Kin4 in yeast. KfsA appeared at septa after actin disappeared, suggesting an additional role of KfsA late during septum formation.     

The effect of gene tubC on the vegetative growth of benomyl-resistant strains of Aspergillus nidulans

Abstract Two benomyl-resistant mutants, benD3 tubC41 and benD4 tubC42 , of Aspergillus nidulans were isolated after UV treatment. The tubC mutations permitted good conidiation of these strains in culture media containing benomyl and were responsible for increasing their benomyl resistance levels. This implies that β3-tubulin, a product of the tubC gene, in addition to being involved in fungal conidiation, participates in the vegetative growth of the fungus. The tubC gene was located in linkage group I.     

The murine stanniocalcin 1 gene is not essential for growth and development

The stanniocalcin 1 (STC1) gene is expressed in a wide variety of tissues, including the kidney, prostate, thyroid, bone, and ovary. STC1 protein is considered to have roles in many physiological processes, including bone development, reproduction, wound healing, angiogenesis, and modulation of inflammatory response. In fish, STC1 is a hormone that is secreted by the corpuscles of Stannius and is involved in calcium and phosphate homeostasis. To determine the role of STC1 in mammals, we generated Stc1-null mice by gene targeting. The number of Stc1-/- mice obtained was in accordance with Mendelian ratios, and both males and females produced offspring normally. No anatomical or histological abnormalities were detected in any tissues. Our results demonstrated that Stc1 function is not essential for growth or reproduction in the mouse.     

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function.

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.     

The Aspergillus nidulans rcoA gene is required for veA-dependent sexual development

The Aspergillus nidulans rcoADelta mutant exhibits growth and developmental defects. We show that the rcoADelta mutant lacks cleistothecia and is self-sterile. In crosses with wild-type strains, rcoADelta nuclei do not contribute to the cleistothecial walls. Furthermore, sexual development resulting from veA overexpression is rcoA dependent, indicating that rcoA lies downstream of veA in the sexual development pathway.