scholarly journals FigA, a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae, Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

2013 ◽  
Vol 13 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Shizhu Zhang ◽  
Hailin Zheng ◽  
Nanbiao Long ◽  
Natalia Carbó ◽  
Peiying Chen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Aspergillus Nidulans ◽  
Sexual Development ◽  
Calcium Uptake ◽  
Fluorescent Protein ◽  
Calcium Influx ◽  
Hyphal Growth ◽  
Uptake System ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTCalcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. InSaccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 fromS. cerevisiae, was functionally characterized in the filamentous fungusAspergillus nidulans. Loss offigAresulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACSmidA. Interestingly, in afigAdeletion mutant, adding extracellular Ca2+rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed thatfigAdeletion sharply decreased the expression ofbrlAandnsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system inA. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

scholarly journals Aspergillus nidulans ArfB Plays a Role in Endocytosis and Polarized Growth

2008 ◽  
Vol 7 (8) ◽  
pp. 1278-1288 ◽  
Author(s):  
Soo Chan Lee ◽  
Sabrina N. Schmidtke ◽  
Lawrence J. Dangott ◽  
Brian D. Shaw
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Fluorescent Protein ◽  
Homo Sapiens ◽  
Hyphal Growth ◽  
Life Cycles ◽  
Small Gtpase ◽  
Polarized Growth ◽  
Sequence Alignments ◽  
Green Fluorescent

ABSTRACT 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 (ArfBG2A::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.

scholarly journals Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+Homeostasis in Aspergillus nidulans

2016 ◽  
Vol 82 (11) ◽  
pp. 3420-3430 ◽  
Author(s):  
Sha Wang ◽  
Xiao Liu ◽  
Hui Qian ◽  
Shizhu Zhang ◽  
Ling Lu
Keyword(s):  
Salt Stress ◽  
Calcium Channel ◽  
Aspergillus Nidulans ◽  
Transient Receptor Potential ◽  
Feedback Inhibition ◽  
Calcium Influx ◽  
Hyphal Growth ◽  
Salt Stress Response ◽  
Content Type ◽  
Growth Defects

ABSTRACTThe eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion ofcchAsuppresses the hyphal growth defects caused by the loss of calcineurin under salt stress inAspergillus nidulans. Additionally, the hypersensitivity of the ΔcnaAstrain to extracellular calcium and cell-wall-damaging agents can be suppressed bycchAdeletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca2+concentration ([Ca2+]c) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of eithercnaAorcchAsignificantly decreased the [Ca2+]c, but a deficiency in bothcnaAandcchAswitches the [Ca2+]cto the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction ofcchAin the ΔcnaAstrain dramatically restored the expression ofenaA(a major determinant for sodium detoxification), which was abolished in the ΔcnaAstrain under salt stress. These results suggest that double deficiencies ofcnaAandcchAcould bypass the requirement of calcineurin to induceenaAexpression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca2+channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption.IMPORTANCEThe feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One7:e46564, 2012,http://dx.doi.org/10.1371/journal.pone.0046564) showed that the deletion ofcchAcould suppress the hyphal growth defects caused by the loss of calcineurin under salt stress inAspergillus nidulans. In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca2+]c. Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca2+channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca2+homeostasis in response to different environments.

scholarly journals Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

2015 ◽  
Vol 14 (9) ◽  
pp. 908-921 ◽  
Author(s):  
Nicole Bühler ◽  
Daisuke Hagiwara ◽  
Norio Takeshita
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
Apical Plasma Membrane ◽  
Important Species ◽  
Content Type

ABSTRACT Polarized growth in filamentous fungi needs a continuous supply of proteins and lipids to the growing hyphal tip. One of the important membrane compounds in fungi is ergosterol. At the apical plasma membrane ergosterol accumulations, which are called sterol-rich plasma membrane domains (SRDs). The exact roles and formation mechanism of the SRDs remained unclear, although the importance has been recognized for hyphal growth. Transport of ergosterol to hyphal tips is thought to be important for the organization of the SRDs. Oxysterol binding proteins, which are conserved from yeast to human, are involved in nonvesicular sterol transport. In Saccharomyces cerevisiae seven oxysterol-binding protein homologues (OSH1 to -7) play a role in ergosterol distribution between closely located membranes independent of vesicle transport. We found five homologous genes ( oshA to oshE ) in the filamentous fungi Aspergillus nidulans . The functions of OshA-E were characterized by gene deletion and subcellular localization. Each gene-deletion strain showed characteristic phenotypes and different sensitivities to ergosterol-associated drugs. Green fluorescent protein-tagged Osh proteins showed specific localization in the late Golgi compartments, puncta associated with the endoplasmic reticulum, or diffusely in the cytoplasm. The genes expression and regulation were investigated in a medically important species Aspergillus fumigatus , as well as A. nidulans . Our results suggest that each Osh protein plays a role in ergosterol distribution at distinct sites and contributes to proper fungal growth.

scholarly journals The Aspergillus nidulans Kinesin-3 UncA Motor Moves Vesicles along a Subpopulation of Microtubules

2009 ◽  
Vol 20 (2) ◽  
pp. 673-684 ◽  
Author(s):  
Nadine Zekert ◽  
Reinhard Fischer
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Cellular Transport ◽  
Mitotic Spindles ◽  
Cytoplasmic Microtubules ◽  
Conventional Kinesin ◽  
Green Fluorescent ◽  
Vesicle Movement

The extremely polarized growth form of filamentous fungi imposes a huge challenge on the cellular transport machinery, because proteins and lipids required for hyphal extension need to be continuously transported to the growing tip. Recently, it was shown that endocytosis is also important for hyphal growth. Here, we found that the Aspergillus nidulans kinesin-3 motor protein UncA transports vesicles and is required for fast hyphal extension. Most surprisingly, UncA-dependent vesicle movement occurred along a subpopulation of microtubules. Green fluorescent protein (GFP)-labeled UncArigor decorated a single microtubule, which remained intact during mitosis, whereas other cytoplasmic microtubules were depolymerized. Mitotic spindles were not labeled with GFP-UncArigor but reacted with a specific antibody against tyrosinated α-tubulin. Hence, UncA binds preferentially to detyrosinated microtubules. In contrast, kinesin-1 (conventional kinesin) and kinesin-7 (KipA) did not show a preference for certain microtubules. This is the first example for different microtubule subpopulations in filamentous fungi and the first example for the preference of a kinesin-3 motor for detyrosinated microtubules.

scholarly journals The Septin AspB in Aspergillus nidulans Forms Bars and Filaments and Plays Roles in Growth Emergence and Conidiation

2012 ◽  
Vol 11 (3) ◽  
pp. 311-323 ◽  
Author(s):  
Yainitza Hernández-Rodríguez ◽  
Susan Hastings ◽  
Michelle Momany
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Germ Tube ◽  
Fluorescent Protein ◽  
Early Growth ◽  
Content Type ◽  
Green Fluorescent ◽  
And Function ◽  
Cytoskeletal Elements ◽  
Germ Tubes

ABSTRACTIn 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 fungusAspergillus nidulansthere are five septin genes,aspA(an ortholog ofSaccharomyces cerevisiae CDC11),aspB(an ortholog ofS. cerevisiae CDC3),aspC(an ortholog ofS. cerevisiae CDC12),aspD(an ortholog ofS. cerevisiae CDC10), andaspE(found only in filamentous fungi). TheaspBgene was previously reported to be the most highly expressedAspergillus nidulansseptin and to be essential. Using improved gene targeting techniques, we found that deletion ofaspBis 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 inΔaspDand lost inΔaspAandΔaspCstrains. Interestingly, in theΔaspEmutant, 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.

scholarly journals Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans

2010 ◽  
Vol 9 (10) ◽  
pp. 1504-1518 ◽  
Author(s):  
América Hervás-Aguilar ◽  
Miguel A. Peñalva
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Nidulans ◽  
Germ Tube ◽  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Gene Replacement ◽  
Actin Binding ◽  
Nitrate Medium ◽  
Content Type ◽  
Tube Emergence

ABSTRACT The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical “comets” of AbpA.

scholarly journals Role of Fig1, a Component of the Low-Affinity Calcium Uptake System, in Growth and Sexual Development of Filamentous Fungi

2012 ◽  
Vol 11 (8) ◽  
pp. 978-988 ◽  
Author(s):  
Brad Cavinder ◽  
Frances Trail
Keyword(s):  
Mating Type ◽  
Sexual Development ◽  
Calcium Uptake ◽  
Transmembrane Protein ◽  
Uptake System ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Content Type ◽  
High Affinity

ABSTRACT The function of Fig1, a transmembrane protein of the low-affinity calcium uptake system (LACS) in fungi, was examined for its role in the growth and development of the plant pathogen Fusarium graminearum . The Δ fig1 mutants failed to produce mature perithecia, and sexual development was halted prior to the formation of perithecium initials. The loss of Fig1 function also resulted in a reduced vegetative growth rate. Macroconidium production was reduced 70-fold in the Δ fig1 mutants compared to the wild type. The function of the high-affinity calcium uptake system (HACS), comprised of the Ca 2+ channels Mid1 and Cch1, was previously characterized for F. graminearum . To better understand the roles of the LACS and the HACS, Δ fig1 Δ mid1 , Δ fig1 Δ cch1 , and Δ fig1 Δ mid1 Δ cch1 double and triple mutants were generated, and the phenotypes of these mutants were more severe than those of the Δ fig1 mutants. Pathogenicity on wheat was unaffected for the Δ fig1 mutants, but the Δ fig1 Δ mid1 , Δ fig1 Δ cch1 , and Δ fig1 Δ mid1 Δ cch1 mutants, lacking both LACS and HACS functions, had reduced pathogenicity. Additionally, Δ fig1 mutants of Neurospora crassa were examined and did not affect filamentous growth or female fertility in a Δ fig1 mating type A strain, but the Δ fig1 mating type a strain failed to produce fertile fruiting bodies. These results are the first report of Fig1 function in filamentous ascomycetes and expand its role to include complex fruiting body and ascus development.

scholarly journals Class III Chitin Synthase ChsB of Aspergillus nidulans Localizes at the Sites of Polarized Cell Wall Synthesis and Is Required for Conidial Development

2009 ◽  
Vol 8 (7) ◽  
pp. 945-956 ◽  
Author(s):  
Kazuharu Fukuda ◽  
Kazunari Yamada ◽  
Ken Deoka ◽  
Shuichi Yamashita ◽  
Akinori Ohta ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Cell Walls ◽  
Deletion Mutant ◽  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Chitin Synthase ◽  
Class Iii ◽  
Chitin Synthases ◽  
Conidial Development ◽  
Green Fluorescent

ABSTRACT Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.

scholarly journals Fig1 Facilitates Calcium Influx and Localizes to Membranes Destined To Undergo Fusion during Mating in Candida albicans

2011 ◽  
Vol 10 (3) ◽  
pp. 435-444 ◽  
Author(s):  
Meng Yang ◽  
Alexandra Brand ◽  
Thyagarajan Srikantha ◽  
Karla J. Daniels ◽  
David R. Soll ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Calcium Influx ◽  
Fold Increase ◽  
Gene Encoding ◽  
Content Type ◽  
Daughter Cells ◽  
Green Fluorescent

ABSTRACTFew mating-regulated genes have been characterized inCandida albicans. C. albicans FIG1(CaFIG1) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. InSaccharomyces cerevisiae, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of CaFIG1was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca2+uptake in mating-competent opaque cells. Uptake was reduced substantially in thefig1Δ null mutant. CaFig1 is therefore involved in Ca2+influx and localizes to membranes that are destined to undergo fusion during mating.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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