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 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 CLIP-170 Homologue and NUDE Play Overlapping Roles in NUDF Localization in Aspergillus nidulans

2006 ◽  
Vol 17 (4) ◽  
pp. 2021-2034 ◽  
Author(s):  
Vladimir P. Efimov ◽  
Jun Zhang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Live Cells ◽  
Physiological Conditions ◽  
Physical Interactions ◽  
Cytoplasmic Microtubules ◽  
Growth Promoting ◽  
Green Fluorescent ◽  
Hyphal Tip

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32°C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150Glued dynactin, and NUDF were all seen as plus-end comets at 32°C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32°C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42°C).

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 Myosin-V, Kinesin-1, and Kinesin-3 Cooperate in Hyphal Growth of the FungusUstilago maydis

2005 ◽  
Vol 16 (11) ◽  
pp. 5191-5201 ◽  
Author(s):  
Isabel Schuchardt ◽  
Daniela Aßmann ◽  
Eckhard Thines ◽  
Christian Schuberth ◽  
Gero Steinberg
Keyword(s):  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Myosin V ◽  
Long Distance ◽  
Long Distance Transport ◽  
Conventional Kinesin ◽  
Green Fluorescent ◽  
Mutant Cells ◽  
Distance Transport

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. Δkin1 and Δkin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in Δkin1rkin3 double mutants, but polarity was lost in Δmyo5rkin1 and Δmyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenkörper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.

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 Dis1/XMAP215 Protein AlpA Localizes to Spindle Pole Bodies and Microtubule Plus Ends and Contributes to Growth Directionality

2007 ◽  
Vol 6 (3) ◽  
pp. 555-562 ◽  
Author(s):  
Cathrin Enke ◽  
Nadine Zekert ◽  
Daniel Veith ◽  
Carolin Schaaf ◽  
Sven Konzack ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Spindle Pole Bodies ◽  
Family Protein ◽  
Associated Proteins ◽  
Hyphal Morphology ◽  
Conventional Kinesin ◽  
Green Fluorescent

ABSTRACTThe dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. InSchizosaccharomyces pombeand in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA fromAspergillus nidulansand show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA.alpAdeletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics.

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 The Kip3-Like Kinesin KipB Moves along Microtubules and Determines Spindle Position during Synchronized Mitoses in Aspergillus nidulans Hyphae

2004 ◽  
Vol 3 (3) ◽  
pp. 632-645 ◽  
Author(s):  
Patricia E. Rischitor ◽  
Sven Konzack ◽  
Reinhard Fischer
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Mitotic Spindles ◽  
Spindle Positioning ◽  
High Concentrations ◽  
Synthetically Lethal ◽  
Green Fluorescent ◽  
Spindle Position

ABSTRACT Kinesins are motor proteins which are classified into 11 different families. We identified 11 kinesin-like proteins in the genome of the filamentous fungus Aspergillus nidulans. Relatedness analyses based on the motor domains grouped them into nine families. In this paper, we characterize KipB as a member of the Kip3 family of microtubule depolymerases. The closest homologues of KipB are Saccharomyces cerevisiae Kip3 and Schizosaccharomyces pombe Klp5 and Klp6, but sequence similarities outside the motor domain are very low. A disruption of kipB demonstrated that it is not essential for vegetative growth. kipB mutant strains were resistant to high concentrations of the microtubule-destabilizing drug benomyl, suggesting that KipB destabilizes microtubules. kipB mutations caused a failure of spindle positioning in the cell, a delay in mitotic progression, an increased number of bent mitotic spindles, and a decrease in the depolymerization of cytoplasmic microtubules during interphase and mitosis. Meiosis and ascospore formation were not affected. Disruption of the kipB gene was synthetically lethal in combination with the temperature-sensitive mitotic kinesin motor mutation bimC4, suggesting an important but redundant role of KipB in mitosis. KipB localized to cytoplasmic, astral, and mitotic microtubules in a discontinuous pattern, and spots of green fluorescent protein moved along microtubules toward the plus ends.

scholarly journals Accumulation of Cytoplasmic Dynein and Dynactin at Microtubule Plus Ends in Aspergillus nidulans Is Kinesin Dependent

2003 ◽  
Vol 14 (4) ◽  
pp. 1479-1488 ◽  
Author(s):  
Jun Zhang ◽  
Shihe Li ◽  
Reinhard Fischer ◽  
Xin Xiang
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Nidulans ◽  
Filamentous Fungus ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Loss Of Function ◽  
Conventional Kinesin ◽  
Green Fluorescent

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end–directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.

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.

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