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1.
Mol Biol Cell ; 19(1): 339-51, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18003978

ABSTRACT

In filamentous fungi, hyphal extension depends on the continuous delivery of vesicles to the growing tip. Here, we describe the identification of two cell end marker proteins, TeaA and TeaR, in Aspergillus nidulans, corresponding to Tea1 and Mod5 in Schizosaccharomyces pombe. Deletion of teaA or teaR caused zig-zag-growing and meandering hyphae, respectively. The Kelch-repeat protein TeaA, the putatively prenylated TeaR protein, and the formin SepA were highly concentrated in the Spitzenkörper, a vesicle transit station at the tip, and localized along the tip membrane. TeaA localization at tips depended on microtubules, and TeaA was required for microtuble convergence in the hyphal apex. The CENP-E family kinesin KipA was necessary for proper localization of TeaA and TeaR, but not for their transportation. TeaA and TeaR localization were interdependent. TeaA interacted in vivo with TeaR, and TeaA colocalized with SepA. Sterol-rich membrane domains localized at the tip in teaA and teaR mutants like in wild type, and filipin treatment caused mislocalization of both proteins. This suggests that sterol-rich membrane domains determine cell end factor destinations and thereby polarized growth.


Subject(s)
Aspergillus nidulans/cytology , Aspergillus nidulans/growth & development , Cell Membrane/metabolism , Cell Polarity , Fungal Proteins/metabolism , Sterols/metabolism , Aspergillus nidulans/drug effects , Biomarkers/metabolism , Cell Membrane/drug effects , Cell Polarity/drug effects , Filipin/pharmacology , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Gene Deletion , Hyphae/drug effects , Hyphae/growth & development , Hyphae/metabolism , Microtubules/drug effects , Microtubules/metabolism , Phenotype , Protein Binding/drug effects , Protein Transport/drug effects
2.
J Neurosci ; 27(37): 9916-27, 2007 Sep 12.
Article in English | MEDLINE | ID: mdl-17855606

ABSTRACT

Long-haul transport along microtubules is crucial for neuronal polarity, and transport defects cause neurodegeneration. Tau protein stabilizes microtubule tracks, but in Alzheimer's disease it aggregates and becomes missorted into the somatodendritic compartment. Tau can inhibit axonal transport by obstructing motors on microtubules, yet tau itself can still move into axons. We therefore investigated tau movement by live-cell fluorescence microscopy, FRAP (fluorescence recovery after photobleaching), and FSM (fluorescence speckle microscopy). Tau is highly dynamic, with diffusion coefficients of approximately 3 microm2/s and microtubule dwell times of approximately 4 s. This facilitates the entry of tau into axons over distances of millimeters and periods of days. For longer distances and times, two mechanisms of tau transport are observed. At low near-physiological levels, tau is cotransported with microtubule fragments from cell bodies into axons, moving at instantaneous velocities approximately 1 microm/s. At high concentrations, tau forms local accumulations moving bidirectionally at approximately 0.3 microm/s. These clusters first appear at distal endings of axons and may indicate an early stage of neurite degeneration.


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Neurons/metabolism , tau Proteins/metabolism , Animals , Cells, Cultured , Chlorocebus aethiops , Microtubule-Associated Proteins/analysis , Microtubules/chemistry , Neurons/chemistry , Protein Transport/physiology , Rats , Vero Cells , tau Proteins/analysis
4.
Eukaryot Cell ; 6(3): 555-62, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17237365

ABSTRACT

The dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. In Schizosaccharomyces pombe and in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA from Aspergillus nidulans and 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. alpA deletion 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.


Subject(s)
Aspergillus nidulans/ultrastructure , Centrosome/ultrastructure , Hyphae/growth & development , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/physiology , Microtubule-Organizing Center/diagnostic imaging , Microtubules/ultrastructure , Spindle Apparatus/ultrastructure , Aspergillus nidulans/genetics , Aspergillus nidulans/growth & development , Aspergillus nidulans/metabolism , Cell Nucleus/metabolism , Cell Polarity , Green Fluorescent Proteins/metabolism , Hyphae/ultrastructure , Microtubule-Organizing Center/metabolism , Microtubules/metabolism , Mitosis , Phylogeny , Protein Binding , Ultrasonography
5.
Nature ; 438(7071): 1151-6, 2005 Dec 22.
Article in English | MEDLINE | ID: mdl-16372009

ABSTRACT

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.


Subject(s)
Allergens/genetics , Aspergillus fumigatus/genetics , Aspergillus fumigatus/pathogenicity , Genome, Fungal , Genomics , Hypersensitivity/microbiology , Aspergillus fumigatus/immunology , Gene Expression Profiling , Gene Expression Regulation, Fungal , Genes, Fungal/genetics , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Sequence Analysis, DNA , Temperature , Virulence/genetics
6.
Mol Biol Cell ; 16(2): 497-506, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15563609

ABSTRACT

Polarized growth in filamentous fungi requires the integrity of the microtubule (MT) cytoskeleton. We found that growing MTs in Aspergillus nidulans merge at the center of fast growing tips and discovered that a kinesin motor protein, KipA, related to Tea2p of Schizosaccharomyces pombe, is required for this process. In a DeltakipA strain, MT plus ends reach the tip but show continuous lateral movement. Hyphae lose directionality and grow in curves, apparently due to mislocalization of the vesicle supply center (Spitzenkörper) in the apex. Green fluorescent protein (GFP)-KipA accumulates at MT plus ends, whereas a KipA rigor mutant protein, GFP-KipA(G223E), coated MTs evenly. These findings suggest that KipA requires its intrinsic motor activity to reach the MT plus end. Using KipA as an MT plus-end marker, we found bidirectional organization of MTs and determined the locations of microtubule organizing centers at nuclei, in the cytoplasm, and at septa.


Subject(s)
Aspergillus nidulans/metabolism , Fungal Proteins/metabolism , Kinesins/metabolism , Microtubule-Organizing Center/metabolism , Microtubules/metabolism , Molecular Motor Proteins/metabolism , Alleles , Aspergillus nidulans/genetics , Aspergillus nidulans/growth & development , Biomarkers , Cell Nucleus/metabolism , Cytoplasm/metabolism , Cytoskeleton/metabolism , Fungal Proteins/genetics , Genes, Fungal , Green Fluorescent Proteins/metabolism , Hyphae/metabolism , Kinesins/genetics , Models, Biological , Mutation , Protein Binding , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Transformation, Genetic
7.
Eukaryot Cell ; 3(3): 632-45, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15189985

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.


Subject(s)
Aspergillus nidulans/genetics , Cell Cycle/physiology , Kinesins/genetics , Microtubules/metabolism , Spindle Apparatus/genetics , Amino Acid Sequence , Aspergillus nidulans/metabolism , Benomyl/pharmacology , Cell Cycle/drug effects , Fungicides, Industrial/pharmacology , Kinesins/metabolism , Microtubule-Associated Proteins/genetics , Microtubules/drug effects , Molecular Motor Proteins/genetics , Molecular Motor Proteins/metabolism , Molecular Sequence Data , Mutation/genetics , Saccharomyces cerevisiae Proteins/genetics , Schizosaccharomyces pombe Proteins/genetics , Spindle Apparatus/drug effects
8.
Curr Genet ; 45(6): 383-9, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15071756

ABSTRACT

The advent of fluorescent proteins as vital dyes had a major impact in many research fields. Different green fluorescent protein (GFP) variants were established in prokaryotic and eukaryotic organisms within the past 10 years, and other fluorescent proteins were discovered and applied. We expressed the Discosoma red fluorescent protein, DsRed (T4), the improved monomeric red fluorescent protein (mRFP1) and the blue fluorescent protein (BFP) in the filamentous fungus Aspergillus nidulans. Whereas DsRed requires tetramer formation for fluorescence, mRFP1 functions as monomer. We used sGFP, DsRed (T4), mRFP1 and BFP for nuclear and/or mitochondrial labelling. To facilitate gene tagging, we established a number of cloning vectors for the efficient, simultaneous fusion of any protein with mRFP1, BFP and sGFP or the haemagglutinin epitope, 3xHA. A PCR-amplified gene of interest can be inserted into the expression vectors without cloning but using homologous recombination in vitro (GATEWAY). The vectors contain the argB gene as a selection marker for A. nidulans and the inducible alcA promoter for control of expression. The system allows labelling of a protein with several tags in one recombination reaction. Both the nutritional marker gene and the promoter are frequently used in other fungi, suggesting that this set of expression vectors will be very useful tools for gene analysis on a genome-wide scale.


Subject(s)
Aspergillus nidulans/genetics , Genes, Reporter/genetics , Genetic Vectors , Luminescent Proteins/genetics , Recombination, Genetic , Cell Nucleus/genetics , Gene Expression Regulation, Fungal , Promoter Regions, Genetic/genetics , Recombinant Fusion Proteins/genetics , Transfection , Red Fluorescent Protein
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