The tip growth apparatus of Aspergillus nidulans.

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1-2 mum behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.

[1]  G. May,et al.  Constitutive Activation of Endocytosis by Mutation ofmyoA, the Myosin I Gene of Aspergillus nidulans* , 1998, The Journal of Biological Chemistry.

[2]  J. Hamer,et al.  The Aspergillus nidulans sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity , 1997, The EMBO journal.

[3]  S. Bartnicki-Garcia,et al.  Computer simulation of fungal morphogenesis and the mathematical basis for hyphal (tip) growth , 1989, Protoplasma.

[4]  P. Novick,et al.  Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiae , 1993, Cell.

[5]  L. Pon,et al.  Live cell imaging of the assembly, disassembly, and actin cable–dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae , 2004, The Journal of cell biology.

[6]  L. Pon,et al.  The life cycle of actin patches in mating yeast. , 2001, Journal of cell science.

[7]  David G. Drubin,et al.  A Pathway for Association of Receptors, Adaptors, and Actin during Endocytic Internalization , 2003, Cell.

[8]  S. Osmani,et al.  Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique , 2006, Nature Protocols.

[9]  S. Bartnicki-García,et al.  Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of Neurospora crassa , 2007, Eukaryotic Cell.

[10]  H. Ronne,et al.  Yeast syntaxins Sso1p and Sso2p belong to a family of related membrane proteins that function in vesicular transport. , 1993, The EMBO journal.

[11]  M. Momany.  Polarity in filamentous fungi: establishment, maintenance and new axes. , 2002, Current opinion in microbiology.

[12]  G. Steinberg,et al.  Hyphal Growth: a Tale of Motors, Lipids, and the Spitzenkörper , 2007, Eukaryotic Cell.

[13]  Yi Xiong,et al.  Fusion PCR and gene targeting in Aspergillus nidulans , 2006, Nature Protocols.

[14]  S. Bartnicki-García,et al.  Apical growth and mitosis are independent processes in Aspergillus nidulans , 2003, Protoplasma.

[15]  G. Steinberg.  On the move: Endosomes in growth and pathogenicity of Ustilago maydis , 2007 .

[16]  G. Gierz,et al.  Dynein and dynactin deficiencies affect the formation and function of the Spitzenkörper and distort hyphal morphogenesis of Neurospora crassa. , 2000, Microbiology.

[17]  David G. Drubin,et al.  A Modular Design for the Clathrin- and Actin-Mediated Endocytosis Machinery , 2005, Cell.

[18]  S. Harris,et al.  Functional characterization and localization of the Aspergillus nidulans formin SEPA. , 2002, Molecular biology of the cell.

[19]  N. Read,et al.  Polarisome Meets Spitzenkörper: Microscopy, Genetics, and Genomics Converge , 2005, Eukaryotic Cell.

[20]  D. Cove.  The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. , 1966, Biochimica et biophysica acta.

[21]  Adam C. Martin,et al.  Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. , 2006, Developmental cell.

[22]  S. Harris,et al.  MesA, a novel fungal protein required for the stabilization of polarity axes in Aspergillus nidulans. , 2004, Molecular biology of the cell.

[23]  L. Du,et al.  Distinct ceramide synthases regulate polarized growth in the filamentous fungus Aspergillus nidulans. , 2005, Molecular biology of the cell.

[24]  H. Pelham,et al.  Slow Diffusion of Proteins in the Yeast Plasma Membrane Allows Polarity to Be Maintained by Endocytic Cycling , 2003, Current Biology.

[25]  M. Peñalva.  Tracing the endocytic pathway of Aspergillus nidulans with FM4-64. , 2005, Fungal genetics and biology : FG & B.

[26]  N. Read,et al.  Does endocytosis occur in fungal hyphae? , 2003, Fungal genetics and biology : FG & B.

[27]  Jiujiang Yu,et al.  Toxins of filamentous fungi. , 2002, Chemical immunology.

[28]  Peter Novick,et al.  Sec3p Is a Spatial Landmark for Polarized Secretion in Budding Yeast , 1998, Cell.

[29]  B. Oakley,et al.  The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans. , 2004, Molecular biology of the cell.

[30]  G. Steinberg,et al.  A dynein loading zone for retrograde endosome motility at microtubule plus‐ends , 2006, The EMBO journal.

[31]  B. Hjelm,et al.  Application of electron tomography to fungal ultrastructure studies. , 2006, The New phytologist.

[32]  Jijun Cheng,et al.  Cell Cycle Progression and Cell Polarity Require Sphingolipid Biosynthesis in Aspergillus nidulans , 2001, Molecular and Cellular Biology.

[33]  M. Bölker,et al.  A putative endosomal t‐SNARE links exo‐ and endocytosis in the phytopathogenic fungus Ustilago maydis , 2000, The EMBO journal.

[34]  H. Pelham.  SNAREs and the secretory pathway-lessons from yeast. , 1999, Experimental cell research.

[35]  J. Hamer,et al.  Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis. , 1994, Genetics.

[36]  S. Osmani,et al.  A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans , 2006, Genetics.

[37]  H. Pelham,et al.  Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes. , 2000, Molecular biology of the cell.

[38]  G. Gierz,et al.  A three-dimensional model of fungal morphogenesis based on the vesicle supply center concept. , 2001, Journal of theoretical biology.

[39]  Thomas D. Pollard,et al.  Myosin Va maneuvers through actin intersections and diffuses along microtubules , 2007, Proceedings of the National Academy of Sciences.

[40]  N. Read,et al.  Confocal microscopy of FM4‐64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae , 2000, Journal of microscopy.

[41]  S. Osmani,et al.  Rapid Production of Gene Replacement Constructs and Generation of a Green Fluorescent Protein-Tagged Centromeric Marker in Aspergillus nidulans , 2004, Eukaryotic Cell.

[42]  J. Gerst.  Conserved α-Helical Segments on Yeast Homologs of the Synaptobrevin/VAMP Family of v-SNAREs Mediate Exocytic Function* , 1997, The Journal of Biological Chemistry.

[43]  Gongshe Han,et al.  The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics , 2001, Current Biology.

[44]  R. Lehmann.  Cell-cell signaling, microtubules, and the loss of symmetry in the drosophila oocyte , 1995, Cell.

[45]  Å. Engqvist-Goldstein,et al.  Actin assembly and endocytosis: from yeast to mammals. , 2003, Annual review of cell and developmental biology.

[46]  M. Raudaskoski,et al.  Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans. , 1998, Microbiology.

[47]  J. Latgé.  The pathobiology of Aspergillus fumigatus. , 2001, Trends in microbiology.

[48]  F. Hughson,et al.  SNARE protein structure and function. , 2003, Annual review of cell and developmental biology.

[49]  R. Fischer,et al.  The role of the kinesin motor KipA in microtubule organization and polarized growth of Aspergillus nidulans. , 2004, Molecular biology of the cell.

[50]  C. Weil,et al.  Cloning of the riboB locus of Aspergillus nidulans. , 1987, Gene.

[51]  J. Gerst,et al.  Involvement of Long Chain Fatty Acid Elongation in the Trafficking of Secretory Vesicles in Yeast , 1998, The Journal of cell biology.

[52]  D. Denning,et al.  Molecular genetics in Aspergillus fumigatus. , 2000, Current opinion in microbiology.

[53]  C. McGoldrick,et al.  myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth , 1995, The Journal of cell biology.

[54]  B. Oakley,et al.  A β-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly , 1981, Cell.

[55]  S. Bartnicki-García.  8 – Role of Vesicles in Apical Growth and a New Mathematical Model of Hyphal Morphogenesis , 1990 .

[56]  E. Espeso,et al.  Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans , 2008, Molecular microbiology.

[57]  J. Doonan,et al.  Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a gamma-actin. , 1988, Gene.