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      The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis, Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

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          Abstract

          Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P 2. In N. crassa hyphae, PtdIns(4,5)P 2 localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P 2 that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.

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          Adaptation of core mechanisms to generate cell polarity.

          Cell polarity is defined as asymmetry in cell shape, protein distributions and cell functions. It is characteristic of single-cell organisms, including yeast and bacteria, and cells in tissues of multi-cell organisms such as epithelia in worms, flies and mammals. This diversity raises several questions: do different cell types use different mechanisms to generate polarity, how is polarity signalled, how do cells react to that signal, and how is structural polarity translated into specialized functions? Analysis of evolutionarily diverse cell types reveals that cell-surface landmarks adapt core pathways for cytoskeleton assembly and protein transport to generate cell polarity.
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            Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism.

            We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.
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              Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis.

              Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2(a)- and Rab-A1(e)-labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                13 December 2012
                : 7
                : 12
                : e51454
                Affiliations
                [1 ]Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Göttingen, Göttingen, Germany
                [2 ]Institute for Microbiology and Genetics, Georg-August-University Göttingen, Göttingen, Germany
                Cinvestav, Mexico
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: TI SS IH. Performed the experiments: AM TI YH IS FH IH. Analyzed the data: AM TI YH IS FH SS IH. Contributed reagents/materials/analysis tools: TI IS SS. Wrote the paper: SS IH.

                [¤a]

                Current address: Molecular Genetics and Cell Biology of Plants, Westfälische Wilhelms-University Münster, Münster (Westf.), Germany

                [¤b]

                Current address: Department for Molecular Systems Biology, University of Vienna, Vienna, Austria

                [¤c]

                Current address: Institute for Biology II - Molecular Plant Physiology, Albert-Ludwigs University Freiburg, Freiburg, Germany

                [¤d]

                Current address: Department of Cellular Biochemistry, Institute for Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany

                Article
                PONE-D-12-22548
                10.1371/journal.pone.0051454
                3521734
                23272106
                6d364ca6-e55c-4eb6-8256-8e50ea198a27
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 31 July 2012
                : 1 November 2012
                Page count
                Pages: 13
                Funding
                This work was supported by the German Research Foundation (DFG) [Emmy-Noether grant He3424/1 to I.H., grant SE1054/3-2 to S.S. and grant GSC 226/1 through the Göttingen Graduate School for Neurosciences and Molecular Biosciences (GGNB)]. The authors acknowledge the FGSC and the Neurospora program grant PO1 GM068087 for continuous support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Lipids
                Fatty Acids
                Lipid Classes
                Lipid Mediators
                Plant Biochemistry
                Microbiology
                Mycology
                Fungal Biochemistry
                Fungal Physiology
                Fungal Structure
                Model Organisms
                Yeast and Fungal Models
                Plant Science
                Botany
                Plant Morphology
                Plant Growth and Development

                Uncategorized
                Uncategorized

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