Determining the function of Myo1p: A novel, unconventional myosin in Tetrahymena thermophila.
Item
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Title
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Determining the function of Myo1p: A novel, unconventional myosin in Tetrahymena thermophila.
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Identifier
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AAI3144151
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identifier
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3144151
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Creator
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Williams, Selwyn A.
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Contributor
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Adviser: Ray H. Gavin
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Date
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2004
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Molecular
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Abstract
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In an earlier study (Garces et al. 1998), MYO1 and its encoded product, Myo1p, were identified by a PCR-based genomic screen of the ciliate protozoan, Tetrahymena thermophila. Head domain homology analysis based on partial genomic sequence data, phylogenetically defined Myo1p as a novel, unconventional myosin heavy chain, not belonging to any of the previously established myosin classes. In the present study, a Basic Local Alignment Search Tool (BLAST) analysis of a searchable database created by the Tetrahymena Genome Project was used to determine the complete genomic sequence of MYO1 and to predict the structure of Myo1p. MYO1 is a 7 kb gene encoding a ∼2000 as-heavy chain polypeptide. Myo1p is predicted to have a 300 aa-N-terminal extension, a 750 aa-conserved motor domain, two IQ motifs, and 190 aa-FERM domain located 250--300 residues from the C-terminal end of the protein. Phenotypic analysis of a MYO1-knockout (DeltaMYO1) strain revealed temperature dependent defects in macronuclear elongation and endocytosis. In dividing knockout cells at 20°C, the macronucleus failed to elongate properly along the anterior-posterior axis resulting in grossly unequal nuclear segregation. At 20°C, MYO1-knockout cells also exhibited reduced rates of food vacuole formation when compared with wild-type. A Tetrahymena strain expressing GFP-actin showed fusion-protein localization to cortical structures (basal body cage) and the contractile ring. Incorporation of GFP-actin into filament-associated structures resulted in the temperature-dependent impairment of macronuclear elongation (phenotypically similar to DeltaMYO1) and cytokinesis. Anti-actin immunolocalization studies demonstrated that actin was closely associated with nascent food vacuoles in the oral regions of both wild-type and MYO1-knockout cells. Electron microscopy, however, revealed significant differences in vacuole morphology between wild-type and MYO1-knockout Tetrahymena . Wild type cells possessed vacuoles that were rapidly expanded through membrane fusions, whereas MYO1-knockout cells accumulated comparatively smaller nascent food vacuoles. These accumulations frequently formed multi-vacuolar aggregates comprised of clusters of small vacuoles loosely connected by slender vacuolar extensions. The evidence presented in this study suggests that an actin-Myo1p contractile system may have a novel function within the cell apparatus facilitating macronuclear division, and may also provide the translocatory component for rapid vacuole-vacuole fusion during early endosome expansion.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
