Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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Item Myosins IIIa and IIIb regulate stereocilia length by transporting espin 1 to the polymerizing end of actin filaments(2009) Merritt, Raymond Clyde; Kachar, Bechara; Popper, Arthur N; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mutations in the myosin IIIa gene are linked to DFNB30 late onset deafness, in which those affected individuals can hear for the first twenty years of life. Mutations in the espin gene are linked to DFNB36 congenital deafness. Both myosin IIIa and espin 1 are expressed in the inner ear hair cells and colocalize at stereocilia tips, the site of actin polymerization. Overexpression of these proteins in hair cells produce an increase in stereocilia length, and when both are co-expressed together they produce a length increase greater than when each one are overexpressed individually. These results suggest that these proteins interact and influence stereocilia length regulation. We confirmed the interaction of these two proteins in heterologous COS-7 cells. We observed that when co-expressed, these proteins promote elongation of filopodial actin protrusions in a synergistic manner. Mutational analyses show that myosin IIIa3THDI binds to the ankyrin repeats of espin 1. Live and fixed cell imaging shows that myosin IIIa transports espin 1 to the filopodia tips where espin 1 promotes actin polymerizations through its WH2 domain. Because of the late onset of deafness associated with myosin IIIa, it has been speculated that the lack of myosin IIIa function is partially compensated by the paralogous protein, myosin IIIb. Myosin IIIb, encoded by a distinct gene, lacks a C-terminal actin-binding domain shown to be essential for myosin IIIa filopodia tip localization. We observed that myosin IIIb localizes at stereocilia tips and is expressed at an earlier stage than myosin IIIa. We confirmed that myosin IIIb transports espin 1 to stereocilia tips and promotes actin polymerization, consistent with the hypothesis that it partially compensates for myosin IIIa. We found that binding to espin 1 is required for myosin IIIb movement and localization. We observed that myosin IIIb can downregulate the myosin IIIa localization in vestibular, but not in cochlear, hair cells. The interplay of myosins IIIa, IIIb, and espin 1 and their influence on stereocilia length unravels a novel molecular complex at the polymerizing end of F-actin and a framework to understand the cause of DFNB30 and DFNB36 deafnesses.Item STRUCTURE, LOCALIZATION AND FUNCTION OF MOUSE MYOSIN XVA IN THE INNER EAR(2007-01-30) Boger, Erich; Friedman, Thomas B; Popper, Arthur N; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recessive mutations of MYO15A encoding unconventional myosin XVA are associated with sensorineural deafness in both humans (DFNB3) and the shaker 2 phenotype in mice, which are deaf and exhibit circling behavior due to a vestibular abnormality. Myosin XVa is extensively alternatively spliced and encodes approximately thirty protein isoforms. In mouse hair cells that are homozygous for the shaker 2 (Myo15asupsh2) missense mutation, stereocilia are abnormally short due to a failure of their actin filamentous core to elongate. The stereocilia bundle of homozygous (Myo15asupsh2) hair cells lack the characteristic staircase architecture found in wild type hair cell stereocilia bundles. I show that the inability of mutant myosin XVa to deliver whirlin, a scaffold protein, to stereocilia tips underlies the stereocilia dysmorphology in hair cells from homozygous (Myo15asupsh2) mice. The introduction of exogenous myosin XVa homozygous (Myo15asupsh2) hair cells results in restoration of stereocilia elongation and staircase bundle formation. These results imply that the delivery of whirlin by myosin XVa is essential for stereocilia elongation and staircase formation. Using a series of GFP tagged myosin XVa expression constructs containing deletions of one or more domains, the regions necessary for localization to filopodia tips in COS-7 cells, as a model system, and to stereocilia tips of inner ear hair cells was determined. In COS-7 cells, the myosin XVa motor plus several combinations of domains of the myosin XVa tail are sufficient for filopodia tip targeting. The myosin XVa motor plus either the MyTh4sub1/FERMsub1 or MyTh4sub2/FERMsub2 domains are sufficient for stereocilia tip localization in inner ear hair cells. A preliminary assessment of the regions of myosin XVa necessary for the re-initiation of stereocilia elongation and staircase formation in homozygous (Myo15asupsh2) mutant inner ear hair cells is provided. These results provide a better understanding of the molecular mechanisms underpinning normal stereocilia morphogenesis.