Date of Award

8-2012

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Sharon Ashworth

Second Committee Member

Robert Gunderson

Third Committee Member

Mary Tyler

Abstract

Actin, the most abundant protein found in mammalian cells, polymerizes to form one of the basic components of the cytoskeleton in the cell. Actin is an essential part of cell structure and is involved in important cell events such as migration and cytokinesis. Actin is found in both a monomeric state (G-actin) and a polymeric form (F-actin). In order to ensure proper regulation of actin dynamics, actin requires actin-binding factors (ABFs). One such ABF is cofilin, an 18 kDa protein that binds along the polymer to weaken lateral contacts and induce actin filament severing and depolymerization (Van Troys and others 2008). Cofilin is regulated by phosphorylation on serine 3 that inhibits binding of cofilin to either G-actin or F-actin (Agnew and others 1995). Danio rerio, commonly known as the zebrafish, is a popular model organism to study human disorders due to the large number of offspring, transparent embryos, ease of genetic manipulation, and similarity to human with both being vertebrates. The zebrafish contains three cofilin isoform genes that include non-muscle localized isoforms cofilin 1 (cfl 1) and cofilin 1- like (Cfl 1-L), and the muscle isoform cofilin 2 (Cfl 2). Our studies utilized morpholino mRNA knockdown of Cfl 1, Cfl 1-L and Cfl 2 zebrafish isoforms. We observed the expression of Cfl 1-L, p-cofilin, tropomyosin, and actin in wildtype zebrafish from 0 to 120 hours post fertilization (Hpf) utilizing immunoblot analysis. Zebrafish Cfl 1-L was expressed at low levels until it begun to increase at 36 Hpf. Two phosphorylated zebrafish cofilin isoforms (p-cofilin) were recognized at different molecular weights. The larger p-cofilin was detected throughout development while expression of the smaller protein was detected at 36 Hpf and it increased at every time point after. Tropomyosin was expressed at low levels during very early hours of development but increased at 36 Hpf and onwards. Cfl 1 mutants and morphants were observed with severe pericardial edema, malformed heart chambers, jaw deformities, a deflated swim bladder, and a slower heartbeat compared to wildtype zebrafish. Cfl 1-L morphants at 48 hpf were observed with decreased pigmentation along the trunk and a larger yolk compared to wildtype. At 72 Hpf and continuing through early development, a bend to the tip of the tail was observed and starting at 96 Hpf the heart cavity was enlarged. Cfl 1-L morphant zebrafish fins are curled and not properly formed compared to wildtype at 120 Hpf. Cfl 2 morphant zebrafish were observed with little defects with only minor alterations to cranialfacial features compared to wildtypes. Immunoblot analysis revealed a decrease in Cfl 1-L expression in Cfl 1-L morphants. The two p-cofilins were regulated differently between cofilin isoform morphants. This information will aid in the development of zebrafish-based disease models relating to the role of cofilin in cytokinesis, neural crest migration, proteinuria, and others.

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