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Characterization of the zebrafish zipper interacting protein kinase homolog

Author

Tamara Lee Basepayne, University of the Pacific

Date of Award

2012

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Douglas Weiser

First Committee Member

Gregg D. Jongeward

Second Committee Member

Craig A. Vierra

Third Committee Member

Lisa A. Wrischnik

Abstract

The regulation and maintenance of normal cell movements and shape play a vital role in the normal development and health of every living thing. The characterization of 6 zebrafish Zipper Interacting Protein Kinase homolog has helped to better understand how changes in cell cytoskeletal elements can lead to changes in cell shape and movement. Zebrafish are ideal model organisms for studying ZIPK because it has been previously shown that zebrafish ZIPK has closer sequence homology to human ZIPK than rodent ZIPK, and because zebrafish embryos are ideal for studying cell shape and movement in vivo. Using Whole Mount In Situ Hybridization we found that the zebrafish ZIPK is expressed during all stages of embryonic development, but most importantly during gastrulation when cell motility and changes in cell shape can best be studied. To determine where zebrafish ZIPK is expressed at the sub-cellular level, GFP-ZIPK and Flag-ZIPK clones were created and used for transfecting into Hek293T cells and Hela Cells. From these transfections, cell counterstaining and confocal microscopy we found that ZIPK is expressed ubiquitously throughout the cell, although mainly cytoplasmic. To study the effects on cell shape various ZIPK mutants were created through site-directed mutagenesis. These mutants were made to study the effects of the kinase domain of the protein, or other functional domains within the protein. From these studies it was shown that ZIPK does affect cell shape through changes in the actomyosin cytoskeleton resulting in aberrant cytoskeletal structures. Finally, we have also shown through phosphorylation assays that ZIPK phosphorylates and thus regulates MYPT-1, a scaffolding protein of the myosin protein phosphate complex and directly phosphorylates myosin light chain, both of which play a role in changes in cell shape and movement.

Pages

120

Recommended Citation

Basepayne, Tamara Lee. (2012). Characterization of the zebrafish zipper interacting protein kinase homolog. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/817