Thesis Title

Analysis of a Proposed Cell Binding Site on Caenorhabditis Elegans Collagen Type Iv

Date of Graduation

Spring 2003

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Colette Witkowski

Subject Categories

Medical Molecular Biology

Abstract

Collagen IV (col IV), a basement membrane protein, provides structural support and regulation of cell migration and binding. C. elegans body wall muscle col IV consists of two alpha 1 chains and one alpha 2 chain. The human col IV molecule has a cell binding site (CBS) composed of aspartate (D) on each alpha 1 chain and arginine (R) on the alpha 2 chain that form a D-D-R sequence recognized by the cell surface receptor integrin alpha 1 beta 1. The D-D-R sequence is evolutionarily conserved in the C. elegans col IV molecule. This proposed CBS was analyzed to determine its importance in C. elegans development. Microinjection techniques were used to produce transgenic animals and generate worm strains with a mutation on the alpha 1 chain in the putative D-D-R site. These strains were produced in a null mutant background (no alpha 1 chain produced) and in a wild type background. Control strains were also produced using the wild type alpha 1 sequence and an upstream control mutation. Homozygous null animals arrest during embryogenesis exhibiting degeneration of muscle structure. Transgenic animals with the CBS mutation also arrest, while those with control mutations rescue the homozygous null phenotype. Nomarski and phase contrast imaging indicate that CBS mutants exhibit a worse morphological phenotype than homozygous null mutants. Immunocytochemical techniques were used to analyze physical defects of body wall muscle structure and function in animals with the CBS mutation. Immunofluorescent images of alpha 2 chain expression and myosin heavy chain A organization reveal that the CBS mutants in this study express col IV in the basement membrane and exert dominant negative effects over sarcomere organization. Overall results show embryos carrying the CBS mutation continue to have muscle cell disruption demonstrating the importance of the proposed CBS.

Copyright

© Lori G Langiano

Citation-only

Dissertation/Thesis

Share

COinS