Degree Type

Dissertation

Date of Award

2000

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Richard M. Robson

Abstract

Synemin is a large (∼182 kDa by sequence, ∼230 kDa by SDS-PAGE) cytoskeletal protein that was originally identified based upon its co-localization and co-purification with the intermediate filament (IF) proteins desmin and vimentin in muscle tissue. Studies in this dissertation have focused on understanding the molecular properties and role of synemin. Cloning and sequencing studies revealed that synemin itself is a large member of the IF protein superfamily because it contains the domain structure typical of IF proteins, i.e., an ∼310 amino acid, coiled-coil rod domain flanked by an N-terminal head domain, and a C-terminal tail domain that is very large in the case of synemin. Based upon analysis of the sequence, we developed the hypothesis that synemin forms heteropolymeric IFs with other IF proteins such as desmin and/or vimentin, and acts to link, via its large tail domain, those EFs to other cytoskeletal structures. Transfection studies utilizing the SW13 IF-mosaic cell line indicate that synemin does, in fact, require the presence of another IF protein to assemble into IFs. These tissue culture findings are supported by the results of in vitro cosedimentation studies using both purified proteins and expressed protein domains, which indicate that synemin forms EFs with desmin via interactions of their rod domains. Additionally, blot-overlay assays have identified interactions between the tail domain of synemin and both the myofibrillar Z-line protein alpha-actinin and the costameric protein vinculin. Furthermore, these interactions have been confirmed, and the protein sub-domains involved in the interactions more specifically defined, by the use of the Gal4 yeast two hybrid system. In toto, these results support a role for synemin as a component of obligate heteropolymeric IFs, which may function to crosslink the IFs to other cytoskeletal structures. In striated muscle cells, synemin may play an important role in directly linking the heteropolymeric IFs to alpha-actinin within the myofibrillar Z-lines and to vinculin within the costameres. By these interactions, synemin-containing IFs may firmly link all adjacent myofibrils within the cell, and attach the peripheral layer of myofibrils to the muscle cell membrane, thereby promoting overall cytoskeletal integrity and contractile function.

DOI

https://doi.org/10.31274/rtd-180813-13775

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Robert Milton Bellin

Language

en

Proquest ID

AAI9962798

File Format

application/pdf

File Size

123 pages

Included in

Biochemistry Commons

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