Formación basada en competencias en el Grado en Medicina

The overall goal of the research presented in this dissertation is to extend our understanding of the cellular functions of the mammalian synemin isoforms. By identifying and studying interactions of synemin isoforms with their specific protein partners, the exact role of synemin within mammalian cells could be gradually clarified. And, these efforts should provide useful information for future investigations on this large unique type VI intermediate filament protein.

Although previous results from our lab showed that avian synemin interacts with vinculin, whether the two major mammalian synemin isoforms interact with vinculin and its muscle specific isoform metavinculin has been unclear. In addition, a recent study demonstrated co-precipitation of human synemin with talin from hepatic stellate cell extracts and co-localization of synemin with talin within cultured cells. However, evidence of a direct interaction of mammalian synemin with talin has still been missing. In order to study direct interactions of mammalian synemins with vinculin/metavinculin and talin, several different approaches testing protein-protein interactions were conducted using bacterially expressed and purified recombinant synemin regions as well as purified recombinant regions from vinculin/metavinculin and talin. These studies discovered that the 312 amino acid insert (SNTIII) present only within α-synemin exhibited specific interactions with both the vinculin/metavinculin tail domain and the talin rod domain in both blot overlay assays and GST pull-down assays, whereas regions of β-synemin did not. Both the vinculin tail domain and the talin rod domain exhibited saturated interaction with SNTIII with similar affinities in ELISA solid phase binding assays. Furthermore, it was shown that vinculin and talin interact with SNTIII in a competitive manner in the ELISA solid phase binding assays, suggesting they do not bind to synemin simultaneously in vivo.

Interactions of SNTIII with both vinculin and talin were further supported by evidence that EGFP tagged SNTIII demostrated specific co-localization with both vinculin and talin in the focal adhesions within transfected mammalian cells. In contrast, β-synemin regions showed distinct localization patterns, but without specific localization at focal adhesion sites. These results in toto indicate that α-synemin, but not β-synemin, is the isoform that interacts

specifically with vinculin/metavinculin and talin. Interestingly, analysis of the primary sequence of the SNTIII region indicates that it is composed of seven ~39 amino acid tandem repeats, with each repeat containing a specific S/T-X-R-Q/H motif. This unique primary sequence arrangement, and possibly of the corresponding unique secondary and tertiary structures, of SNTIII may be the underlying basis for its specific interactions with both vinculin and talin.

In order to extend our understanding of the cellular function of the smaller β-synemin, novel interactions of β-synemin with its protein partners were discovered by yeast two-hybrid screening assays. By using the entire β-synemin tail domain (SNβT) as the bait in screening against a human adult skeletal muscle total cDNA library, the LIM domain protein zyxin was pulled out as one of the β-synemin interacting partners. Mapping the respective binding site within these two molecules using β-galactosidase liquid culture assays indicated that synemin binds specifically to the LIM domains of zyxin, whereas the zyxin binding site within the β-synemin molecule was located within the C-terminal ~300 amino acid (SNβTII) sequence at the end of the β-synemin tail domain. Because α-synemin shares with β-synemin the same sequence containing the binding site for zyxin, it is therefore not surprising to observe interaction of α-synemin with zyxin as well. However, the presence of the unique 312 amino acid insert within α-synemin may interfere with the interaction, as reflected by a lower affinity of the α-synemin tail domain for zyxin than that of the β-synemin tail domain in the β-galactosidase liquid culture assays. In vitro protein-protein interaction assays, such

as GST pull-down assays and surface plasmon resonance assays, provided further support for a direct interaction of the β-synemin tail domain with zyxin. In addition, synemin co-precipitated with zyxin from mammalian cell extracts. When expressed within mammalian cells, EGFP-tagged SNβTII led to a markedly reduced protein level of zyxin and the loss of zyxin localization at the focal adhesion sites. These results suggest synemin and zyxin associate with each other in vivo. Zyxin has been implicated as being essential in

modulating actin cytoskeletons at actin-membrane interactions as well as in cell adhesion and migration. Thus, synemin may link the heteropolymeric intermediate filaments to these cellular processes within synemin-expressing cells. Indeed, novel functions of synemin in

cell adhesion and cell motility is presented in this dissertation. RNAi studies knocking down synemin expression within Hela cells, a human cervix epithelial adenocarcinoma cell line, led to markedly compromised cell adhesion and migration.

Taken in toto, the research presented in this dissertation demonstrates that mammalian

synemins interact differentially with the cytoskeletal proteins vinculin/metavinculin, talin, and zyxin, all of which are present in cell-cell and/or cell-substratum adhesion sites, and are essential in regulating cell adhesion and migration. Thus, it is our hypothesis that synemin plays important roles in linking the heteropolymeric intermediate filaments to these adhesion sites within synemin-expressing cells. Different ratios of α- versus β-synemin may modulate the interactions of mammalian synemins with their protein partners, and thereby regulate the strength of the linkages between the synemin-containing heteropolymeric intermediate filaments and the adhesion sites in mammalian muscle cells, and more generally, in all mammalian cells expressing synemin.