Studies on the Cell Surface and Adhesion of Myoblasts During Early Myogenesis

Broadley, Kenneth N (1987) Studies on the Cell Surface and Adhesion of Myoblasts During Early Myogenesis. PhD thesis, University of Glasgow.

Abstract

During myogenesis mononucleated cells, myoblasts, fuse with one another to give rise to multinucleated cells, known as myotubes. This event has been shown to be highly cell specific, in that myoblasts will only fuse with other skeletal muscle cells. It was this specificity that was under investigation. Clearly, cell-cell adhesion and recognition must take place prior to fusion, and it has been suggested that specific adhesion molecule(s) present at the myoblast surface at the time of fusion mediate the specificity of the event. Four main approaches were undertaken:- (a) Monitoring transitions in mannose containing glycoproteins as myogenesis procedes. (b) Affinity chromatography using plasma-membrane proteins coupled to Sepharose. (c) Comparing the adhesion of 24 hour old (in culture) myoblasts and 48 hour old (in culture) myoblasts to myoblasts and myotubes of varying age. (d) Raising antibodies against 48 hour old (in culture) myoblasts. (a) It has been reported that the lectin concanavalin A (Con A) will inhibit the fusion of myoblasts. Therefore, the appearance of a mannose containing moiety at the time of fusion might be involved in mediating fusion. Changes in glycoprotein synthesis were monitored using both one and two-dimensional polyacrylamide gel electrophoresis and iodinated Con A. Numerous complex differences were observed, too many for this approach to be particularly helpful. (b) A novel approach to affinity chromatography was employed. Plasma-membranes from 48hr old (in culture) myoblasts (fusion competent) were isolated by centrifugation. The proteins were then coupled to Sepharose in the presence of octyl-glucoside. Iodinated cell-surface proteins, also from 48 hour old (in culture) myoblasts, were then added to the column, and allowed to circulate overnight. The column was then washed extensively, and bound proteins were eluted with a salt gradient. This approach should give rise to the isolation of proteins with the ability to bind to each other, either homophilically, or heterophilically. Two peaks were isolated. These were dialysed to a small volume, and prepared for SDS-PAGE. The resulting autoradiograph detected the presence of nine distinct bands, at 250KD, 225KD, 170KD, 140KD, 100KD, 76KD, 58KD, 41KD, and 29KD. (c) No difference could be detected between the adhesion of 24 hour old myoblasts and 48 hour old myoblasts to myoblasts and myotubes of varying age. The idea behind this approach being that if a developmentally regulated antigen were responsible for the specificity of fusion, and that if its expression were limited to the time of fusion, then one might expect to see an increase in cell-cell and cell-myotube adhesion. (d) Antibodies to the 48hr old (in culture) myoblasts were raised in a rabbit. IgG was isolated and tested for reactivity. The antibody was then extensively adsorbed against 24 hour old (in culture) myoblasts, until the antibody reacted only with the 48 hour old myoblasts. The 48 hour old myoblasts are fusion competent, whereas the 24hr old ones are not. An Fab was prepared from this IgG, and tested to see whether it could inhibit cell-cell aggregation, as measured by Coulter counter assay. Results show that while aggregation proceded as normal for 30-45 minutes, the Fab was able to prevent further aggregation, and indeed caused the breakdown of existing aggregates. A non-specific rabbit Fab had no effect on aggregation, as did IgG. This tends to suggest that myoblast adhesion/recognition is at least a two step event.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Cellular biology
• Date of Award: 1987
• Depositing User: Enlighten Team
• Unique ID: glathesis:1987-77608
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jan 2020 11:53
• Last Modified: 14 Jan 2020 11:53
• URI: https://theses.gla.ac.uk/id/eprint/77608