טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentEzaguy Noa
SubjectCharacterization of the Role of LOXL3 during the
Differentiation of C2c12 Myoblasts
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Gera Neufeld
Full Thesis textFull thesis text - English Version


Abstract

The musculoskeletal system provides form, support, stability, and movement to the body. The formation of the musculoskeletal system represents an intricate process of tissue assembly involving heterotypic inductive interactions between tendons, muscles and cartilage. The tendons integrate with the muscle through specialized structure termed the myotendinous junction (MTJ). Very little is known about the molecular processes involved in the formation of the MTJ.

Lysyl oxidase-like protein 3 (LOXL3) is a member of the Lysyl oxidase gene family. These proteins are copper amine oxidases that initiate the covalent cross linking between subunits of elastin and collagen by oxidizing peptidyl lysine residues in these proteins to form allysine. The lysyl-oxidase gene family consists of five members that are highly conserved in their C-terminal domains. Lysyl oxidases play a critical role in extracellular matrix crosslinking and remodeling and were recently found to induce tumor progression, tissue fibrosis, and various developmental processes. Very little is known about the LOXL3 protein. Preliminary results obtained in the laboratory of Dr. Hasson from the faculty of Medicine indicate that LOXL3 is expressed at the tip of developing muscles in mice embryos at the region in which tendons attach to muscle cells. In this research project we tried to establish an in vitro assay system based upon the C2C12 mouse myoblast cell line in order to study in more detail the mechanisms which regulate the localization of Loxl3 to the muscle-tendon junction. The C2C12 cell was chosen because it undergoes induced differentiation in cell culture to produce striated muscle fibers.

We found that LOXL3 in expressed in C2C12 cells. Its protein levels seems to be elevated as differentiation progresses but the mRNA levels stays stable at all differentiation stages. We also silenced LOXL3 in C2C12 cells in order to determine its importance in the differentiation process. Silenced cells exhibit morphological changes and did not form mature fibers.

For a better understanding of the localization of LOXL3 in C2C12 cells we performed immunofluorescence experiments. In non-differentiated cells LOXL3 was localized in peri-nuclear vessicles. In differentiated cells that form fibers LOXL3 was localized at the tip of the elongated striated C2C12 derived muscle cells. LOXL3 was also localized in the nucleus of differentiated and non-differentiated cells but the amount of LOXL3 in cell nuclei seemed much higher in differentiated cells.

Our results indicate that C2C12 cells are suitable as an in-vitro system for understanding the role of LOXL3 in C2C12 differentiation and muscle development.