טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSheleg Ortal
SubjectLocalized LoxL3-Dependent Fibronection Oxidation Regulates
Myofiber Stretch and Integrin-Mediated Adhesion
DepartmentDepartment of Medicine
Supervisor Professor Peleg Hasson
Full Thesis textFull thesis text - English Version


Abstract

During embryonic development, muscle fibers attach to tendons to relay muscle force to the skeleton. The interface region connecting muscles and tendons, the myotendinous junction (MTJ), is a highly specialized region in which the extracellular matrix (ECM) differs from that of the muscle and the tendon. Defects in MTJ activity and formation lead to muscle dystrophies, further emphasizing the key roles this junction plays in muscle function. Surprisingly, despite being such a crucial component of the musculoskeletal system, our understanding of the processes that regulate MTJ development and maintenance are still unclear. The ECM has classically been thought of as a largely inert scaffold onto which the tissues are organized. However, recent work has clearly demonstrated that this is far from being the case. Multiple processes including cell migration, adhesion, and differentiation are regulated and affected by the ECM in distinct manners ranging from matrix elasticity to cleavage of matrix constituents that affect cellular outcomes. Yet the mechanisms that underlie the matrix’s instructive properties and matrix proteins’ activity are largely unknown. It has been implicated that integrin signaling, a crucial component relaying signals from the ECM to the cells, plays a key role in MTJ formation and mutations affecting the cascade cause MTJ deformities leading to muscular dystrophies in mice and humans. Underlying mechanisms for integrin activation at the MTJ and ECM modifications regulating its signaling are unclear. Post-translational modifications are frequently used in the regulation of protein function. Numerous cellular and secreted proteins undergo distinct modifications that are crucial for their function. However, the vast majority of known post-translational modifications of both intra- and extracellular proteins occur intracellularly. One example of a known post translational modifier family is the Lysyl-oxidase (Lox) family of enzymes; acting on both the ECM and intracellular proteins. The five genes of the Lox family encode secreted enzymes that are considered to be key regulators of ECM organization. We show that Lysyl oxidase-Like 3 (LoxL3) is specifically expressed at myofiber extremities. Knockout mice for LoxL3 exhibit myofiber anchorage defects in the early stages of myotome formation at the somitic boundaries as well as MTJ defects in fully developed embryos and new born mice. We further find that in LoxL3 mutant embryos, integrin signaling is downregulated. We demonstrate that LoxL3 complexes with and directly oxidizes Fibronectin (FN), a key ECM scaffold protein and integrin ligand enriched at the MTJ. We show that this modification enhances FN-induced, integrin-mediated signal transduction allowing for a properly formed and maintained MTJ. Altogether, in my work, we have identified a mechanism whereby the posttranslational modification of a matrix protein potentiates integrin- mediated signal transduction which is induced specifically by the modified matrix protein. The localized expression of LoxL3 at the myofiber tips activates spatially restricted integrin signaling at the MTJ that is required for the precise localization of myofiber anchorage points and for the assembly of the FN matrix at the somitic boundaries.