COLLABORATIVE RESEARCH: Development of the Triad Junction inSkeletal Muscle

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The triad junction is a complex structure involving plasma and sarcoplasmic reticulum membranes and at least seven proteins, and is the site where electrical excitation is transduced to release intracellular calcium, and thus has a critical role in coupling electrical excitation to muscle contraction. Additionally, it may interact with the cellular cytoskeleton to form muscle-specific morphology. Two key components of the triad junction, the dihydropyridine (DHPR) and ryanodine receptors (RR), are expressed early during embryonic chick skeletal muscle differentiation. The aim of this project is to determine how these proteins are incorporated into the triad junction and become active in the release of intracellular calcium in developing skeletal muscle, and whether the calcium release events mediated by the triad junction influence other aspects of muscle development. The specific aims are to: (1), define the experimental in detail by establishing the time course of the expression of the DHPR and RR in developing embryonic chick skeletal muscle; and (2), investigate whether these proteins influence skeletal muscle development by testing the hypothesis that DHPR and RR serve as organizers for the assembly of the triad junction and its integration into mature muscle structure. This will be done by defining the time course of incorporation of these and other junctional components into the junction, and assess the significance of interactions between junctional proteins and cytoskeletal elements. Dr. Ellisman's contribution to the project will be primarily with regard to the morphological aspects. Skeletal muscle provides the contractile force to move bones and other structures, effecting organ or whole organism movement. The signal which "tells" the muscle to contract and ensures synchronous contraction along the length of the muscle involves an electrical wave generated at the neuromuscular junction, which travels down the length of the muscle fiber along the plasma membrane. The conversion of the electrical signal to contraction occurs by transduction of the electrical signal to a "second messenger" signal, namely release of calcium from intracellular stores (sarcoplasmic reticulum) into the cytoplasm. At the "triad junction" of the muscle cell, the membrane of the sarcoplasmic reticulum is closely apposed to a specialized structure of the plasma membrane known as the transverse tubule. This highly specialized region contains a set of unique sarcoplasmic reticulum membrane proteins which interact with the closely apposed transverse tubule membrane to effect the coupling of the electrical signal to the opening of a calcium channel which allows the release of calcium from the sarcoplasmic reticulum to the cytoplasm. Neither the mechanism of this signal transduction event, nor the cellular development of this intricate subcellular structure, is well understood. The results of this research will increase our understanding of both of these very important processes in vertebrate muscle.