The candidate's long-term goal is to elucidate the molecular mechanisms by which cytoskeletal proteins participate in the formation of functionally specialized membrane and cytoplasmic domains in developing embryos and differentiating cells. This will involve application of antisense RNA technology initially in Xenopus, but later in transgenic mice. Attached to the inner surface of the membrane of cells is a skeleton of proteins. The major protein in this membrane skeleton is spectrin, which is a very large, flexible, rod-like dimeric protein. It is clear that spectrin is important in human diseases since defects in the alpha-subunit of spectrin in red blood cells are linked to hereditary hemolytic anemia. A related spectrin, termed fodrin, is encoded by different genes and is present in muscle. It is likely that defects in fodrin also occur, and are linked to muscle diseases. The applicant's short term goal is to directly test the hypothesis that alpha-fodrin is important in the development muscle cells. Our approach is to use antisense RNA techniques to cause defects in alpha-fodrin during muscle development in the frog Xenopus laevis. This technique involves cloning an alpha-fodrin cDNA in an antisense orientation under control of one of several promoters. The recombinant plasmids are then microinjected into the cytoplasm of fertilized Xenopus eggs, which are cultured in vitro. The plasmids are transcribed into antisense RNA at the blastula stage of development. At various times during development into tadpoles, the embryos will be assayed at the level of RNA and protein to determine whether expression of antisense RNA from the injected plasmid leads to specific inhibition of alpha-fodrin mRNA and protein. We have already shown that this general approach works, and the present study will focus specifically on the muscle embryos defective in alpha-fodrin. If defects in muscle fodrin lead to defects in normal muscle structure and function, this will support our hypothesis. Once we understand how alpha-fodrin is involved in muscle defects in frogs, our long-term goal is to identify human muscle diseases which also involve defective alpha-fodrin. Related to this goal, we are presently developing full-length human cloned fodrin probes which may be useful in the diagnosis of human muscle diseases which involve fodrin. Dr. Moon is a tenure-track Assistant Professor of Pharmacology at the University of Washington School of Medicine. The Department of Pharmacology expects to provide the space and support necessary for development of the full potential of his research program. Award of an RCDA will enhance Dr. Moon's career development by increasing the portion of time that he can devote to development of his novel antisense RNA method for probing the roles of specific cytoskeletal proteins in morphogenesis and formation of specialized membrane and cytoplasmic domains. This work has important implications in cell biology, molecular pharmacology, and human disease will have broad impact.
Showing the most recent 10 out of 15 publications
