My laboratory has been investigating the organization of the postsynaptic membrane of the developing neuromuscular junction, in order to elucidate the cellular and molecular processes involved in synapse formation. Many of our experiments focus on the acetylcholine receptor (AChR), which accumulates at high density in the postsynaptic membrane. We have found that an unusual cytoskeletal structure, consisting in part of actin and spectrin, and resembling the membrane skeleton of the human erythrocyte, is important in binding and immobilizing receptors in the muscle membrane. We have also identified several extracellular and integral membrane proteins that are associated with accumulations of AChR. Our results suggest that some of these proteins are bound together in a large macromolecular complex through their associations with a spectrin-rich membrane skeleton. We are now characterizing the complex formed by these proteins and AChR using intracellular injection of antibodies and fusion proteins, and with the yeast two-hybrid screen.
Related experiments have examined the composition and organization of the plasma membrane of muscle where it makes connections to the contractile apparatus, in structures termed "costameres". Dystrophin, the protein missing in Duchenne's Muscular Dystrophy, is concentrated at costameres. Current efforts are aimed at identifying and characterizing the spectrins and spectrin-associated proteins at costameres and learning how these are affected in DMD. Our results suggest widespread changes in the organization of the sarcolemma in dystrophic muscle.
We are also studying an unusually small form of ankyrin that is highly enriched in the sarcoplasmic reticulum of skeletal and cardiac muscle fibers. We are using cellular transfection, site-directed mutagenesis, the yeast two-hybrid screen, and biochemical techniques to learn how small ankyrin is targeted to calcium-sequestering membrane compartments in muscle, and how it mediates the interactions of these membranes with surrounding structures.
Bezakova, G., and R.J. Bloch (1998) The zinc finger domain of the 43K receptor-associated protein, rapsyn: Role in acetlycholine receptor clustering. Molec. Cell. Neurosci.. 11, 274-288.
DeDeyne, P., A. O'Neill, W.G. Resneck, G.M. Dmytrenko, D.W. Pumplin, and R.J. Bloch (1998) Vitronectin receptor associated with clathrin-coated membrane domains through the cytoplasmic sequence of the beta 5 subunit. J. Cell. Sci., 111, 229-2740.
Zhou, D., C.S. Birkenmeier, M. Williams, J.J. Sharp, J.E. Barker and R.J. Bloch (1997) Small, Membrane-Bound, Alternatively Spliced Forms of Ankyrin 1 Associated with the Sarcoplasmic Reticulum of Mammalian Skeletal Muscle. J. Cell Biol. 136, 621-631.
Zhou, D., J.A. Ursitti and R.J. Bloch (1998) Developmental Expression of Spectrins in Rat Skeletal Muscle. Molec. Biol. Cell 9, 47-61
Williams, M.W., and R.J. Bloch (1999) Extensive but Coordinated Reorganization of the Membrane Skeleton in Myofibers of Dystrophic (mdx) Mice. J. Cell Biol. 144,1259-1270.