Membrane Domains and Membrane Organization in Nerve and Muscle

Neurons receive, process and relay electrical signals by using highly specialized structures in their intracellular and plasma membranes, including pre- and postsynaptic membranes, the axon hillock, and intracellular compartments that sequester and release Ca2+. How do these structures form? What determines their subcellular locations? And how do mistakes in their assembly or localization affect the functions of excitable cells? We have been addressing these and related questions by examining the membrane systems of cells that are excitable but that are much more highly organized than neurons -- striated muscle.

We have focused on three sets of structural proteins that organize membranes in other cell types but whose roles in excitable cells have not yet been elucidated -- the spectrin, ankyrin and titin superfamilies. We are especially interested in how members of these protein families influence the organization of the plasmalemma and intracellular membranes, how they participate in the clustering of acetylcholine receptors at the postsynaptic membrane of the developing neuromuscular junction, how they help organize the cytoplasm of striated muscle cells, and how changes in these proteins are linked to human diseases, including muscular dystrophy.

Postsynaptic Membrane Domains

Our studies of a model for the postsynaptic membrane of the neuromuscular junction show that the acetylcholine receptors are bound to a spectrin-rich membrane-associated cytoskeleton (membrane skeleton). This skeleton is unusual, however, because it contains several giant members of the spectrin superfamily, including dystrophin, utrophin and ACF7. Current studies focus on how these related molecules interact with each other to form a membrane skeleton and with the receptors to anchor them in the postsynaptic region. Related studies are in progress to determine if similar molecules are involved in the formation of synapses in the mammalian hippocampus.

Organization of Intracellular Membranes

We have recently discovered an unusually small form of ankyrin that is highly enriched in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Similar molecules are present in select neuronal populations in the brain. We are using cellular transfection, site-directed mutagenesis, and biochemical techniques to learn how small ankyrin is targeted to Ca2+-sequestering membranes in muscle and nerve, and the yeast two hybrid screen to learn how it mediates the interactions of these membranes with surrounding structures. We have shown that small ankyrin anchors the SR to the contractile apparatus in striated muscle by binding directly to two giant proteins, titin and obscurin. Both these proteins also play important roles in organizing the contractile apparatus in developing heart and skeletal muscle (see below).

In another, related project, we have been studying the relationship of the SR to the transverse tubules of cardiac muscle. Our results are consistent with a model in which spectrins help link the SR to the transverse tubules through complexes formed via ankyrins with integral membrane proteins in both membrane compartments. These integral proteins include the Na,Ca-exchanger and the Na,K-ATPase in the transverse tubule membrane and the IP3 and ryanodine receptors in the SR membrane.

Organization of the Sarcolemma into Costameres

The plasma membrane, or "sarcolemma", of skeletal and cardiac muscle cells is organized into structures called "costameres" that are highly enriched in a number of integral and peripheral membrane proteins serving a variety of structural and functional roles. We are particularly interested in these structures because they contain high concentrations of spectrin and dystrophin, the protein that is missing in young boys with Duchenne Muscular Dystrophy (see below). Our studies of the biochemical complexes present at costameres suggest that, in addition to actin, desmin-based and cytokeratin-based intermediate filaments link costameres to the contractile apparatus. We have established that cytokeratins specifically associate with the N-terminal region of dystrophin, and that overexpression of cytokeratin 19 specifically disrupts the organization of cytoplasmic and costameric structures. We are currently using transfection of cells in culture to learn how cytokeratins interact with other proteins of costameres, including dystrophin-associated proteins, spectrin and its associated proteins, and vinculin.

The Role of Obscurin in Striated Muscle

Obscurin is the third member of the titin superfamily to be identified in striated muscle. Like its "cousins", titin and nebulin, obscurin is a giant protein (~800 kDa) made up of many structural (mostly IG) domains, linked to several domains involved in cellular signaling pathways. Unlike its cousins, which are fully integrated into the contractile apparatus, obscurin surrounds the contractile structures, serving to link them to the surrounding SR (see above). Our recent experiments suggest that obscurin also interacts closely with several proteins, including myosin and myosin binding protein C, that are essential for assembling the contractile apparatus. We are continuing to characterize these interactions of obscurin as well as its binding to small ankyrin in the SR. We are also studying the signaling activities linked to the Rho-GEF domain of obscurin, with the aim of elucidating its role in myofibrillogenesis.

Studies of Muscular Dystrophy

A major focus of many of our studies has been to learn how changes in the composition and organization of the membrane systems of skeletal muscle can lead to muscular dystrophy. We have learned that the organization of structural proteins at the sarcolemma, and especially at costameres, is affected in several forms of muscular dystrophy. Although most of our studies to date have focused on an animal model for Duchenne Muscular Dystrophy, we are now applying these findings to Facioscapulohumeral Muscular Dystrophy, an autosomal dominant disease that affects 1 in 20,000 Americans