We study the cellular and molecular mechanisms involved in development of the mammalian neuromuscular junction, central nervous system synapses and the differentiation of skeletal muscle cells, utilizing cell culture, microscopy and molecular techniques. Postsynaptic acetylcholine receptor aggregation is a critical early event in neuromuscular junction formation. Agrin, a proteoglycan secreted by motoneurons, is required for postsynaptic differentiation in muscle. A transmembrane form of agrin is widely expressed in the central nervous system and there is evidence for its involvement in neurite outgrowth as well as synapse formation and function. We are studying the program for packaging, transport and secretion of agrin in motoneurons and hippocampal neurons by expression of recombinant agrin and agrin-green fluorescent protein (agrin-GFP). We previously found that the transmembrane form of agrin-GFP is transported into both dendrites and axons in a different compartment from synaptic vesicle proteins. However, like endogenous agrin, it is targeted predominantly to the membranes of distal portions of axons. The secreted form of recombinant agrin is similarly targeted. We have now compared the targeting of full-length and truncated forms of recombinant agrin in both motor neurons and hippocampal neurons in order to determine the sequences involved. We found that the N-terminal moiety of agrin is required for targeting to the axon growth cone, with a contribution from the N-terminal cytoplasmic tail. The C-terminal moiety is not sufficient for targeting to the axon growth cone but may contain additional targeting information. We previously found that the expression of the integral membrane form of agrin in skeletal muscle and other cultured cells induces the formation of filopodia. We are using truncated agrin-GFP constructs and mutant muscle cell lines to examine the mechanism of this effect. Our results suggest that the N-terminal moiety of transmembrane agrin is sufficient to induce filopodia formation. We are now investigating the roles of agrin?s glycosaminoglycan side chains and the possible interaction with growth factors or their receptors in filopodia induction. We previously showed that the synthesis and assembly of slow myosin heavy chains in cultured skeletal muscle is dependent on depolarization/contractile activity and on the activity of the calcium/calmodulin activated phosphatase calcineurin. This effect appears to be largely mediated through dephosphorylation of the transcription factor NFAT by calcineurin, but other effects of calcineurin are probably mediated through other phosphatase substrates in skeletal muscle. We have now localized calcineurin to novel sites in skeletal muscle cells where it may influence development and contractility through the dephosphorylation of these substrates.
