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. An integral membrane form of agrin is widely expressed in the central nervous system but little is known about its function. We previously found that isolated motoneurons initially secrete agrin indiscriminately but progressively accumulate agrin around axons as they mature, indicating a developmentally regulated program for targeting of agrin secretion. We have been 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 found that the integral membrane form of agrin-GFP is transported into both dendrites and axons in a different compartment from synaptic vesicle proteins. However, it is targeted predominantly to the membranes of distal portions of axons. The secreted form of recombinant agrin is similarly targeted. We are now comparing the transport, targeting and secretion of full-length and truncated forms of recombinant agrin in order to determine the sequences involved in agrin trafficking. We have recently found that the N-terminal moiety of transmembrane agrin, but not the short cytoplasmic tail, is required for targeting to the axon growth cone. 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 now using truncated agrin-GFP constructs and mutant cell muscle cell lines to examine the mechanism of this effect. 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 calcineurin. This effect appears to be largely mediated through dephosphorylation of the transcription factor NFAT by calcineurin, but other effects of calcineurin are not mediated through NFAT. The calcium-calmodulin activated phosphatase calcineurin has multiple substrates in skeletal muscle. Dephosphorylation of these substrates may modulate development and contractile activity. We are now investigating the localization of calcineurin at sites containing some putative substrates in skeletal muscle and other cell types.
