We study the cellular and molecular mechanisms involved in development of the mammalian neuromuscular junction 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 its function is unknown. 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 are now studying the program for packaging, transport and secretion of agrin in motoneurons by expression of recombinant agrin and agrin-green fluorescent protein (agrin-GFP). We have 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 found that the expression of the integral membrane form of agrin in skeletal muscle and other cultured cells induces the formation of filopodia. We now plan to examine the mechanism of this effect and the possible role of integral membrane agrin in the remodeling of cell processes. 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 association of calcineurin with some putative target molecules in skeletal muscle and other cell types.
