We do basic research on the cellular and molecular mechanisms involved in development of the mammalian neuromuscular junction and the differentiation of skeletal muscle cells, utilising cell culture systems, microscopy and molecular techniques. Postsynaptic acetylcholine receptor aggregation is a critical early event in neuromuscular junction formation. Agrin is a proteoglycan that is secreted by motor neurons and is required for normal postsynaptic differentiation in muscle. 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 intrinsic program for packaging, transport and secretion of agrin in motoneurons with recombinant agrin-green fluorescent proteins (agrin-GFP) that we have generated. We have found that agrin-GFP is transported into both dendrites and axons, in a different compartment from synaptic vesicle proteins. However, secreted agrin-GFP is found predominantly on the distal portions of axons. The expression of specific myosin heavy chains (MHCs) is a key indicator of muscle fiber differentiation into the fast- or slow-twitch phenotype. We previously showed that the synthesis and assembly of slow MHCs is dependent on depolarization/contractile activity and on the activity of calcineurin. Transfection of myotubes with a constitutively active form of calcineurin markedly increases slow MHC protein while the calcineurin inhibitor, cyclosporin, blocks this effect. We have now demonstrated that the effect of calcineurin activation on slow MHC expression is largely mediated through the transcription factor NFAT, since transfection with VIVIT, a polypeptide that specifically blocks calcineurin?s activation of NFAT, largely blocks the effect of constitutively active calcineurin. However, other effects of calcineurin activation, such as the regulation of fast MHC expression, appear to be mediated through a different signal transduction pathway involving calcineurin.
