The postsynaptic cytoskeleton is a specialized feature of neuronal cytoarchitecture that participates in the organization of synapses, both at the muscle endplate and in the central nervous system (CNS). During the previous funding period, we have definitively characterized a structural component at the Torpedo electroplax cholinergic synapse which appears to be present on the cytoplasmic face of the membrane. This protein, which is designated Upsilon1, is 43,000 Mr when isolated from the electroplax. The homologous protein in muscle may play a role in limiting acetylcholine receptor vulnerability to the effects of myasthenic autoimmune antibodies. However, two independent but preliminary lines of investigation in our laboratory suggest that Upsilon1 is synthetized as a polypeptide of at least 53,000 Mr. We propose to verify that Upsilon1 is biosynthesized in a high molecular weight form by immunoaffinity partitioning of polyribosomes that synthesize Upsilon1, followed by their in vitro translation. We will utilize a highly sensitive assay, currently under development, to detect picogram quantities of Upsilon1 in order to monitor the levels of Upsilon1 in amphibians whose endplates have been observed to undergo alternating stages of sprouting and retraction, respectively, during months of winter hibernation and summer activity. We will also attempt to establish whether Upsilon1 exists in established tissue culture lines or primary muscle explants, and to evaluate the biosynthetic processing of Upsilon1 during experimental conditions that are relevant to muscle endplate development and maintenance. In the case of the CNS synapses, definition of the molecular constituents in the postsynaptic cytoskeleton or density still remains unclear. We will first evaluate three hypothetical structural constituents of both cerebral and cerebellar postsynaptic densities, i.e. tubulin, calmodulin and a 51,000-dalton calmodulin binding protein that we observe in the forebrain but find virtually absent in the cerebellum. Based upon these studies, we will attempt to identify which proteins are physically attached to tubulin, calmodulin and calmodulin binding proteins in deoxycholate extracted fraction from forebrain and cerebellum in order to identify novel putative markers for the CNS.
| Gysin, R; Flanagan, S D (1987) Alkylation of free sulfhydryls fortifies electroplax subsynaptic structures. J Neurochem 49:452-9 |
