The goal of this project is to understand how mRNAs, proteins and subcellular organelles are distributed and organized in nerve and muscle cells. In muscle, where cells are multinucleated, the retention of some mRNAs and proteins near the nucleus that produces them seems to contribute to the formation of functional domains. We are examining the role of mRNA lifetime in this process are using the mouse muscle cell line C2. In this experimental system, we are planning to manipulate the lifetime of specific endogenous mRNAs and of foreign mRNAs introduced by DNA transfection. In parallel, we plan to examine the parameters that contribute to the segregation of specific mRNAs between cell body, axon and dendrites in polarized nerve cells. We are characterizing primary cell cultures and neuronal cell lines that acquire polarity in culture and developing protocols to express foreign genes in these cells. We are also investigating the mechanism of vesicle traffic responsible in muscle for the increase in glucose transport following stimulation by insulin or exercise. It is generally believed that, upon stimulation, the intracellular vesicles carrying the glucose transporter GT4 are translocated to and fuse with the cell membrane. We have been studying the localization of GT4 at high resolution in single fibers of the rat soleus muscle, by a combination of classical and confocal immunofluorescence microscopy and by immunogold electron microscopy. We have developed a protocol for pre~embedding immunogold staining of the fibers with which we have detected about a 10~fold increase in the number of GT4 immuno~reactive sites in the membrane of insulin~stimulated fibers. Our results clarify several discrepancies in this field. Since several proteins involved in the traffic of synaptic vesicles have counterparts in vesicles in non~neural cells, we are now searching muscle, both at RNA and protein levels, for expression of synaptic vesicle proteins. Initial results suggest, surprisingly, that two synaptobrevins, hitherto considered neural~specific, are actually expressed in muscle, making a stronger case for a general vesicle traffic mechanism.
