The targeted, differential localization of specific mRNAs and proteins into distinct domains is important for the functional specialization of cells in the nervous system. The establishment of this differential localization is a crucial part of the development of the cells of the nervous system. The goal of this project is to understand how domains are organized in muscle and neurons. Over the past years we have carried out experiments aimed at understanding how mRNAs are distributed. Although some mRNAs are found in neuronal dendrites, most mRNAs seem to be confined to the cell bodies, by a process which seems to require ongoing protein synthesis. We have examined the localization of ferritin mRNA in cultured rat hippocampal neurons. In control cultures, ferritin mRNA observed by in situ hybridization is confined to the neuronal cell bodies, but in cultures treated by protein synthesis inhibitors, it extends into neuronal dendrites. Experiments carried out on cultures treated with either of two protein synthesis inhibitors (cycloheximide and puromycin) on their own or together with an inhibitor of RNA transcription (DRB or actinomycin D) pointed to ribosome binding as the mechanism for retaining mRNAs in the cell bodies. However, experiments taking advantage of the specific sensitivity of ferritin mRNA translation to the iron concentration in the medium, pointed to the existence of an additional, saturable mRNA sink. These results provide a working model with which to interpret mRNA localization data. We are now focusing our efforts on the investigation of the changes in the distribution of the Golgi complex that take place during muscle differentiation. cDNA constructs encoding Golgi- localized enzymes tagged with the fluorescent protein GFP have been introduced into myoblasts of the muscle cell line C2. Permanently transfected cell lines have been cloned and characterized and will be used to follow the Golgi complex in live cells. Because the Golgi complex, in most cells, is positioned by microtubules converging onto the centrosome, we also plan to follow changes in the distribution of the centrosome during muscle differentiation. To that effect, C2 cell lines have been cloned that express a GFP-tagged centrosomal protein, pericentrin, under control of an inducible promoter. We are presently characterizing these cell lines. Preliminary results suggest that the Golgi complex changes that occur during differentiation resemble the changes that take place during mitosis, a very interesting parallel.
