"A Study of the Localization and Transport of a Tissue Plasminogen Activator (tPA) / Green Florescent Protein (GFP) Hybrid in Rat Hippocampal Neurons."

Student Researchers: Aaron Blackwell and John Hodges.

Faculty Advisors: Janis Lochner and Bethe Scalettar.

John Hodges, Janis Lochner and Aaron Blackwell in the biochemistry laboratory.

Tissue plasminogen activator is a serine-protease that has been implicated in axonal elongation and long-term potentiation. In order for tPA to perform either of these functions, it must be targeted to specific areas of the cell. In an effort to elucidate the targeting mechanism, we have constructed florescent hybrids consisting of a variety of tPA mutants coupled with either Enhanced Green Florescent Protein, Enhanced Yellow Florescent Protein, or Enhanced Cyan Florescent Protein. These constructs will be used in future studies to simultaneously visualize two proteins using florescence microscopy, and therefore compare relative distributions and dynamics within the cell.

We have also infected cultured rat hippocampal neurons with a tPA/EGFP hybrid and have begun characterizing the distribution of tPA/EGFP within these cells at different stages of development. We have found that tPA/EGFP distribution is similar to that seen previously in PC12 cells without the same level of build-up in the growth cone. There is a slight build-up in the growth cone of dendrites after 4 days in culture. We have also found that tPA/EGFP levels in the processes of 17 day old neurons appear to be lower than expected. Furthermore, we have found that tPA/EGFP does not appear to associate with the pre-synaptic protein synaptophysin.

Using live-imaging, we have also begun to analyze the dynamics of tPA/EGFP transport in regulated secretory granules in the neurites of rat hippocampal neurons. The overall motion within neurites we found to be non-random and while differences were observed between the rat hippocampal transport dynamics and those previously studied in model neuronal PC12 cells, the overall dynamics were comparable.