1. Localization of a GFP/Botulinum Neurotoxin A Gene Fusion Product Within Mouse N2A Cells Matt Linsey York College of Pennsylvania, Department of Biological Sciences Abstract The extremely lethal Botulinum Neurotoxin type A (Botox A) is produced by the bacteria Clostridium botulinum. This neurotoxin’s mechanism of cellular binding is not fully known and its specific receptor has not yet been identified. In this study, mouse N2A (neuronal) cells were transfected with a GFP/Botox A gene fusion product to study the neurotoxin’s localization within the cell. The successfully transfected cells were viewed under a fluorescent microscope for the presence of the GFP, and thus Botox A. Preliminary data suggested the cells maintained a normal morphology, but the fusion protein was localized within the cell around the cellular membrane. Our results suggest that the GFP tag did not compromise the native function of the Botox A protein. This new fusion protein is, thus, a safer alternative to studying Botox A than using a radioactive labeled intact neurotoxin. Introduction Botulinum neurotoxins are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses (Singh 1999). Botulinum neurotoxins are composed of a lethal light chain (50 kDa) and a binding/internalizing heavy chain (100 kDa) linked by a disulfide bond. Binding of the toxin involves attachment of the carboxy-terminus of the heavy chain of the toxin molecule to receptors on the presynaptic side of the synapse (Habermann 1986). Although a large amount of data is available on the binding properties of Botox A, specific proteinaceous receptors with functional significance have not yet been identified in the nervous system for the toxin (Herreros 1997). Our research study was aimed at supplying some new information on this aspect of the toxin by using a Green Fluorescent Protein (GFP)/Botox A carboxy-terminus gene fusion product to study the binding of the neurotoxin in vitro in mouse N2A cells. The GFP serves only as a molecular tag to visualize where Botox A is within the neuron. Since the function of the Botox A carboxy- terminus is to bind to the cell membrane, we hypothesized that the translated fusion protein would localize to the cell membrane. The following questions were addressed by this study: 1)Does the GFP/Botox A translated protein localize within the cell, and if so, where? 2) Does the GFP alter the function of the carboxy- terminus of the heavy chain from its native function? Methods Culture N2A Cells Prepare GFP/Botox A Gene Fusion Vector Transiently Transfect N2A Cells Fix With Paraformaldehyde and DAPI Take Pictures of Successful Transfectants Results The transient transfections of the N2A cells were successful. When viewed under the fluorescent microscope, the negative control group (N2A cells transfected with water) did not show any fluorescent glow (data not pictured). However, the GFP transfected control group did show fluorescence and this fluorescing light was seen throughout the entire cytoplasm of the cell (Figure 1). The GFP/Botox A transfected cells showed the presence of fluorescence as well, but these cells seemed to have the fluorescence maintained around the cell membrane as opposed to throughout the entire cell (Figure 2). Future Studies Follow-up studies of this research could address several aspects of this promising gene fusion product. One could be to stably transfect the N2A cells instead of just transiently transfecting the cells, which could aid in determining how efficiently the fusion protein is expressed. Another study could be done using the GFP/Botox A gene fusion product in an attempt to identify the proteinaceous receptor that the toxin binds to. If this knowledge was obtained, subsequent research could be started on drugs to inhibit the toxin’s binding to the receptor, thus eliminating its lethal effects. This would eliminate the threat of Botox A as a potential biological weapon. Figure 1: GFP transfected control N2A cells showing total cytoplasmic fluorescence Figure 2: GFP/Botox transfected N2A cells showing “halo” fluorescence around the cell membrane. Discussion The obtained results from the transfections suggest a few things. First of all, by comparing the presence of fluorescence in the GFP control group and the GFP/Botox A experimental group cells to the negative control group cells, it is plausible to conclude that the cells did in fact get successfully transfected and were expressing the gene vectors. Secondly, by comparing the patterns of fluorescence between the GFP transfected N2A cells and the GFP/Botox A transfected cells, we have concluded that the translated GFP/Botox A protein did indeed localize to the cell membrane of the cells. This is different than the fluorescence pattern of just the GFP transfected cells, which suggests that the “halo” localization pattern seen within the GFP/Botox A transfected cells is due to the presence of the Botox A carboxy-terminus. This “halo” localization pattern also suggests that the function of the Botox A carboxy-terminus was not compromised by the GFP. These results could be used to the advantage of researchers studying Botox A. Currently, many of the binding and internalizing studies of the Botox A toxin are done using a radioactive probe to visualize the Botox A within the cell. Working with radioactive probes present many major health risks to researchers. But by utilizing this GFP/ Botox A gene fusion product, researchers could obtain the same results without the health dangers because the GFP is a non-toxic molecule. Conclusions 1) The N2A cells were successfully transfected by the GFP/Botox A gene fusion product. 2) The translated GFP/Botox A protein did localize within the N2A cells and this was seen to be to the cell membrane. 3) The GFP did not alter the native function of the Botox A carboxy- terminus. Literature Cited Haberman, E., Dreyer, F. 1986. Clostridial Neurotoxins: Handling and Action at the Cellular and Molecular Level. Curr. Top. Microbiol. Immunol. 129; 93-179. Herreros, J., Marti, E. 1997. Localization of Putative Receptors for Tetanus Toxin and Botulinum Neurotoxin Type A in Rat Central Nervous System. European Journal of Neuroscience 9; 2677-2686. Singh, BR., Li, L. 1999. In Vitro Translation of Type A Clostridium Botulinum Neurotoxin Heavy Chain and Analysis of Its Binding to Rat Synaptosomes. Journal of Protein Chemistry 18; 89-95. Acknowledgements Dr. Jeffrey Thompson (Dr. T) Ph.D