Colocalization of synapsin I and Munc13 within presynaptic axon terminals of the earthworm neuromuscular junction. Michael P. Tekin and William L. Coleman Department of Biological and Allied Health Sciences Bloomsburg University, 400 E. 2 nd St. Bloomsburg PA Abstract: Synapsins are a group of proteins classically thought to regulate synaptic vesicle cycling well “upstream” of vesicle exocytosis. Several previous studies however have suggested that synapsin I may have multiple regulatory roles, including functions closer in time to the actual release event. This led to the hypothesis that synapsin I may interact with the major vesicle priming protein Munc13. Synapsin I / Munc13 interaction was investigated using double immunofluorescence staining at earthworm (Lumbricus terrestris) neuromuscular junctions. Earthworms were anesthetized by soaking in 95% ethanol until cessation of motor activity and pinned ventral side down to a dissecting tray. The dorsal integument was bisected longitudinally and the ventral nerve cord and longitudinal muscle fibers were exposed by removing any overlaying organs. Sections of tissue were then transferred and pinned to a Sylgard filled dish. Throughout dissection, tissue was moistened with a saline solution that mimics normal extracellular fluid. Tissue was then fixed with 2% paraformaldehyde and processed using standard methods for fluorescence immunohistochemistry. Fluorescent images were taken using an inverted epifluorescence microscope, and analyzed using freely available ImageJ software with the colocalization plug-in JACoP to obtain the Pearson coefficient. The Pearson coefficient indicated strong colocalization at rest (average R = 0.883±0.005). This high degree of colocalization suggests that synapsin I and Munc13 may interact at rest. Future studies will investigate how this colocalization may change during synaptic activity. Background:  Synapsins are a very abundant synaptic protein and regulate the release of synaptic vesicles. Rationale/Hypothesis: General Methods: Result: Conclusion: Future Directions:  In future studies these techniques will be used to determine if colocalization of synapsin I and Munc13 changes during synaptic activity.  The interactions of Munc13 with other synapsins, such as synapsin II, can be investigated using similar methods. References: Acknowledgements:  Department of Biological and Allied Health Sciences, Bloomsburg University. The Pearson Coefficient demonstrates a high level of colocalization in the neuromuscular junction in the unexcited state (average R = 0.883±0.005). ABAB Figure 3. The images of these synapses from worm tissue were obtain from using a Photometrics CoolSNAP ES2 monochromatic camera and an inverted epifluorescence microscope. The tissue was stained using fluorescence immunohistochemistry. The red fluorescent dye targeted Munc13 (I), and the green fluorescent dye targeted Synapsin I (II). The yellow images (III) are overlays of the corresponding synapsin I and Munc13 staining. Both sets of images demonstrate strong colocalization in the neuromuscular junction at the unexcited state (Fig3A, R=0.915, Fig3B, R= 0.956) I II III There is a high level of colocalization of synapsin I and Munc13 in the unexcited state, suggesting that these proteins may interact at rest. Both Munc13 and synapsin I play an important role in the releasing of neurotransmitters. Munc13 regulates exocytosis through vesicle priming. Synapsin I regulate vesicles cycling and may alter the timecourse of vesicle release. Synapsin I may mediate these effects on neurotransmitter release by interacting with Munc13. Live tissue was obtained from earthworms (Lumbricus terrestris). The worm was anesthetized with 95% ethanol solution and then longitudinally opened on the dorsal side. Next, the digestive and reproductive organs were removed, leaving the longitudinal muscle fibers and ventral nerve cord intact. After the live tissue was thoroughly cleaned using a modified Drewes-Pax solution sections of tissue were pinned to a sylgard filled dish. The tissue was then fixed using 2% paraformaldehyde solution. Fluorescence immunohistochemical techniques were then used to stain Munc13 and synapsin I. Pictures of the results of these florescence immunohistochemistry methods were taken with a Photometrics CoolSNAP ES2 monochromatic camera and an inverted fluorescent microscope. The JACoP colocalization plug-in (Bolte and Cordelieres 2006) in ImageJ (Rasband ) was used to measure the colocalization of Munc13 and synapsin I by obtaining the Pearson coefficient of individual synapses. The average Pearson coefficient and standard was calculated with Microsoft Excel.  Munc13 regulates the release of neurotransmitters by priming the synaptic vesicles. synapsin N.T. actin synaptic vesicle presynaptic terminal Ca2+ CaM CaM kinase phosphorylated synapsin p p p Figure 1. Left- When the presynaptic terminal is at rest, synapsin binds to both actin and the synaptic vesicles. When the presynaptic terminal is excited voltage gated Ca 2+ channels open, and Ca 2+ enters the cell causing the release of neurotransmitter through exocytosis. Ca 2+ also activates secondary messengers like calcium/calmodulin dependent protein kinases. Right- CaM kinase then phosphorylates synapsin, which allows the protein and the synaptic vesicles to dissociate. The synaptic vesicles then can release their neurotransmitters through exocytosis. Figure 2. Synaptic vesicles are tethered to the presynaptic membrane by various SNARE proteins that are essential for vesicle fusion. The “fusion readiness” of synaptic vesicles is determined by the priming state of the SNARE complex- this is mediated in part by Munc13. Bolte S and Cordelieres P (2006). A guided tour into subcellular colocalization analysis in light microscopy. Journal of Microscopy 224, Rasband WS, ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,