UNIVERSITY OF ILLINOIS
Grant: $454,344 - National Institutes of Health - Sep. 30, 2009
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Award Description: The following two aims are designed to supplement our ongoing investigation into the role of tomosyn, which we have shown negatively regulates synaptic transmission at C. elegans synapses. Aim 1) Generate and characterize Drosophila tomosyn mutants. To validate and extend our previous findings from C. elegans, we will generate and characterize Drosophila tomosyn mutants. This characterization will address important questions not easily answered in C. elegans. Specifically, we feel that understanding the basis of the altered evoked response kinetics in Drosophila tomosyn mutants, will provide important insights into the spatiotemporal mode of regulation by tomosyn. At the completion of this aim we will have defined the synaptic phenotype of both up and down-regulating tomosyn levels at the Drosophila NMJ. We anticipate that the results of this study will provide invaluable new insights into the mechanism by which tomosyn regulates synaptic release by providing a more detailed electrophysiological analysis, including the evaluation of the calcium-dependence and kinetic changes of several release parameters as well as changes in short-term plasticity, that can not readily be performed in C. elegans. Furthermore, comparison between C. elegans and Drosophila tomosyn mutant phenotypes will allow us to identify the pertinent synaptic changes common to both organisms, thereby providing a framework upon which to build a solid mechanistic model of tomosyn function. Aim 2) Interestingly, similar changes in EJC kinetics and vesicle docking observed in flies subjected to D-tomosyn RNAi, have been observed in learning-defective Drosophila dunce mutants, in which cAMP-phosphodiesterase function is disrupted. Given the similarity in the synaptic phenotypes of D-tomosyn RNAI and dunce mutants, and the fact that the binding affinity of tomosyn for SNARE proteins is modulated by cAMP-dependent kinases (PKA and ROCK), we hypothesize that tomosyn may be an important effector of cAMP-dependent synaptic plasticity and learning. This hypothesis will be explored in the second aim by first examining the role of tomosyn in Drosophila learning using Drosophila tomosyn mutants generated in aim 1. The experiments proposed in aim 2 should provide a clear indication as to whether tomosyn plays a role in olfactory-associative learning, and will begin to address whether tomosyn functions in the cAMP signaling pathway. In addition to providing important insights in to the regulation of synaptic transmission and the basis for synaptic plasticity, this funding will provide employment opportunities for several highly qualified but otherwise unfunded personnel with Drosophila expertise, very well suited to complete the supplemental aims. These personnel have already generated significant preliminary data for this project.
Project Description: In the 10 days since receiving the ARRA funding, we have begun aim 1 of the proposed research, to generate a Drosophila (D-tomosyn) mutant which will be used to characterize the role of D-tomosyn in synaptic transmission and to explore it's possible role in cAMP-dependent learning. Two strategies have been developed to maximize the speed and likelihood of generating at least one and hopefully several D-tomosyn mutants in the next quarter of the funding period. 1) In the first approach, Drosophila tomosyn mutants will be generated by imprecise P-element excision, using standard methods 40,41. Briefly, we will generate loss-of-function mutants in Drosophila tomosyn by imprecise excision of P{EP}tomosyn[EP1359] or other transposon insertions already generated by the Berkeley Drosophila Gene Disruption Project, which has the goal of generating transposon insertions in every Drosophila gene 42. P{EP}tomosyn[EP1359] was chosen for our initial efforts because it is in the middle of the tomosyn gene, near an exon present in every predicted splice variant. Excision of this element therefore has high likelihood of disrupting tomosyn without removing any portion of the flanking genes. For excision, P-elements are mobilized by crossing insertion strains to a strain carrying transposase, and subsequent generations are screened by eye color and PCR for gene-specific deletions. We will screen for appropriate deletions using 3 pairs of PCR primers. 2) The second strategy uses FLP recombinase to excise FRT-bearing insertion in D-tomosyn and a flanking region to generate a large isogenic deletion. This approach enables the generation of small custom deletions with predictable endpoints, and should expedite the isolation of a D-tomosyn mutant. In addition, we are preparing wild-type Drosophila specimens for high-pressure freeze fixation in order to optimize fixation conditions in preparation for the morphometric analysis and immunoEM of wild-type and D-tomosyn mutant flies.
Jobs Summary: As a direct result of this ARRA funding, in the first 10 days since receiving the award, we have been able to reinstate Dr. Kevin Chen, a talented and experienced Drosophilist with the requisite expertise in electrophysiology, and immunohistochemistry and one of only a handful of researchers in the world able to dissect and record from synapses of fly embryos. Without this supplement, Dr. Chen's employment would have been terminated Sept 30th, ending what has been a very fruitful research contribution in Dr. Featherstone's research group. In addition in this, the first 10 days of the ARRA award, we have also asigned a research assistanship to a graduate student, Szi-Chieh Yu who has expertise in electron miscroscopy. Finally, two undregraduate students have been hired as lab aids to assist in the passgaing of fly stocks and in the preparation of reagents needed for fly husbandry. (Total jobs reported: 0)
Project Status: Not Started
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