This project is awarded under the Minority Postdoctoral Research Fellowships and Supporting Activities Program for 2006.
Genetic and Biological Analysis of AMPKa Signaling on Drosophila Peripheral Nervous System Development and Interaction with upstream modulator LKB1
Information processing in the nervous system occurs as external and electro-chemical signals are received and propagated through neuronal dendrites and axons, respectively. The dendrite subcellular compartment contains 2 largely separate cytoskeletal domains; a dendrite shaft enriched for microtubules, and later forming dendritic filopodia enriched for filamentous actin (F-actin). In order to identify in vivo molecular regulators of dendrite structure, a forward genetic screen for mutants defective during dendrite development was performed. The Drosophila sensory neuron dendrites of the peripheral nervous system was analyzed due to the unparalleled ability to visualize these large, stereotyped and highly branched dendrites. A high-resolution visual screen of greater than 4,000 distinctly mutagenized lines expressing an actin-GFP fusion protein in subsets of sensory neurons in transgenic animals was conducted. 2nd instar larvae from independent recessive lethal lines were visually screened for dendrite development defects to isolate mutants. Multiple alleles with distinct mutations in the single Drosophila gene encoding the a subunit of AMP-activated protein kinase (dAMPKa) were isolated. AMPKa is an evolutionarily conserved protein and belongs to a family of serine/threonine protein kinases that regulate cell polarity and microtubules. AMPKa itself is regulated by upstream kinases including CaM kinase kinase (CaMKK) and LKB1. One of the goals of this project is the characterization of the first known Drosophila mutants of AMPKa through genetic and cell biological analyses of mutant and transgenic animals. In depth characterization of the dendritic phenotypes of these mutants will be performed with particular focus on microtubule regulation. The role of LKB1 signaling on dendrite development independently and genetically with AMPKa mutants will also be explored. In order to fully understand the breadth of LKB1 signaling loss-of-function, constitutively active and inactive Drosophila lkb1 (dlkb1) mutants for possible dendritic phenotypes, as well as lines over-expressing LKB1 (UAS-dlkb1), will be investigated first. Each of these lines will then be used to test if there is a genetic interaction between dlkb1 and dAMPKa by examining the resulting trans-heterozygous dendritic phenotypes. A test for a genetic interaction between dAMPKa and dlkb1 by studying Drosophila mosaic eye clones will also be carried out. During the implementation of this project Dr. Swick will gain experience beyond laboratory training in Drosophila molecular genetics and neuroscience. The sponsoring lab was chosen precisely for its expertise in molecular neuroscience and Drosophila genetics, however, numerous core facilities and seminar series exist at this institution to support his research, training, and professional goals. It is envisioned that this postdoctoral fellowship will help Dr. Swick in becoming a successful neuroscientist. This post-doctoral research at UNC will help in getting him skilled at more techniques and broaden his knowledge base and view of the developmental neuroscience field. As his career progresses he plans to remain in neuroscience and continue to do research with long term applications in neurodevelopment and brain dysfunction.
