Role of the ubiquitin system in Drosophila muscle synaptogenesis and function

Role of the ubiquitin system in Drosophila muscle synaptogenesis and function ******BACKGROUND: Proper muscle development, innervation and function are critical to animal survival, including Drosophila. Our recent work has demonstrated an essential role for the ubiquitin system in regulating muscle innervation (neuromuscular (NM) synaptogenesis) and muscle function in flies. ***The ubiquitin system plays a pivotal role in controlling protein breakdown and trafficking, and thus affects most cellular functions. Key components of the ubiquitination cascade are the E3 ubiquitin ligases, responsible for ubiquitin conjugation to proteins, therefore tagging them for destruction or other fates, such as movement/traffic within cells. We first isolated the E3 ubiquitin ligase Nedd4 from flies (Drosophila, dNedd4). We then showed that while dNedd4 (dNedd4-short, dNedd4S) promotes NM synaptogenesis, a longer splice isoform of the protein (dNedd4-long, dNedd4Lo) inhibits synaptogenesis and impairs larval locomotion. Using genetic and biochemical analyses in flies we demonstrated that two unique regions in dNedd4Lo are responsible for these inhibitory effects. To identify proteins that interact with these unique regions, we performed mass spectrometry analysis using fly embryo extracts, and identified Drosophila amphiphysin (dAmph) and SH3PX1 as binding partners to dNedd4Lo. We then showed that dNedd4Lo regulates dAmph in muscle T-tubules to control muscle function. SH3PX1, a SH3-PX-BAR containing protein that is expressed both pre- and post- synaptically and in muscles, is known to regulate nerve function. Our preliminary results show that postsynaptic expression of dNedd4Lo leads to disappearance of SH3PX1, but How dNedd4-long regulates SH3PX1 function is unknown. We HYPOTHESIZE that dNedd4Lo inhibits SH3PX1 function pre and post synaptically and in muscles by ubiquitination and degradation of SH3PX1, thus impairing vesicular trafficking in nerve and muscle cells. Our OBJECTIVE is to decipher the biochemical and physiological nature of interactions between dNedd4Lo and SH3PX1 and how they regulate muscle innervation and function. ***Our SPECIFIC AIMS are:***1) To analyze Nedd4Lo-mediated binding to, ubiquitination of, and regulation of SH3PX1 stability in the synapse and muscle.***2) To compare the effect of dNedd4Lo overexpression to SH3PX1 loss in the pre and post synapse by analyzing synapse ultrastructure & function, and muscle function. ***3) To analyze the physiological significance of dNedd4Lo - SH3PX1 interactions by analyzing their genetic interactions.******SIGNIFICANCE: These studies will delineate the role of dNedd4 proteins and ubiquitination in regulating muscle innervation and function, effects essential for animal life.

Role of the ubiquitin system in Drosophila muscle synaptogenesis and function ******BACKGROUND: Proper muscle development, innervation and function are critical to animal survival, including Drosophila. Our recent work has demonstrated an essential role for the ubiquitin system in regulating muscle innervation (neuromuscular (NM) synaptogenesis) and muscle function in flies. ***The ubiquitin system plays a pivotal role in controlling protein breakdown and trafficking, and thus affects most cellular functions. Key components of the ubiquitination cascade are the E3 ubiquitin ligases, responsible for ubiquitin conjugation to proteins, therefore tagging them for destruction or other fates, such as movement/traffic within cells. We first isolated the E3 ubiquitin ligase Nedd4 from flies (Drosophila, dNedd4). We then showed that while dNedd4 (dNedd4-short, dNedd4S) promotes NM synaptogenesis, a longer splice isoform of the protein (dNedd4-long, dNedd4Lo) inhibits synaptogenesis and impairs larval locomotion. Using genetic and biochemical analyses in flies we demonstrated that two unique regions in dNedd4Lo are responsible for these inhibitory effects. To identify proteins that interact with these unique regions, we performed mass spectrometry analysis using fly embryo extracts, and identified Drosophila amphiphysin (dAmph) and SH3PX1 as binding partners to dNedd4Lo. We then showed that dNedd4Lo regulates dAmph in muscle T-tubules to control muscle function. SH3PX1, a SH3-PX-BAR containing protein that is expressed both pre- and post- synaptically and in muscles, is known to regulate nerve function. Our preliminary results show that postsynaptic expression of dNedd4Lo leads to disappearance of SH3PX1, but How dNedd4-long regulates SH3PX1 function is unknown. We HYPOTHESIZE that dNedd4Lo inhibits SH3PX1 function pre and post synaptically and in muscles by ubiquitination and degradation of SH3PX1, thus impairing vesicular trafficking in nerve and muscle cells. Our OBJECTIVE is to decipher the biochemical and physiological nature of interactions between dNedd4Lo and SH3PX1 and how they regulate muscle innervation and function. ***Our SPECIFIC AIMS are:***1) To analyze Nedd4Lo-mediated binding to, ubiquitination of, and regulation of SH3PX1 stability in the synapse and muscle.***2) To compare the effect of dNedd4Lo overexpression to SH3PX1 loss in the pre and post synapse by analyzing synapse ultrastructure & function, and muscle function. ***3) To analyze the physiological significance of dNedd4Lo - SH3PX1 interactions by analyzing their genetic interactions.******SIGNIFICANCE: These studies will delineate the role of dNedd4 proteins and ubiquitination in regulating muscle innervation and function, effects essential for animal life.