Noreen Reist - US grants

Most communication between neurons in the brain occurs by the regulated secretion of a chemical transmitter at sites of contact, called synapses. Functional studies have shown that the synaptic protein synaptotagmin in lethal. Therefore, many biochemical studies have been conducted to characterize the interactions of synaptotagmin with other synaptic proteins in test tubes. However, before one can understand how synaptotagmin regulates synaptic transmission, the functional significance of these biochemical interactions must be determined in animals. Since synaptic proteins are highly conserved in fruit fly, Drosophila, this project combines the advantages of Drosophila genetic mutations know to disrupt specific protein interactions in test tubes, the investigators will establish the functional importance of the specifically mutated regions of synaptotagmin through a detailed analysis of synaptic structure and function in transgenic Drosophila.

Learning and memory, as well as various neurological disorders are thought to involve changes in the efficacy of transmitter secretion. A thorough understanding of the molecular interactions mediating synaptic transmission is prerequisite for the development of therapies to enhance neural function. This study will disclose the functional importance of specific interactions of synaptotagmin, a protein vital for regulating synaptic transmission.

DESCRIPTION (provided by applicant): Communication among neurons occurs primarily at chemical synapses. To identify the molecular mechanisms mediating synaptic transmitter release, biochemical techniques have been used to screen nerve terminal proteins for interactions. Our long term goal is to test the physiological relevance of hypotheses based on these biochemical interactions, using site-directed mutagenesis with electrophysiological and ultrastructurat techniques. This knowledge will be prerequisite to understanding neurodegenerative diseases involving defective neurotransmission. We use the model system of Drosophila, since synaptic proteins are highly conserved, yet Drosophila provide an inexpensive, rapid, in vivo system, for mutagenic studies. Here we propose a detailed structure/function analysis of synaptotagmin, a key presynaptic molecule for regulating synaptic transmission. Synaptotagmin is an integral membrane protein located on synaptic vesicles and has a vast literature describing its biochemical interactions with other nerve terminal molecules in vitro. Initial studies have demonstrated certain binding motifs of synaptotagmin are essential for synaptic function. Using molecular techniques to construct specific mutations, we will establish the function of these motifs in vivo through a detailed morphological and physiological analysis in transgenic Drosophila. Specifically, we will address the following issues: 1) Which residues of the C2B Ca2+-binding motif are critical for synaptic transmission? By mutating individual amino acids within this motif, we will determine the relative importance of each for synaptic function. 2) Are any of the residues of the C2A Ca2+-binding motif necessary for synaptic transmission? By mutating multiple amino acids within this motif, we will determine whether any are important for synaptic transmission. 3) Is phospholipid binding by the C2B domain required for synaptotagmin function? By mutating residues that mediate phospholipid binding by C2B, we will determine the relevance of this interaction in vivo. 4) Do the C2A and C2B polylysine motifs mediate independent functions? By mutating both motifs simultaneously, we will determine whether these motifs mediate independent or coordinated functions.