FUNCTIONS OF DYNAMIN AND DYNAMIN RELATED PROTEINS

University Of California Los Angeles, Los Angeles CA

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Abstract

Our research is focused on the roles of dynamin and dynamin-related proteins. In the first funding period, we identified and characterized the C. elegans dyn-1 locus, which encodes a homologue of human dynamin. A temperature-sensitive mutation in the GTPase domain causes rapid and reversible paralysis. Dynamin is enriched in synapses where it is important for synaptic vesicle recycling. The membrane localization of dynamin has been attributed to interactions with SH3 proteins, such as amphiphysins found in mammals. GFP-dynamin chimeras were used to study the mechanisms of synaptic localization. Surprisingly, the dynamin GTPase domain is sufficient for strong localization, which suggests that novel interactions contribute to synaptic localization. This raises the question which of the many dynamin binding interactions are important for dynamin localization and function in vivo. In Specific Aim 1, Characterization of C. elegans Dynamin Binding Partners, we propose to investigate the interactions with the dynamin GTPase domain and the possible contributions of C. elegans amphiphysin homologues to membrane localization. Our use of C. elegans allows us to test the biological relevance of these interactions. The research was expanded to include functions of dynamin-related proteins in C. elegans and humans. We discovered that Drp1 is an important regulator of mitochondrial morphology. We hypothesize that Drp1 is a pinchase that severs mitochondria. This new role for a dynamin family member is very exciting, because the mechanisms that control mitochondrial distribution are largely unknown. In Specific Aim 2, Classic and Reverse Genetic Analysis of C. elegans drp-1, we propose to investigate how loss-of-function and dominant interference of drp-1 affect mitochondria in live animals. We aim to see drp-1 activity in real time. In Specific Aim 3, Cellular and Biochemical Analysis of Human Drp1, we propose to investigate the functions of human Drp1 and eventually reconstitute Drp1 with isolated mitochondria. Thus, we will test whether mitochondria and clathrin coated vesicles utilize similar constriction mechanisms.