Functional analyses of dynamin GTPase on early steps of endocytosis

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The following results were obtained in the term of current project. 1. Combined morphological and biochemical analyses of dynamin mutant cells : We have analyzed three dominant-negative mutants of dynamin, K44A, S45N, and T65F. All three mutants lacked binding capacity for both GTP and GDP, and biochemically indistinguishable. However, electron microscopic analyses revealed the each mutant showed the accumulation of unique morphological intermediates in transformed cells. These results indicated that these mutations in dynamin may inhibit the endocytosis through unknown dynamin's function not through well-known GTP/GDP-dependent fission steps. 2. Ultrastructural study of lysenin : Lysenin is a sphingomyelin-specific binding protein which induce hemolysis. We have identified the unique hexagonal oligomer on sphingomyelin-containing liposomes. The similar structures were found on the surface of lysenin-treated erythrocytes. These results indicated that lysenin induced hemolysis by pore-forming oligomer on the cell surface. 3. Analyses of in vitro reconstitution of synaptic vesicle formation : We have succeeded in reconstitution of the synaptic vesicle formation by incubating liposomes with brain cytosol, ATP and GTPγS. We then analyzed the effect of various lipid composion of liposomes on the rate of vesicle formation. We found that PIP2 accumulate the formation of vesicles. The accumulation of vesicle formation persisted after rapid degradation of PIP2. These results indicated that PIP2 may recruited various coat proteins to the liposomes required for vesicle budding.