PERTURBATIONS OF THE VESICLE CYCLE IN RETICULOSPINAL SYNAPSES IN LAMPREY AFTER PRESYNAPTIC MICROINJECTIONS OF
GTPγS
N. V. Tomilin, E. V. Vasilyeva, A. V. Gilarov, T. F. Chernyak
Institute of Toxicology of Federal Medico-Biological Agency, St. Petersburg;
e-mail: Nikolay_Tomilin@rambler.ru
The synaptic vesicle cycle sustains neurotransmission and keeps pace between exo- and endocytosis in synapses.
GTP-binding proteins function as key regulators of this cycle. The large GTPγSe dynamin is implicated in fission of
clathrin-coated vesicles from the presynaptic membrane during endocytosis. The present study addresses the effect of
the non-hydrolysable GTP analog, GTPγS, on the assembly of the dynamin fission complex in situ. Intraaxonal
microinjections of GTPγS induced distinct ultrastructural changes in synapses: the number of synaptic vesicles at
active zones was reduces, and the number of docked vesicles was increased; at the same time the number of
clathrin-coated intermediates at the synaptic endocytic zone was increased, indicating that synaptic vesicle recycling
was inhibited. Clathrin-coated intermediates with unusual shape were found. At low concentrations of GTPγS they
were represented by long tubules decorated by spirals containing dynamin and clathrin-coated vesicles on the top. At
high concentrations of GTPγS the tubulular structures were shorted and branched. The pitch of the spiral and
tubule's diameter were significantly reduced (23.1 ± 0.4 and 19.0 ± 0.5 nm, respectively, as compared to those at
low concentration of GTPγS, 26.6 ± 0.4 and 23.3 ± 0.4 nm; P < 0.001). We suggest that these structural changes
correspond to distinct steps in the fission reaction. A model is proposed. It implies that the fast GTP hydrolysis
leads to an increase in length of the spiral due to the straightening of the dynamin dimmers, composing the spiral.
This leads to a fast increase both in the pitch and the diameter of the helix. The shift in diameter breaks the local
hydrophobic interactions between the inner and the outer leaflets of the lipid membrane at the sites of dynamin
binding. Stretching of the spiral leads to an expansion of the neck in the longitudinal direction and promotes
severing of the membrane that subsequently results in the release of the clathrin-coated vesicle.
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