| Popis: |
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 GTPase 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, GTPgammaS, on the assembly of the dynamin fission complex in situ. Intraaxonal microinjections of GTPgammaS 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 GTPgammaS they were represented by long tubules decorated by spirals containing dynamin and clathrin-coated vesicles on the top. At high concentrations of GTPgammaS 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 GTPgammaS, 26.6 +/- 0.4 and 23.3 +/- 0.4 nm; P0.001). 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. |
