Cytoskeletal mechanisms in synaptic vesicle recycling

Abstract: This thesis explores cytoskeletal mechanisms involved in the recycling of synaptic vesicles. As an initial step it characterizes synapses formed by giant reticulospinal axons in lamprey and defines their use as a model system. The most common type of reticulospinal synapse, "simple synapse", appeared to be most suitable as an experimental model. The majority had active zones with a diameter of 0.8-1.8 gm. The number of synaptic vesicles in the middle section of serially cut synapses correlated with the active zone length within this size range. This type of synapse comprised about 75- 80% of the total number of reticulospinal synapses in the trunk region of the lamprey spinal cord. It was axo-dendritic and composed of a single active zone. Two other types of reticulospinal synapse were also identified. They were less suitable as experimental models due to their complex organization. The "simple" type of synapse was further used in microinjection studies focused on the role of the cytoskeleton in the region surrounding the active zone ("endocytic zone") where synaptic vesicles are recycled via clathrin-mediated endocytosis. Microinjection of GTPgammaS in axons followed by stimulation induced clathrin-coated endocytic intermediates with elongated necks extending from the plasma membrane of the endocytic zone. The necks of these structures were decorated with dynamin- like rings or spirals. A thin filamentous cytomatrix, which was visible in the endocytic zone of stimulated control synapses, also showed a marked proliferation after microinjection of GTPgammaS. Microinjection of compounds expected to increase the levels of phosphorylated inositolphospholipids (antibodies and a peptide perturbing synaptojanin function) also inhibited synaptic vesicle recycling and induced accumulation of free clathrincoated vesicles around the synapse. In addition, they caused proliferation of a cytomatrix in the endocytic zone similar to the one induced by GTPgammaS. The filamentous cytomatrix was characterized by microinjecting compounds directly interfering with actin function. Microinjection of phalloidin caused the appearance of brightly fluorescent rings around synapses at the light microscopic level. At the EM level, in unstimulated phalloidin-injected axons filaments formed a thin cytomatrix adjacent to the plasma membrane of the endocytic zone. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. After injection of the catalytic subunit of C. botulinum C2 toxin the filaments could not be detected. Injection of NEM-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. In addition, both phalloidin and C2 toxin affected the shape of coated pits, suggesting that actin may also be involved in clathrin-mediated membrane retrieval. This thesis provides further insight into the ultrastructural organization of the lamprey reticulospinal synapse. Using this model synapse, it shows an involvement of the actin cytoskeleton in synaptic vesicle recycling, and indicates that GTPases and phosphoinositides take part in regulation of its function in the endocytic zone.