Ph.D Thesis

Ph.D StudentShapira-Rootman Mika
SubjectFormation of Presynaptic Active Zones - Involvement of
Precurser Vesicles
DepartmentDepartment of Medicine
Supervisor PROF. Noam Ziv


The active zone (AZ) is a highly specialized region of the presynaptic plasma membrane where synaptic vesicles dock, fuse and release their neurotransmitters into the synaptic cleft. 

In spite of enormous progress made toward the molecular elucidation of AZ structure and function, the cellular and molecular mechanisms that underlie the formation of AZs during synapse formation process, as well as the mechanisms that underlie the recruitment of additional presynaptic molecules to the nascent AZs are not well understood.

Biochemical studies led to the identification of an 80 nm dense-core vesicle that carries a comprehensive set of presynaptic proteins (Zhai et al, 2001). It has been hypothesized that the vesicle (termed PTV) is in fact a preassembled 'ready for use' synapse. Accordingly, its fusion with the plasma membrane may lead to immediate formation of the presynaptic active zone. Time-lapse experiments revealed that PTVs are highly mobile and may move in axons at velocities of up to 0.35 µm/sec (average velocity ~0.1 µm/sec). In addition, PTVs were observed to travel along axons in both directions. They occasionally stopped, merged with one another to form a larger cluster, split into smaller clusters and so on. Next we examined how many PTVs are required for the formation of a single active zone. Immunocytochemical studies revealed that two to three PTVs are involved in the formation of a single active zone. We also found the PTVs' population is heterogenous in nature: the quantities of different proteins carried on PTVs are not fixed and not all PTVs necessarily contain all of the active zone proteins.

Afterwards we examined the exclusiveness of the PTV in active zone formation. Accordingly, we chose two proteins known to be carried on PTVs: RIM1 and Rab3A. For each of these proteins we tried to answer the following questions: 1. Can the PTV deliver this protein to most of the synapses? 2. Can the PTV deliver enough copies of the protein to synapses? The answer for both of the proteins was positive. In the final chapter of this work we examined PTV's involvement in synaptic maintenance. In order to do so, we expressed the chimeric protein GFP:Bassoon in live hippocampal neurons, and used fluorescence recovery after photobleaching to study the dynamics of active zone replenishment. Interestingly, we did not manage to demonstrate the involvement of PTV's in synaptic maintenance. Apparently, PTVs are involved mainly  in synapse formation.