Results

Synaptotagmin Antfbodies B&k Evoked Doplunlne Bslyd= We Sw To explore the function of synaptic vesicle proteins, initial experiments focused on the use of antibodies raised against the cytoplasmic domains of vamp, rabSA, synaptcphysin, synaptotagmin and SV2. lmmunoprecipitation experiments on a synaptic vesicle enriched fraction prepared from rat brain revealed that, in the absence of detergents, all five synaptic vesicle protein antibodii were independently capable of immunoprecipkatlng the full complement of vesicle proteins present in the starting material. The inability of a control antibody directed against an Aplysia antigen and a particular anti-rab9A potyclonal antiserum to precipitate the synaptic vesicte markers further confirmed the specificity of the immunoprecipitation results. The data, together with the previous characteritation of these antibodies, demonstrated that the antibodies recognized their respective epitopes on the cytoplasmic face of intact vesicles. In view of these properties, the antibodies were used in subsequent microinjection experiments to investigate the role of these proteins in vesicle function in PC12 cells.

Nerve growth factor (NGF) differentiated PC12 cells were microinjected with each of the synaptic vesicle antibodies, and their effects on synaptic vesicle trafficking were assayed by immunofluorescence microscopy 1 hr after injection. Microinjected cetts were identified by coinjection with either Cascade blue-conjugated bovine serum albumin (BSA) or Texas red-conjugated dextran. Figure 1 shows an example of a PC12 cell coinjected with Cascade blue and a polyclonal antibody raised against synaptotagmin. The injected PC1 2 cell displays extensive