Visualization and trafficking of the vesicular acetylcholine transporter in living cholinergic cells

The present experiments investigated the trafficking of the vesicular acety lcholine transporter (VAChT) tagged with the enhanced green fluorescent pro tein (EGFP) in living cholinergic cells (SN56). The EGFP-VAChT chimera was located in endosomal-like compartments in the soma of SN56 cells, and it wa s also targeted to varicosities of neurites. In contrast, EGFP alone in cel ls was soluble in the cytoplasm. The C-terminal cytoplasmic tail of VAChT h as been implicated in targeting of VAChT to synaptic vesicles; thus, we hav e examined the role of the C-terminal region in the trafficking to varicosi ties. A C-terminal fragment tagged with EGFP appeared to be selectively acc umulated in varicosities when expressed in SN56 cells. Interestingly, the p rotein was not freely soluble in the cytosol, and it presented a punctate p attern of expression. However, EGFP-C terminus did not present this peculia r pattern of expression in a nonneuronal cell line (HEK 293). Moreover, the C-terminal region of VAChT did not seem to be essential for VAChT traffick ing, as a construct that lacks the C-terminal tail was, similar to EGFP-VAC hT, partially targeted to endocytic organelles in the soma and sorted to va ricosities. These experiments visualize VAChT for the first time in living cells and suggest that there might be multiple signals that participate in trafficking of VAChT to sites of synaptic vesicle accumulation.