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Visualization and Trafficking of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells
Author(s) -
Santos Magda S.,
Barbosa José,
Kushmerick Christopher,
Gomez Marcus V.,
Prado Vania F.,
Prado Marco A. M.
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0742425.x
Subject(s) - vesicular acetylcholine transporter , green fluorescent protein , microbiology and biotechnology , cholinergic , biology , synaptic vesicle , cytoplasm , cholinergic neuron , zebrafish , endosome , neurite , vesicle , biochemistry , neuroscience , choline acetyltransferase , membrane , intracellular , gene , in vitro
The present experiments investigated the trafficking of the vesicular acetylcholine transporter (VAChT) tagged with the enhanced green fluorescent protein (EGFP) in living cholinergic cells (SN56). The EGFP‐VAChT chimera was located in endosomal‐like compartments in the soma of SN56 cells, and it was also targeted to varicosities of neurites. In contrast, EGFP alone in cells was soluble in the cytoplasm. The C‐terminal cytoplasmic tail of VAChT has been implicated in targeting of VAChT to synaptic vesicles; thus, we have examined the role of the C‐terminal region in the trafficking to varicosities. A C‐terminal fragment tagged with EGFP appeared to be selectively accumulated in varicosities when expressed in SN56 cells. Interestingly, the protein was not freely soluble in the cytosol, and it presented a punctate pattern of expression. However, EGFP‐C terminus did not present this peculiar 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 trafficking, as a construct that lacks the C‐terminal tail was, similar to EGFP‐VAChT, partially targeted to endocytic organelles in the soma and sorted to varicosities. 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.