Labeling and dynamic imaging of synaptic vesicle-like microvesicles in PC12 cells using TIRFM

Total internal reflection fluorescence microscopy (TIRFM) was employed to study the trafficking and exocytosis of synaptic vesicle-like microvesicles (SLMVs) in PC12 cells. SLMVs were labeled with vesicular acetylcholine transporter (VAChT) tagged with enhanced green fluorescent protein (EGFP), which displayed punctuate distribution under TIRFM and confocal microscopy. Immunofluorescence analysis confirmed the colocalization of EGFP and VAChT. No significant difference was observed in the distribution or sorting of VAChT when fused either at the N- or the C-terminus. Thus, tagging with GFP does not appear to impair or change the traffic of the VAChT in PC12 cells. Under TIRFM, EGFP-labeled spots moved in a restrained fashion, which resembled that of secretory granules and underwent exocytosis upon stimulation. Together, these data indicate that EGFP-tagged VAChT can be used to explore SLMVs trafficking using TIRFM.