Dual-color visualization of trans-Golgi network to plasma membrane trafficalong microtubules in living cells

The mechanisms and carriers responsible for exocytic protein trafficking be tween the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticu lum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma me mbrane transport were visualized in real time by confocal and video microsc opy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 mu m long) are used as transport containers (TCs), These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of simil ar to 0.7 mu m/second. Automatic computer tracking objectively determined t he dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To d irectly determine the role of microtubules in post-Golgi traffic, rhodamine -tubulin was microinjected and both labeled cargo and microtubules were sim ultaneously visualized in living cells. These studies demonstrated that exo cytic cargo moves along microtubule tracks and reveals that carriers are ca pable of switching between tracks.