Toomre D, Keller P, White J, Olivo J C, Simons K
Cell Biology/Biophysics Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, Germany.
J Cell Sci. 1999 Jan;112 ( Pt 1):21-33. doi: 10.1242/jcs.112.1.21.
The mechanisms and carriers responsible for exocytic protein trafficking between 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 reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.
负责反式高尔基体网络（TGN）与质膜之间胞吐蛋白运输的机制和载体仍不清楚。为了研究TGN到质膜运输的动力学以及细胞骨架在这些过程中的作用，我们用绿色荧光蛋白融合蛋白、标记有绿色荧光蛋白的水泡性口炎病毒G蛋白（VSVG3-GFP）转染细胞。在内质网中利用温度变化阻断VSVG3-GFP并随后使其在TGN中积累后，通过共聚焦和视频显微镜实时观察TGN到质膜运输的动力学。小囊泡（<250 nm）和较大的囊泡管状结构（>1.5微米长）均用作运输容器（TCs）。这些TCs以平均约0.7微米/秒的速度沿着曲线路径迅速移出高尔基体。自动计算机跟踪客观地确定了不同载体的动力学。观察到TCs的分裂和融合，表明这些晚期胞吐过程具有高度的相互作用。为了直接确定微管在高尔基体后运输中的作用，显微注射罗丹明微管蛋白，并在活细胞中同时观察标记的货物和微管。这些研究表明，胞吐货物沿着微管轨道移动，并揭示载体能够在轨道之间切换。
