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Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein,Irene Wacker,Christoph Kaether,Andre

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein   (Citations: 73)
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Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB- GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20°C, fluorescent hCgB-GFP colocal- ized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37°C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorom- etry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t1/2 of 20-30 minutes. Video- microscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 μ μm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of flu- orescent vesicles ceased. Concomitantly, secretion of hCgB- GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter. SUMMARY
Published in 1997.
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