Datensatz

Zusammenfassung

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 degrees C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37 degrees 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 fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Video-microscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 microm/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 fluorescent 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.