Studying exit and surface delivery of post-golgi transport intermediates using in vitro and live-cell microscopy-based approaches

This chapter examines exit and surface delivery of post-golgi transport intermediates using in vitro and live-cell microscopy-based approaches. The most important advance provided by cDNA microinjection and adenoviral-mediated gene transfer in examining protein-sorting events is the ability to co-introduce two or more genes into cells and express simultaneously multiple secretory cargoes that follow divergent routes out of the TGN. This allows one to evaluate the role(s) of potential molecular effectors of protein sorting and targeting to different cellular domains. While cDNA microinjection is limited with respect to the number of cells that can be evaluated, it is exquisitely suited to live cell imaging studies aimed at evaluating highly dynamic membrane trafficking events occurring at specific points throughout the biosynthetic pathway. Semi-intact MDCK cells are prepared after accumulating marker proteins in the TGN. Cells are first swollen in a low salt buffer and are subsequently scraped from the substratum, which produces large tears in the plasma membrane.