Membrane Traffic In The Early Secretory Pathway
Transport of material between two adjacent membranes in the secretory pathway of mammalian cells involves at least four basic steps. First, secretory cargo is segregated from residents and concentrated into newly synthesized transport carriers. These are then transported towards the target membrane, where they subsequently dock and fuse with the target membrane to deliver their cargo. Finally, the transport machinery is recycled back to the donor membrane to be used for further rounds of transport. All four steps have been individually reconstituted in the past in simplified in vitro systems. This, together with yeast genetics and ultra structural analyses by electron microscopy has helped to identify the basic transport machinery of each step and characterize its biochemistry. The tight temporal and spatial coupling and organization of these four steps as it occurs in a living cell has however remained largely elusive. A further complication of the in vitro reconstitution systems is that carriers and membrane domains involved in transport have a transient lifetime in cells and are therefore difficult to isolate for the in vitro assays in a state that resembles that of their cellular context.
In order to overcome these problems we are using advanced fluorescence microscopy methods to characterize the kinetics and interactions of the molecules involved in each transport step in intact living cells. To complement this, we also use large scale approaches in living cells, such as the systematic localization of novel GFP-tagged proteins in living cells together with systematic gene knockdowns by RNA interference (RNAi), to identify and characterize comprehensively the molecules involved in the temporal and spatial regulation of the secretory pathway.