The ER-Golgi-Intermediate compartment : dynamics and cargo sorting studied by time-lapse video microscopy

Membrane trafficking in mammalian cells proceeds through several steps including sorting the correct components to be transported, packaging them into appropriate containers and conveying the carriers to the proper organelles with which they fuse. All these steps are tightly regulated by several machineries like coats, fusion proteins, motors, tethers, Rabs and other regulatory components. The investigation of the molecular mechanisms of these machineries unraveled the trafficking events taking place between different compartments; but these findings did not elucidate how organelles can persist and maintain their integrity in a constantly dynamic environment. Two major controversial models are constantly debated: the stability and the maturation of compartments. The stability model favors the notion that compartments are long-lived stations in which cargo material is sorted from the resident components and transits from one organelle to the other in vesicular carriers. The maturation hypothesis suggests that organelles of the secretory pathway are transient stations that form at the level of the ER; once they leave it, they continuously homo-fuse and recycle back machinery components to the previous station. A particular discordance resides in defining the ER-Golgi-intermediate compartment (ERGIC) features: is it a stable or a maturing organelle? The ERGIC defined by the cycling lectin ERGIC-53 consists of tubulovesicular clusters. Here, I show by live imaging that GFP-ERGIC-53 mainly localizes to long-lived stationary and some short-lived highly mobile elements. Unlike the anterograde marker VSV-G-GFP, GFP-ERGIC-53 does not vectorially move to the Golgi upon exit from the ERGIC, as assessed by a novel quantitative vector field method. Dual color imaging of GFP-ERGIC-53 and a secretory protein (signal-sequence-tagged DsRed) reveals that the stationary elements are sites of repeated sorting of retrograde and anterograde cargo, and are interconnected by highly mobile elements. Based on these results, I conclude that the ERGIC is a membrane compartment in the true sense and not simply a collection of mobile carriers mediating protein traffic from endoplasmic reticulum to Golgi. The finding that the ERGIC is a true compartment opens new perspectives for the dissection of ERGIC functions and the molecular machineries that are recruited.