The role of the intermediate compartment in membrane traffic during different stages of the cell cycle

Abstract

The biosynthetic-secretory pathway delivers newly synthesized molecules from the endoplasmic reticulum (ER) to the plasma membrane (PM), as well as to endomembrane compartments. The intermediate compartment (IC) is an interconnected membrane system, which mediates bidirectional trafficking at the ER-Golgi boundary. Besides operating in the classical secretory pathway, the IC also appears to participate in unconventional, Golgi-independent trafficking. Recent live-cell imaging studies have revealed that the IC is stably anchored next to the centrosome via its central domain, the pericentrosomal IC (pcIC). This novel structure, defined by the GTPase Rab1A, separates from the Golgi during cellular events involving centrosome relocation. This IC network also maintains its pericentrosomal positioning and dynamics after treatment with brefeldin A (BFA), a drug that results in the release of membrane-bound COPI coats and reversible Golgi disassembly. Thus, it was proposed that the pcIC acts as an important sorting site in both Golgi-dependent and -independent trafficking and therefore may be an acidic organelle. Based on the above findings, as well as recent results showing the persistence of the IC during mitosis, this study addressed the trafficking functions of this organelle throughout the cell cycle, with a special focus on its possible lumenal acidification. Previous studies have provided evidence that the Golgi enzyme mannosidase II (Man II) continuously cycles between the IC and the Golgi apparatus. Treatment of interphasic cells with ammonium chloride (NH4Cl) resulted in a delay in BFA-induced redistribution of Man II to the ER, leading to its temporary arrest in the pcIC, thus indicating that IC acidification plays a role in retrograde trafficking. By contrast, exposure of mitotic cells to NH4Cl effectively blocked Man II redistribution into the Golgi haze, resulting in its accumulation in large Rab1A-containing membrane clusters at metaphase. Importantly, other acidification inhibitors (chloroquine and bafilomycin A1), as well as BFA, gave similar effects, supporting the conclusion that the IC is an acidic organelle. Moreover, as the IC maintains its BFA-resistant character during cell division, these results raise the interesting possibility that mitotic IC clusters operate as way stations during the redistribution of Golgi enzymes during mitosis. Accordingly, lumenal acidification appears to be required for the COPI vesicle-mediated dispersal of Golgi enzymes from the IC elements into the mitotic Golgi haze. Low temperature (15°C) was also observed to affect the mitotic redistribution of Man II; however, these preliminary results need to be verified by further experiments. Overall, the results in this thesis provide evidence that the sorting and trafficking functions of the acidic IC are regulated in a cell cycle-dependent manner.