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Abstract

The Golgi is a membranous organelle that forms a hub of the secretory pathway in eukaryotic cells. The materials synthesized in ER are transported to the Golgi where they processed further and sorted to their cellular destinations including endosomes, lysosomes and plasma membrane or secreted to the extracellular space. In lower eukaryotes the Golgi is found as disk-shaped cisternae, which can be combined to a ministack. In higher eukaryotes like mammalian cells the Golgi ministacks fuse with each other to form a single complex called the Golgi ribbon, which often localizes to the pericentrosomal area. The Golgi matrix proteins, the centrosome and the microtubule cytoskeleton have a critical role in the assembly and integrity of the Golgi ribbon and this pericentrosomal positioning. The ribbon organization and positioning of the Golgi are important in many cellular processes including cell migration, polarization and differentiation and subject to dynamic regulation. However, the molecular mechanism of dynamic regulation of the Golgi organization and positioning is not well understood. In this study we performed an RNAi screen to find new candidates that would enable us to understand the regulation of Golgi organization and positioning. The screen targeted 680 genes that encode for peripheral proteins. Visual analysis of the Golgi phenotypes revealed 70 genes whose depletion affected Golgi morphology and positioning. Depletion of one of the screening hits, a centrosomal protein named RPGRIP1, lead to an elongated and uncondensed Golgi ribbon organization. Characterization of RPGRIP1 revealed that RPGRIP1 depletion leads to decreased centrosomal nucleation of microtubules and increased microtubule stability, and we showed that Golgi reorganization is due to this increased microtubule stabilization. High-resolution imaging revealed that the Golgi ribbon is bound to stable microtubule protofilaments. Our experiments also show that ectopic microtubule stabilization leads to a loss of compact organization and pericentrosomal positioning of the Golgi. In summary, we propose a new model in which Golgi ribbon organization and positioning is regulated by stable microtubules. We also show for the first time the role of RPGRIP1 in microtubule dynamics in addition to its scaffolding role in organizing the primary cilium.