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Identification of Rab GTPase cascades in the post-Golgi trafficking of mammalian cells

Final Report Summary - RAB GTPASE CASCADES (Identification of Rab GTPase cascades in the post-Golgi trafficking of mammalian cells)

Rab GTPases are the largest family of small GTPases, comprising more than 60 members in humans. They virtually regulate all steps involved in intracellular trafficking: from cargo sorting, vesicle formation, transport along cytoskeletal tracks to docking and fusion with acceptor compartments. These proteins cycle between an inactive (GDP-bound) and an active (GTP-bound) state. Recent findings indicate that Rab proteins not only regulate individual transport steps, but they also function in a coordinated fashion, orchestrating sequential steps along transport pathways. Such a mechanism appears to be crucial to ensure continuity and directionality in membrane traffic (Hutagalung, A.H. & P. J. Novick (2011) Physiol Rev).

Integrins are a group of heterodimeric molecules (α/β subunits) responsible for the adhesion of cells to the extracellular matrix (ECM). These molecules undergo cycles of endocytosis and exocytosis, being predominantly recycled to the plasma membrane. Their recycling is dependent on Rab4 (short-loop recycling) and Rab11 (long-loop recycling). Integrin trafficking is essential for efficient cell migration as well as cell adhesion (Arjonen, A et al (2012) Traffic).

The retrograde trafficking is characterized by the transport of proteins and lipids from the plasma membrane towards the biosynthetic/secretory compartments, like the Golgi and the endoplasmic reticulum. Several proteins have been identified to be transported by this route, such as several protein toxins (e.g. Shiga toxin) as well as endogenous proteins like kinases, receptors and transporters (Shi, G. et al, (2012) Traffic). The retrograde route is important for several cellular processes, from signaling and nutrient uptake to development.

After verifying that Rab6 depletion influenced cell adhesion, we defined the following tasks in order to understand the mechanism behind this observation: 1) Characterize the cell adhesion and migration after Rab6 depletion; 2) Investigate the role of Rab6 on the recycling of integrin β1; 3) Investigate the role of Rab6 in the polarized secretion of integrin β1 in migrating cells

In this project we show, for the first time, that the heterodimer integrin α5β1 is recycled through the retrograde route. Integrin α5β1 is transported from the plasma membrane to the Golgi, through the retrograde transport, in a Rab6 dependent way. Rab6 is the most abundant Rab protein in the Golgi, broadly documented to be involved in both retrograde and anterograde trafficking (Goud B, Akmanova A (2012) Rab6 GTPase. In: Rab GTPases and membrane trafficking. Bentham Science, pp 34-46). We also show that integrin β1 interacts directly or indirectly with Rab6 (confirmed by co-IP of β1 and Rab6). Moreover, if Rab6 is depleted, a lower amount of β1 integrin can be found at the cell surface and migrating cells lose the capacity of polarizing integrin β1 towards the leading edge, which significantly impairs their persistence during migration. We propose that integrin β1 needs to take the retrograde route towards the Golgi in order to be secreted in a polarized way, back to the plasma membrane, assuring cell persistence and directionality, during migration. Finally, Rab6 depleted cells do not spread properly and exert significantly lower forces on the substrate compared with the control. At the moment, we are performing in vivo experiments using a Cre-LoxP Rab6 mouse, available in our laboratory, in order to validate, at the organism level, some of the phenotypes that we observed in cells and that are the consequence of an impaired integrin β1 trafficking, due to Rab6 depletion.
This work constitutes a major breakthrough in the understanding of integrins recycling and their polarized secretion, presenting a significant interest for many areas of biology and biomedicine. Up to now only Rab4 dependent (short-loop) and Rab11-dependent (long-loop) recycling pathways were attributed to integrins recycling. Integrins are essential for many biological processes, from embryonic development to cancer and the understanding of their traffic regulation and dynamics is essential to increase our knowledge about these molecules and develop/improve strategies to deal with integrin related pathologies, like cancer.

Glycosylphosphatidylinositol – anchored proteins (GPI-APs) are plasma membrane proteins, mainly found in the apical membrane of polarized cells and that comprise a group of 150 functionally distinct proteins in mammalian cells (Fujita, M. (2012) Biochimica et Biophysica Acta). Alterations in these proteins are responsible for several diseases, like prion disease or paroxysmal nocturnal hemoglobinuria (Taylor & Hooper (2011) Post-Translational Modifications in Health and Disease). Although essential proteins for the homeostasis of the cells and organisms, little is known about the molecular players involved in GPI-APs secretory trafficking, namely RabGTPases proteins.

In order to access only the secretory pathway of GPI-APs (EGFP-GPI, CD59, Alkaline Phosphatase), we used a two-state secretion assay designed to synchronize the trafficking of any protein of the secretory pathway: Retention Using Selective Hooks (RUSH). Our results show that Rab6, the most abundant Rab protein in the Golgi, is involved in the post-Golgi secretion of GPI-APs (RUSH EGFP-GPI, RUSH EGFP-CD59, RUSH EGFP-Alkaline Phosphatase) in non-polarized cells. Intracellular organization of EGFP-GPI post-Golgi carriers and Rab6 steady state vesicles, assessed by probabilistic density maps in micropatterned RPE1 cells, are very similar. Moreover, co-localization of EGFP-GPI post-Golgi carriers with both overexpressed and endogenous Rab6 was observed in live cells. After Rab6 depletion, GPI-APs arrival to the plasma membrane is delayed. Finally we could also observe that Rab8, another secretory Rab GTPase, colocalizes with the post-Golgi carriers of GPI-APs suggesting that these two Rab proteins may cooperate in the post-Golgi trafficking of GPI-APs, as observed for other cargos, like neuropetptide Y. Further studies are being performed to investigate in deeper detail the cooperation between Rab6 and Rab8 in the secretion of GPI-APs, both in non-polarized and polarized cells, and the role of myosin V as an intermediate of this cooperation. GPI-APs constitute a group of proteins essential for cells and organisms survival and homeostasis. Therefore, understanding their trafficking and the Rab proteins that regulate their transport is of crucial importance.