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Role of Arl13b in endocytic trafficking

Final Report Summary - ARL13B TRAFFICKING (Role of Arl13b in endocytic trafficking)

To move cargo between specific membrane-bound intracellular compartments, eukaryotic cells have evolved numerous mechanisms of membrane trafficking regulation. We found that CD1a, an MHC Class I-like lipid antigen presenting molecule follows an endocytic recycling pathway similar to that used by MHC Class I and other cargo internalized independently of clathrin [1]. In an attempt to discover new regulators of this pathway, we screened a shRNA library for changes in CD1a surface expression and found that the small GTPase Arl13b is involved in the regulation of endocytic recycling traffic [2]. The silencing of Arl13b caused the clustering of early endosomes and the accumulation in this organelle of recycling cargo, such as transferrin, as well as cargo destined for late endosomes and lysosomes, such as dextran. Moreover, the recycling rate of CD1a was decreased when Arl13b was silenced. Together, these results indicate that Arl13b regulates a sorting step from the early/sorting endosome. Furthermore, we found that Arl13b colocalizes and interacts with actin. Arl13b belongs to the Arf-like (Arl) family of small GTPases, which remains poorly characterized. In humans, mutations in ARL13B cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia and mental retardation [3]. In mice, the mutation of Arl13b was found to be embryonic-lethal due to defects in sonic hedgehog signaling [4]. In both cases, these defects were caused by abnormalities in the structure of primary cilia, a mechanosensory organelle to which Arl13b localizes [4,5].
The aims of this project were to: a) determine the function of Arl13b in primary cilium cargo trafficking; b) define the mechanism by which Arl13b regulates endocytic trafficking; and c) identify Arl13b effectors.
We found that Arl13b interacts with the non-muscle myosin heavy chain IIA, or Myh9 and that this interaction is necessary for the binding of Arl13b to actin. Since Myh9 interacts with Arl13b in its GTP-bound form, we can conclude that Myh9 is the first bona fide effector found for Arl13b. Moreover, we found that Arl13b colocalizes with Myh9 in circular dorsal ruffles, which are actin-dependent structures that are involved in macropinocytosis and recycling of molecules such as integrins, as well as in cell migration. Interestingly, we showed that Arl13b is necessary for cell migration both in vitro and in vivo.

Strikingly, we discovered another effector for Arl13b, namely a subunit of the exocyst, a complex involved in tethering vesicles from the Golgi and the endocytic recycling compartment (ERC). Interestingly, it has recently been found that the exocyst is required for ciliogenesis and proper localization of ciliary cargo [6,7]. Hence, the interaction of Arl13b with the exocyst could represent the mechanism by which Arl13b regulates the trafficking of ciliary cargo between the ERC and cilia.
In conclusion, we found two bona fide effectors of Arl13b that could link its function in endocytic recycling, described by us, and in ciliogenesis, described by others.


References:

1. Barral DC, Cavallari M, McCormick PJ, Garg S, Magee AI, et al. (2008) CD1a and MHC class I follow a similar endocytic recycling pathway. Traffic 9: 1446-1457.
2. Barral DC, Garg S, Casalou C, Watts GF, Sandoval JL, et al. (2012) Arl13b regulates endocytic recycling traffic. Proc Natl Acad Sci U S A 109: 21354-21359.
3. Cantagrel V, Silhavy JL, Bielas SL, Swistun D, Marsh SE, et al. (2008) Mutations in the Cilia Gene ARL13B Lead to the Classical Form of Joubert Syndrome. Am J Hum Genet.
4. Caspary T, Larkins CE, Anderson KV (2007) The graded response to Sonic Hedgehog depends on cilia architecture. Dev Cell 12: 767-778.
5. Hori Y, Kobayashi T, Kikko Y, Kontani K, Katada T (2008) Domain architecture of the atypical Arf-family GTPase Arl13b involved in cilia formation. Biochem Biophys Res Commun 373: 119-124.
6. Fogelgren B, Lin SY, Zuo X, Jaffe KM, Park KM, et al. (2011) The Exocyst Protein Sec10 Interacts with Polycystin-2 and Knockdown Causes PKD-Phenotypes. PLoS Genet 7: e1001361.
7. Zuo X, Guo W, Lipschutz JH (2009) The exocyst protein Sec10 is necessary for primary ciliogenesis and cystogenesis in vitro. Mol Biol Cell 20: 2522-2529.