6d, center). bar=10 m. ncomms11919-s9.avi (5.4M) GUID:?D94B16C6-6228-41FF-A3EF-CFB91898E7C2 Supplementary Movie 9 Spinning-disk confocal live imaging of the KO cell of movie 8 after 34 minutes of PAO treatment, scale bar=10 m. ncomms11919-s10.avi (6.2M) GUID:?8B4F8239-324F-4857-AA08-82F101321C66 Data Availability StatementThe authors declare that all the data supporting the findings of this study are available within the article and its Supplementary Information files or are available from the corresponding authors on request. mice generated in this study have been made available to the Jackson Laboratory. Abstract Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of KO MEFs results from mistrafficking of NiemannCPick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer. Intracellular transport routes are under strict regulatory control in eukaryotic cells to ensure proper sorting of cargoes, maintain organelle identity and ultimately guarantee cell K-Ras(G12C) inhibitor 6 homeostasis. Among the key regulators of intracellular trafficking pathways, small GTPases, such as ADP ribosylation factor (Arf) family members, play a fundamental role in a compartment-specific manner. Similar K-Ras(G12C) inhibitor 6 to other GTPases, Arf proteins cycle between an inactive GDP-bound form and an active GTP-bound form1. Unlike the other Arf family members (that is, Arf1-5), Arf6 is uniquely localized to the plasma membrane and to endosomes2,3, where it influences membrane trafficking. The role of Arf6 in various clathrin-dependent and -independent endocytic pathways as well as in recycling to the plasma membrane has been extensively studied4,5. A role for Arf6 in multivesicular body formation has also been recently described6. In addition, Arf6 regulates actin remodeling in such contexts as cell spreading, migration, cytokinesis, phagocytosis and neurite outgrowth5,7. ablation is embryonically lethal in the mouse8 but a conditional knockout (KO) model revealed a non-cell autonomous role for LAMC1 neuronal Arf6 in oligodendrocyte precursor cell migration and myelination9. One of the major mechanisms of action of Arf6 occurs through the control of lipid metabolism. Indeed, Arf6 binds and activates phosphatidylinositol-4-phosphate 5-kinases (PI4P5Ks), also known as type I PIPKs (PIPKIs), which phosphorylate PI4P into PI(4,5)P2 (ref. 10, 11). In addition, Arf6 can activate phospholipase D (PLD)12, whose product phosphatidic acid can in turn activate PIPKIs (ref. 13). Overexpressing a constitutively active mutant of Arf6 (Arf6 Q67L) also results in enlarged endosomes that K-Ras(G12C) inhibitor 6 contain high levels of PI(4,5)P2 (ref. 14). More recently, Arf6 has been implicated in the regulation of cellular cholesterol distribution. In cultured cells, most cholesterol is derived from cholesteryl ester-rich LDL particles present in the media. LDL-particles are internalized by the LDL receptor (LDLR) and trafficked to the lumen of late endosomes/lysosomes (LE/LYS). Cholesteryl esters are first hydrolysed by lysosomal acid lipase to free cholesterol, which is believed to be transferred by NPC2, a small soluble protein of the LE/LYS lumen, to the.