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Rab11 activity and PtdIns(3)P turnover removes recycling cargo from endosomes

Abstract

Directional transport of recycling cargo from early endosomes (EE) to the endocytic recycling compartment (ERC) relies on phosphatidylinositol 3-phosphate (PtdIns(3)P) hydrolysis and activation of the small GTPase Rab11. However, how these events are coordinated is yet unclear. By using a novel genetically-encoded FRET biosensor for Rab11, we report that generation of endosomal PtdIns(3)P by the clathrin-binding phosphoinositide 3-kinase class 2 alpha (PI3K-C2α) controls the activation of Rab11. Active Rab11, in turn, prompts the recruitment of the phosphatidylinositol 3-phosphatase myotubularin 1 (MTM1), eventually enabling the release of recycling cargo from the EE and its delivery toward the ERC. Our findings thus define that delivery of recycling cargo toward the ERC requires spatial and sequential coupling of Rab11 activity with PtdIns(3)P turnover.

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Fig. 1: The FRET biosensor AS-Rab11 specifically measures Rab11 nucleotide binding status.
Fig. 2: Juxtanuclear and peripheral localization of active Rab11 on distinct endosome populations.
Fig. 3: Rab11 activation kinetics on PtdIns(3)P-positive endosomes.
Fig. 4: PI3K-C2α-dependent Rab11 activation on PtdIns(3)P-positive endosomes.
Fig. 5: The PtdIns(3)P phosphatase MTM1 is a Rab11 effector.
Fig. 6: Trafficking of recycling cargo from peripheral endosome to ERC requires Rab11 activation and PtdIns(3)P turnover.

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Acknowledgements

We are grateful to C. Bucci from University of Salento, K. Sato from University of Gunma, G. Apodaca from University of Pittsburgh, M. Bonazzi from University of Montpellier, J. Laporte from Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), L. Lanzetti from Candiolo Cancer Institute and S. Sigismund, G. Scita and A. Palamidessi from Fondazione Istituto FIRC di Oncologia Molecolare (IFOM) for providing reagents. We are grateful to M. Gai from University of Turin for providing technical support in confocal microscopy. This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) (161813), Compagnia di San Paolo, Wold Wide Cancer Research Association (151324) and “Futuro e Ricerca 2010” (RBFR10HP97_004). C.C.C. was supported by a FIRC (Fondazione italiana ricerca sul cancro) research fellowship. J.P.M. was supported by a UIF (Università Italo-Francese) co-tutele Phd programme.

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C.C.C., J.P.M., and E.H. conceived and designed the experiments. C.C.C., M.C.D.S., L.G., and F.C. performed in vitro experiments and analyzed the data; J.P.M., C.C.C., and A.D., performed in vitro experiments and analyzed the data; M.D.G. and C.B. analyzed imaging data; C.C.C. and E.H. wrote the manuscript. All authors contributed to data interpretation. All authors reviewed the paper and provided comments.

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Correspondence to Carlo Cosimo Campa or Emilio Hirsch.

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E.H. is a co-founder of Kither Biotech, a company involved in the development of PI3K inhibitors.

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Campa, C.C., Margaria, J.P., Derle, A. et al. Rab11 activity and PtdIns(3)P turnover removes recycling cargo from endosomes. Nat Chem Biol 14, 801–810 (2018). https://doi.org/10.1038/s41589-018-0086-4

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