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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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References

  1. Maxfield, F. R. & McGraw, T. E. Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132 (2004).

    CAS  PubMed  Google Scholar 

  2. Cullen, P. J. Endosomal sorting and signalling: an emerging role for sorting nexins. Nat. Rev. Mol. Cell Biol. 9, 574–582 (2008).

    CAS  PubMed  Google Scholar 

  3. Mellman, I. & Nelson, W. J. Coordinated protein sorting, targeting and distribution in polarized cells. Nat. Rev. Mol. Cell Biol. 9, 833–845 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Jovic, M., Sharma, M., Rahajeng, J. & Caplan, S. The early endosome: a busy sorting station for proteins at the crossroads. Histol. Histopathol. 25, 99–112 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Takahashi, S. et al. Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. J. Cell Sci. 125, 4049–4057 (2012).

    CAS  PubMed  Google Scholar 

  6. Winter, J. F. et al. Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and apicobasal cell polarity. Nat. Cell Biol. 14, 666–676 (2012).

    CAS  PubMed  Google Scholar 

  7. Jović, M. et al. Endosomal sorting of VAMP3 is regulated by PI4K2A. J. Cell Sci. 127, 3745–3756 (2014).

    PubMed  PubMed Central  Google Scholar 

  8. Ketel, K. et al. A phosphoinositide conversion mechanism for exit from endosomes. Nature 529, 408–412 (2016).

    CAS  PubMed  Google Scholar 

  9. Franco, I. et al. PI3K class II α controls spatially restricted endosomal PtdIns3P and Rab11 activation to promote primary cilium function. Dev. Cell 28, 647–658 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Franco, I. et al. Phosphoinositide 3-kinase-C2α regulates polycystin-2 ciliary entry and protects against kidney cyst formation. J. Am. Soc. Nephrol. 27, 1135–1144 (2016).

    CAS  PubMed  Google Scholar 

  11. Horgan, C. P., Hanscom, S. R., Jolly, R. S., Futter, C. E. & McCaffrey, M. W. Rab11-FIP3 binds dynein light intermediate chain 2 and its overexpression fragments the Golgi complex. Biochem. Biophys. Res. Commun. 394, 387–392 (2010).

    CAS  PubMed  Google Scholar 

  12. Horgan, C. P., Hanscom, S. R., Jolly, R. S., Futter, C. E. & McCaffrey, M. W. Rab11-FIP3 links the Rab11 GTPase and cytoplasmic dynein to mediate transport to the endosomal-recycling compartment. J. Cell Sci. 123, 181–191 (2010).

    CAS  PubMed  Google Scholar 

  13. Ren, M. et al. Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc. Natl. Acad. Sci. USA 95, 6187–6192 (1998).

    CAS  PubMed  Google Scholar 

  14. Ullrich, O., Reinsch, S., Urbé, S., Zerial, M. & Parton, R. G. Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135, 913–924 (1996).

    CAS  PubMed  Google Scholar 

  15. Traer, C. J. et al. SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment. Nat. Cell Biol. 9, 1370–1380 (2007).

    CAS  PubMed  Google Scholar 

  16. Sönnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J. & Zerial, M. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol. 149, 901–914 (2000).

    PubMed  PubMed Central  Google Scholar 

  17. Balla, T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol. Rev. 93, 1019–1137 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Simonsen, A. et al. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 394, 494–498 (1998).

    CAS  PubMed  Google Scholar 

  19. Braccini, L. et al. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling. Nat. Commun. 6, 7400 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Campa, C. C., Franco, I. & Hirsch, E. PI3K-C2α: one enzyme for two products coupling vesicle trafficking and signal transduction. FEBS Lett. 589, 1552–1558 (2015).

    CAS  PubMed  Google Scholar 

  21. Backer, J. M. The regulation and function of class III PI3Ks: novel roles for Vps34. Biochem. J. 410, (1–17 (2008).

    Google Scholar 

  22. Jean, S., Cox, S., Schmidt, E. J., Robinson, F. L. & Kiger, A. Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling. Mol. Biol. Cell 23, 2723–2740 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Cao, C., Backer, J. M., Laporte, J., Bedrick, E. J. & Wandinger-Ness, A. Sequential actions of myotubularin lipid phosphatases regulate endosomal PI(3)P and growth factor receptor trafficking. Mol. Biol. Cell 19, 3334–3346 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Cao, C., Laporte, J., Backer, J. M., Wandinger-Ness, A. & Stein, M. P. Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on endosomes. Traffic 8, 1052–1067 (2007).

    CAS  PubMed  Google Scholar 

  25. Hnia, K., Vaccari, I., Bolino, A. & Laporte, J. Myotubularin phosphoinositide phosphatases: cellular functions and disease pathophysiology. Trends Mol. Med. 18, 317–327 (2012).

    CAS  PubMed  Google Scholar 

  26. Velichkova, M. et al. Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions. J. Cell Biol. 190, 407–425 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Wandinger-Ness, A. & Zerial, M. Rab proteins and the compartmentalization of the endosomal system. Cold Spring Harb. Perspect. Biol. 6, a022616 (2014).

    PubMed  PubMed Central  Google Scholar 

  28. Campa, C. C. & Hirsch, E. Rab11 and phosphoinositides: A synergy of signal transducers in the control of vesicular trafficking. Adv. Biol. Regul. 63, 132–139 (2017).

    CAS  PubMed  Google Scholar 

  29. Lu, Q. et al. Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat. Cell Biol. 17, 531 (2015).

    CAS  PubMed  Google Scholar 

  30. Welz, T., Wellbourne-Wood, J. & Kerkhoff, E. Orchestration of cell surface proteins by Rab11. Trends Cell Biol. 24, 407–415 (2014).

    CAS  PubMed  Google Scholar 

  31. Eathiraj, S., Mishra, A., Prekeris, R. & Lambright, D. G. Structural basis for Rab11-mediated recruitment of FIP3 to recycling endosomes. J. Mol. Biol. 364, 121–135 (2006).

    CAS  PubMed  Google Scholar 

  32. Miyawaki, A. & Tsien, R. Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500 (2000).

    CAS  PubMed  Google Scholar 

  33. Pertz, O., Hodgson, L., Klemke, R. L. & Hahn, K. M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006).

    CAS  PubMed  Google Scholar 

  34. Sakaguchi, A. et al. REI-1 Is a guanine nucleotide exchange factor regulating RAB-11 localization and function in C. elegans embryos. Dev. Cell 35, 211–221 (2015).

    CAS  PubMed  Google Scholar 

  35. Gallo, L. I. et al. TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells. Mol. Biol. Cell 25, 3779–3797 (2014).

    PubMed  PubMed Central  Google Scholar 

  36. Chen, W., Feng, Y., Chen, D. & Wandinger-Ness, A. Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. Mol. Biol. Cell 9, 3241–3257 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Firestone, A. J. et al. Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein. Nature 484, 125–129 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Delevoye, C. et al. Recycling endosome tubule morphogenesis from sorting endosomes requires the kinesin motor KIF13A. Cell Reports 6, 445–454 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Devereaux, K. et al. Regulation of mammalian autophagy by class II and III PI 3-kinases through PI3P synthesis. PLoS One 8, e76405 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Gaidarov, I., Smith, M. E., Domin, J. & Keen, J. H. The class II phosphoinositide 3-kinase C2α is activated by clathrin and regulates clathrin-mediated membrane trafficking. Mol. Cell 7, 443–449 (2001).

    CAS  PubMed  Google Scholar 

  41. Gulluni, F. et al. Mitotic spindle assembly and genomic stability in breast cancer require PI3K–C2α scaffolding function. Cancer Cell 32, 444–459.e7 (2017).

    CAS  PubMed  Google Scholar 

  42. Marat, A. L. & Haucke, V. Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic. EMBO J. 35, 561–579 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Hoepfner, S. et al. Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B. Cell 121, 437–450 (2005).

    CAS  PubMed  Google Scholar 

  44. Whitlow, M. et al. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng. 6, 989–995 (1993).

    CAS  PubMed  Google Scholar 

  45. DiPilato, L. M. & Zhang, J. The role of membrane microdomains in shaping β2-adrenergic receptor-mediated cAMP dynamics. Mol. Biosyst. 5, 832–837 (2009).

    CAS  PubMed  Google Scholar 

  46. Broussard, J. A., Rappaz, B., Webb, D. J. & Brown, C. M. Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt. Nat. Protoc. 8, 265–281 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kardash, E., Bandemer, J. & Raz, E. Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nat. Protoc. 6, 1835–1846 (2011).

    CAS  PubMed  Google Scholar 

  48. Jares-Erijman, E. A. & Jovin, T. M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).

    CAS  PubMed  Google Scholar 

  49. de Chaumont, F. et al. Icy: an open bioimage informatics platform for extended reproducible research. Nat. Methods 9, 690–696 (2012).

    PubMed  Google Scholar 

  50. Chenouard, N., Bloch, I. & Olivo-Marin, J. C. Multiple hypothesis tracking for cluttered biological image sequences. IEEE Trans. Pattern Anal. Mach. Intell. 35, 2736–3750 (2013).

    PubMed  Google Scholar 

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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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