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.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Maxfield, F. R. & McGraw, T. E. Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132 (2004).
Cullen, P. J. Endosomal sorting and signalling: an emerging role for sorting nexins. Nat. Rev. Mol. Cell Biol. 9, 574–582 (2008).
Mellman, I. & Nelson, W. J. Coordinated protein sorting, targeting and distribution in polarized cells. Nat. Rev. Mol. Cell Biol. 9, 833–845 (2008).
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).
Takahashi, S. et al. Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. J. Cell Sci. 125, 4049–4057 (2012).
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).
Jović, M. et al. Endosomal sorting of VAMP3 is regulated by PI4K2A. J. Cell Sci. 127, 3745–3756 (2014).
Ketel, K. et al. A phosphoinositide conversion mechanism for exit from endosomes. Nature 529, 408–412 (2016).
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).
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).
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).
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).
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).
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).
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).
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).
Balla, T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol. Rev. 93, 1019–1137 (2013).
Simonsen, A. et al. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 394, 494–498 (1998).
Braccini, L. et al. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling. Nat. Commun. 6, 7400 (2015).
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).
Backer, J. M. The regulation and function of class III PI3Ks: novel roles for Vps34. Biochem. J. 410, (1–17 (2008).
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).
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).
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).
Hnia, K., Vaccari, I., Bolino, A. & Laporte, J. Myotubularin phosphoinositide phosphatases: cellular functions and disease pathophysiology. Trends Mol. Med. 18, 317–327 (2012).
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).
Wandinger-Ness, A. & Zerial, M. Rab proteins and the compartmentalization of the endosomal system. Cold Spring Harb. Perspect. Biol. 6, a022616 (2014).
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).
Lu, Q. et al. Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat. Cell Biol. 17, 531 (2015).
Welz, T., Wellbourne-Wood, J. & Kerkhoff, E. Orchestration of cell surface proteins by Rab11. Trends Cell Biol. 24, 407–415 (2014).
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).
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).
Pertz, O., Hodgson, L., Klemke, R. L. & Hahn, K. M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006).
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).
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).
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).
Firestone, A. J. et al. Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein. Nature 484, 125–129 (2012).
Delevoye, C. et al. Recycling endosome tubule morphogenesis from sorting endosomes requires the kinesin motor KIF13A. Cell Reports 6, 445–454 (2014).
Devereaux, K. et al. Regulation of mammalian autophagy by class II and III PI 3-kinases through PI3P synthesis. PLoS One 8, e76405 (2013).
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).
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).
Marat, A. L. & Haucke, V. Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic. EMBO J. 35, 561–579 (2016).
Hoepfner, S. et al. Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B. Cell 121, 437–450 (2005).
Whitlow, M. et al. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng. 6, 989–995 (1993).
DiPilato, L. M. & Zhang, J. The role of membrane microdomains in shaping β2-adrenergic receptor-mediated cAMP dynamics. Mol. Biosyst. 5, 832–837 (2009).
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).
Kardash, E., Bandemer, J. & Raz, E. Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nat. Protoc. 6, 1835–1846 (2011).
Jares-Erijman, E. A. & Jovin, T. M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).
de Chaumont, F. et al. Icy: an open bioimage informatics platform for extended reproducible research. Nat. Methods 9, 690–696 (2012).
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).
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.
E.H. is a co-founder of Kither Biotech, a company involved in the development of PI3K inhibitors.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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
Clinical and Translational Medicine (2020)
PI3KC2α-dependent and VPS34-independent generation of PI3P controls primary cilium-mediated autophagy in response to shear stress
Nature Communications (2020)
Nature Reviews Molecular Cell Biology (2019)
Nature Communications (2019)
Nature Communications (2018)