Primary cilia regulate mTORC1 activity and cell size through Lkb1

Abstract

The mTOR pathway is the central regulator of cell size1. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood2,3,4. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD)5,6,7, where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins8. We therefore investigated if cilia have a role in mTOR regulation. Here, we show that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. Our results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling.

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Figure 1: Cilia regulate cell size under flow conditions.
Figure 2: Cilia and flow modulate mTORC1 activity.
Figure 3: Flow-induced cell-size regulation is mTOR dependent and ablation of flow-induced calcium transients has no effect on cell size.
Figure 4: Lkb1 modulates flow-dependent mTOR and cell-size regulation and is localized in the basal body and the cilium.
Figure 5: Phosphorylated AMPK is localized at the basal body and increases under flow.

References

  1. 1

    Wullschleger, S., Loewith, R. & Hall, M. N. TOR signaling in growth and metabolism. Cell 124, 471–484 (2006).

    CAS  Article  Google Scholar 

  2. 2

    Laplante, M. & Sabatini, D. M. mTOR signaling at a glance. J. Cell Sci. 122, 3589–3594 (2009).

    CAS  Article  Google Scholar 

  3. 3

    Polak, P. & Hall, M. N. mTOR and the control of whole body metabolism. Curr. Opin. Cell Biol. 21, 209–218 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Ma, X. M. & Blenis, J. Molecular mechanisms of mTOR-mediated translational control. Nat. Rev. Mol. Cell Biol. 10, 307–318 (2009).

    Article  Google Scholar 

  5. 5

    Tao, Y., Kim, J., Schrier, R. W. & Edelstein, C. L. Rapamycin markedly slows disease progression in a rat model of polycystic kidney disease. J. Am. Soc. Nephrol. 16, 46–51 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Wahl, P. R. et al. Inhibition of mTOR with sirolimus slows disease progression in Han:SPRD rats with autosomal dominant polycystic kidney disease (ADPKD). Nephrol. Dial. Transplant 21, 598–604 (2006).

    CAS  Article  Google Scholar 

  7. 7

    Shillingford, J. M. et al. The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease. Proc. Natl Acad. Sci. USA 103, 5466–5471 (2006).

    CAS  Article  Google Scholar 

  8. 8

    Fliegauf, M., Benzing, T. & Omran, H. When cilia go bad: cilia defects and ciliopathies. Nat. Rev. Mol. Cell Biol. 8, 880–893 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Jansen, M., Ten Klooster, J. P., Offerhaus, G. J. & Clevers, H. LKB1 and AMPK family signaling: the intimate link between cell polarity and energy metabolism. Physiol. Rev. 89, 777–798 (2009).

    CAS  Article  Google Scholar 

  10. 10

    Pedersen, L. B. & Rosenbaum, J. L. Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling. Curr. Top. Dev. Biol. 85, 23–61 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Berbari, N. F., O'Connor, A. K., Haycraft, C. J. & Yoder, B. K. The primary cilium as a complex signaling center. Curr. Biol. 19, R526–535 (2009).

    CAS  Article  Google Scholar 

  12. 12

    Praetorius, H. A. & Spring, K. R. Bending the MDCK cell primary cilium increases intracellular calcium. J. Membr. Biol. 184, 71–79 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Corbit, K. C. et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Haycraft, C. J. et al. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet. 1, e53 (2005).

    Article  Google Scholar 

  15. 15

    Corbit, K. C. et al. Kif3a constrains β-catenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. Nat. Cell Biol. 10, 70–76 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Torres, V. E. & Harris, P. C. Autosomal dominant polycystic kidney disease: the last 3 years. Kidney Int. 76, 149–168 (2009).

    Article  Google Scholar 

  17. 17

    Nauli, S. M. et al. Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat. Genet. 33, 129–137 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Serra, A. L. et al. Sirolimus and kidney growth in autosomal dominant polycystic kidney disease. N. Engl. J. Med. 363, 820–829 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Walz, G. et al. Everolimus in patients with autosomal dominant polycystic kidney disease. N. Engl. J. Med. 363, 830–840 (2010).

    CAS  Article  Google Scholar 

  20. 20

    Distefano, G. et al. Polycystin-1 regulates extracellular signal-regulated kinase-dependent phosphorylation of tuberin to control cell size through mTOR and its downstream effectors S6K and 4EBP1. Mol. Cell Biol. 29, 2359–2371 (2009).

    CAS  Article  Google Scholar 

  21. 21

    Grantham, J. J., Geiser, J. L. & Evan, A. P. Cyst formation and growth in autosomal dominant polycystic kidney disease. Kidney Int. 31, 1145–1152 (1987).

    CAS  Article  Google Scholar 

  22. 22

    Patel, V. et al. Acute kidney injury and aberrant planar cell polarity induce cyst formation in mice lacking renal cilia. Hum. Mol. Genet. 17, 1578–1590 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Kotsis, F. et al. Ciliary calcium signaling is modulated by kidney injury molecule-1 (Kim1). Pflugers Arch. 453, 819–829 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Kotsis, F., Nitschke, R., Doerken, M., Walz, G. & Kuehn, E. W. Flow modulates centriole movements in tubular epithelial cells. Pflugers Arch. 456, 1025–1035 (2008).

    CAS  Article  Google Scholar 

  25. 25

    Nishimura, T. et al. Role of the PAR-3–KIF3 complex in the establishment of neuronal polarity. Nat. Cell Biol. 6, 328–334 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Pazour, G. J. et al. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737, are required for assembly of cilia and flagella. J. Cell Biol. 151, 709–718 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Murcia, N. S. et al. The oak ridge polycystic kidney (orpk) disease gene is required for left-right axis determination. Development 127, 2347–2355 (2000).

    CAS  PubMed  Google Scholar 

  28. 28

    Chiang, G. G. & Abraham, R. T. Phosphorylation of mammalian target of rapamycin (mTOR) at Ser 2448 is mediated by p70S6 kinase. J. Biol. Chem. 280, 25485–25490 (2005).

    CAS  Article  Google Scholar 

  29. 29

    Kim, D. H. et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Polak, P. et al. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration. Cell Metab. 8, 399–410 (2008).

    CAS  Article  Google Scholar 

  31. 31

    Yan, L. et al. Hyperactivation of mammalian target of rapamycin (mTOR) signaling by a gain-of-function mutant of the Rheb GTPase. J. Biol. Chem. 281, 19793–19797 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Kottgen, M. et al. TRPP2 and TRPV4 form a polymodal sensory channel complex. J. Cell Biol. 182, 437–447 (2008).

    Article  Google Scholar 

  33. 33

    Alessi, D. R., Sakamoto, K. & Bayascas, J. R. LKB1-dependent signaling pathways. Annu. Rev. Biochem. 75, 137–163 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Granot, Z. et al. LKB1 regulates pancreatic β cell size, polarity and function. Cell Metab. 10, 296–308 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Sebbagh, M., Santoni, M. J., Hall, B., Borg, J. P. & Schwartz, M. A. Regulation of LKB1/STRAD localization and function by E-cadherin. Curr. Biol. 19, 37–42 (2009).

    CAS  Article  Google Scholar 

  36. 36

    Xie, Z. et al. Identification of the serine 307 of LKB1 as a novel phosphorylation site essential for its nucleocytoplasmic transport and endothelial cell angiogenesis. Mol. Cell Biol. 29, 3582–3596 (2009).

    CAS  Article  Google Scholar 

  37. 37

    Mammoto, T. & Ingber, D. E. Mechanical control of tissue and organ development. Development 137, 1407–1420 (2010).

    CAS  Article  Google Scholar 

  38. 38

    Liu, W. et al. Effect of flow and stretch on the [Ca2+]i response of principal and intercalated cells in cortical collecting duct. Am. J. Physiol. Renal Physiol. 285, F998–F1012 (2003).

    CAS  Article  Google Scholar 

  39. 39

    Maekawa, F. et al. Localization of glucokinase-like immunoreactivity in the rat lower brain stem: for possible location of brain glucose-sensing mechanisms. Endocrinology 141, 375–384 (2000).

    CAS  Article  Google Scholar 

  40. 40

    Zhang, Q., Davenport, J. R., Croyle, M. J., Haycraft, C. J. & Yoder, B. K. Disruption of IFT results in both exocrine and endocrine abnormalities in the pancreas of Tg737(orpk) mutant mice. Lab. Invest. 85, 45–64 (2005).

    CAS  Article  Google Scholar 

  41. 41

    Wiznerowicz, M. & Trono, D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J. Virol. 77, 8957–8961 (2003).

    CAS  Article  Google Scholar 

  42. 42

    Rekas, A., Alattia, J. R., Nagai, T., Miyawaki, A. & Ikura, M. Crystal structure of venus, a yellow fluorescent protein with improved maturation and reduced environmental sensitivity. J. Biol. Chem. 277, 50573–50578 (2002).

    CAS  Article  Google Scholar 

  43. 43

    Hara, K. et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 (2002).

    CAS  Article  Google Scholar 

  44. 44

    Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J. & Cantley, L. C. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell 10, 151–162 (2002).

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by grants DFG KFO 201 (to E.W.K.), WA597 and SFB 592 (to G.W.), SFB 592 Z2 and by the Excellence Initiative of the German Federal and State Governments (EXC 294 to R.N.), and NIH grants P30DK79328, R01DK67565 and T32DK07257 (to P.I.). We thank B. Yoder for anti-sera, R. Lamb, H. Clevers, Y. Mimori-Kiyosue, M. Sebbagh and A. Miyawaki for constructs, S. Arnold, M. Simons, D. Bennet and M. Bloech for critical appraisal of the manuscript and S. Lienkamp, F. Grahammer and members of the Walz lab for helpful discussions.

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C.B., F.K., V.P., Si.B., H.V., Sa.B, T.B., H.J., C.H., K.M., Ma.H., Mi.H., M.D. and R.N. performed experiments. M.G. provided reagents. M.K., R.N., P.I., G.W. and E.W.K. conceived and planned the experiments and interpreted data. C.B., F.K. and E.W.K. wrote the manuscript.

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Correspondence to E. Wolfgang Kuehn.

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Boehlke, C., Kotsis, F., Patel, V. et al. Primary cilia regulate mTORC1 activity and cell size through Lkb1. Nat Cell Biol 12, 1115–1122 (2010). https://doi.org/10.1038/ncb2117

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