Lipoprotein particles are required for Hedgehog and Wingless signalling

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Abstract

Wnt and Hedgehog family proteins are secreted signalling molecules (morphogens) that act at both long and short range to control growth and patterning during development. Both proteins are covalently modified by lipid, and the mechanism by which such hydrophobic molecules might spread over long distances is unknown. Here we show that Wingless, Hedgehog and glycophosphatidylinositol-linked proteins copurify with lipoprotein particles, and co-localize with them in the developing wing epithelium of Drosophila. In larvae with reduced lipoprotein levels, Hedgehog accumulates near its site of production, and fails to signal over its normal range. Similarly, the range of Wingless signalling is narrowed. We propose a novel function for lipoprotein particles, in which they act as vehicles for the movement of lipid-linked morphogens and glycophosphatidylinositol-linked proteins.

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Figure 1: Lipid-linked proteins co-fractionate with lipophorin.
Figure 2: Wingless and Hedgehog co-localize with Alexa488lipophorin.
Figure 3: Lipophorin–RNAi perturbs lipid transport.
Figure 4: Lipophorin–RNAi alters Hedgehog distribution and signalling.
Figure 5: Hedgehog signalling is unaffected by lipid-depletion.
Figure 6: Lipophorin–RNAi narrows the range of Wingless signalling.

References

  1. 1

    Strigini, M. & Cohen, S. M. A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing. Development 124, 4697–4705 (1997)

  2. 2

    Neumann, C. J. & Cohen, S. M. Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124, 861–870 (1997)

  3. 3

    Porter, J. A. et al. Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86, 21–34 (1996)

  4. 4

    Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452 (2003)

  5. 5

    Pepinsky, R. B. et al. Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14037–14045 (1998)

  6. 6

    Williams, K. P. et al. Functional antagonists of sonic hedgehog reveal the importance of the N terminus for activity. J. Cell Sci. 112, 4405–4414 (1999)

  7. 7

    Chamoun, Z. et al. Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science 293, 2080–2084 (2001)

  8. 8

    Micchelli, C. A.,, The, I., Selva, E., Mogila, V. & Perrimon, N. Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. Development 129, 843–851 (2002)

  9. 9

    Lee, J. D. et al. An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development. Dev. Biol. 233, 122–136 (2001)

  10. 10

    Liu, T. et al. Intercellular transfer of the cellular prion protein. J. Biol. Chem. 277, 47671–47678 (2002)

  11. 11

    Dunn, D. E. et al. A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(-)hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins. Proc. Natl Acad. Sci. USA 93, 7938–7943 (1996)

  12. 12

    Anderson, S. M., Yu, G., Giattina, M. & Miller, J. L. Intercellular transfer of a glycosylphosphatidylinositol (GPI)-linked protein: release and uptake of CD4-GPI from recombinant adeno-associated virus-transduced HeLa cells. Proc. Natl Acad. Sci. USA 93, 5894–5898 (1996)

  13. 13

    Greco, V., Hannus, M. & Eaton, S. Argosomes: a potential vehicle for the spread of morphogens through epithelia. Cell 106, 633–645 (2001)

  14. 14

    Denzer, K., Kleijmeer, M. J., Heijnen, H. F., Stoorvogel, W. & Geuze, H. J. Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 113, 3365–3374 (2000)

  15. 15

    Arrese, E. L. et al. Lipid storage and mobilization in insects: current status and future directions. Insect Biochem. Mol. Biol. 31, 7–17 (2001)

  16. 16

    van der Horst, D. J., van Hoof, D., van Marrewijk, W. J. & Rodenburg, K. W. Alternative lipid mobilization: the insect shuttle system. Mol. Cell. Biochem. 239, 113–119 (2002)

  17. 17

    Escola, J. M. et al. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J. Biol. Chem. 273, 20121–20127 (1998)

  18. 18

    Wubbolts, R. et al. Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation. J. Biol. Chem. 278, 10963–10972 (2003)

  19. 19

    Sundermeyer, K., Hendricks, J. K., Prasad, S. V. & Wells, M. A. The precursor protein of the structural apolipoproteins of lipophorin: cDNA and deduced amino acid sequence. Insect Biochem. Mol. Biol. 26, 735–738 (1996)

  20. 20

    Kutty, R. K. et al. Molecular characterization and developmental expression of a retinoid- and fatty acid-binding glycoprotein from Drosophila. A putative lipophorin. J. Biol. Chem. 271, 20641–20649 (1996)

  21. 21

    Hortsch, M. & Goodman, C. S. Drosophila fasciclin I, a neural cell adhesion molecule, has a phosphatidylinositol lipid membrane anchor that is developmentally regulated. J. Biol. Chem. 265, 15104–15109 (1990)

  22. 22

    Nose, A., Mahajan, V. B. & Goodman, C. S. Connectin: a homophilic cell adhesion molecule expressed on a subset of muscles and the motoneurons that innervate them in Drosophila. Cell 70, 553–567 (1992)

  23. 23

    Butler, S. J., Ray, S. & Hiromi, Y. klingon, a novel member of the Drosophila immunoglobulin superfamily, is required for the development of the R7 photoreceptor neuron. Development 124, 781–792 (1997)

  24. 24

    Incardona, J. P. & Rosenberry, T. L. Replacement of the glycoinositol phospholipid anchor of Drosophila acetylcholinesterase with a transmembrane domain does not alter sorting in neurons and epithelia but results in behavioral defects. Mol. Biol. Cell 7, 613–630 (1996)

  25. 25

    Cagan, R. L., Kramer, H., Hart, A. C. & Zipursky, S. L. The bride of sevenless and sevenless interaction: Internalization of a transmembrane ligand. Cell 69, 393–399 (1992)

  26. 26

    Klueg, K. M. & Muskavitch, M. A. Ligand-receptor interactions and trans-endocytosis of Delta, Serrate and Notch: members of the Notch signalling pathway in Drosophila. J. Cell Sci. 112, 3289–3297 (1999)

  27. 27

    Culi, J. & Mann, R. S. Boca, an endoplasmic reticulum protein required for wingless signaling and trafficking of LDL receptor family members in Drosophila. Cell 112, 343–354 (2003)

  28. 28

    Greenspan, P., Mayer, E. P. & Fowler, S. D. Nile red: a selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100, 965–973 (1985)

  29. 29

    Britton, J. S., Lockwood, W. K., Li, L., Cohen, S. M. & Edgar, B. A. Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2, 239–249 (2002)

  30. 30

    Vervoort, M. hedgehog and wing development in Drosophila: a morphogen at work? Bioessays 22, 460–468 (2000)

  31. 31

    Vervoort, M., Crozatier, M., Valle, D. & Vincent, A. The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing. Curr. Biol. 9, 632–639 (1999)

  32. 32

    Torroja, C., Gorfinkiel, N. & Guerrero, I. Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction. Development 131, 2395–2408 (2004)

  33. 33

    Tabata, T. & Kornberg, T. B. Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs. Cell 76, 89–102 (1994)

  34. 34

    Chen, Y. & Struhl, G. Dual roles for patched in sequestering and transducing Hedgehog. Cell 87, 553–563 (1996)

  35. 35

    The, I., Bellaiche, Y. & Perrimon, N. Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol. Cell 4, 633–639 (1999)

  36. 36

    Takei, Y., Ozawa, Y., Sato, M., Watanabe, A. & Tabata, T. Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans. Development 131, 73–82 (2004)

  37. 37

    Mahley, R. W. & Ji, Z. S. Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J. Lipid Res. 40, 1–16 (1999)

  38. 38

    Camejo, G., Hurt-Camejo, E., Wiklund, O. & Bondjers, G. Association of apo B lipoproteins with arterial proteoglycans: pathological significance and molecular basis. Atherosclerosis 139, 205–222 (1998)

  39. 39

    McCarthy, R. A., Barth, J. L., Chintalapudi, M. R., Knaak, C. & Argraves, W. S. Megalin functions as an endocytic sonic hedgehog receptor. J. Biol. Chem. 277, 25660–25667 (2002)

  40. 40

    Wehrli, M. et al. arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 407, 527–530 (2000)

  41. 41

    Tamai, K. et al. LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535 (2000)

  42. 42

    Burke, R. et al. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell 99, 803–815 (1999)

  43. 43

    Nakano, Y. et al. A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched. Nature 341, 508–513 (1989)

  44. 44

    Cooper, M. K. et al. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nature Genet. 33, 508–513 (2003)

  45. 45

    Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993)

  46. 46

    Strigini, M. & Cohen, S. M. Wingless gradient formation in the Drosophila wing. Curr. Biol. 10, 293–300 (2000)

  47. 47

    Taylor, A. M., Nakano, Y., Mohler, J. & Ingham, P. W. Contrasting distributions of patched and hedgehog proteins in the Drosophila embryo. Mech. Dev. 42, 89–96 (1993)

  48. 48

    Capdevila, J., Pariente, F., Sanpedro, J., Alonso, J. & Guerrero, I. Subcellular localization of the segment polarity protein Patched suggests an interaction with the Wingless reception complex in Drosophila embryos. Development 120, 987–988 (1994)

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Acknowledgements

We thank T. Kurzchalia for advice regarding lipid depletion. We thank S. Cohen, I. Guerrero, P. Ingham, Y. Hiromi, M. Horscht, John Incardona and R. White for gifts of antibodies, and G. Griffiths for the CD63:GFP fusion construct. We are grateful to A. Mahmoud and S. Bowman for developing CFP:Rab5-expressing flies, and to V. Greco for helping to initiate these studies. We thank D. Backasch for performing embryo injections. K. Simons, M. Zerial, T. Kurzchalia and C. Dahmann provided comments on the manuscript.Author contributions This work has been a collaborative effort between the groups of C. Thiele and S. Eaton, the Thiele laboratory contributing biochemical expertise and the Eaton laboratory expertise in working with Drosophila. First authors appear in alphabetical order.

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Correspondence to Christoph Thiele or Suzanne Eaton.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure S1

Supplementary Figure S1 describes and characterizes the marker used for exosomes (CD63:GFP) and Lipophorin (anti-ApoLI and anti-ApoLII). (JPG 81 kb)

Supplementary Figure S2

Hedgehog and Fasciclin I can be co-immunoprecipitated with Lipophorin from the top, low-density fraction of KBr gradients. (PDF 240 kb)

Supplementary Figure S3

The rate of reduction of ApoLI protein levels by Lipophorin-RNAi. (PDF 196 kb)

Supplementary Figure S4

Insulin signalling is not reduced in Lipophorin RNAi discs and that apoptosis is not significantly elevated. (JPG 47 kb)

Supplementary Figure S5

Hedgehog and Patched in Lipophorin-RNAi larvae accumulate in endosomes. (JPG 44 kb)

Supplementary Figure S6

Quantification of Distalless staining intensity in individual wild type and Lipophorin-RNAi discs. (JPG 24 kb)

Supplementary Figure S7

Hedgehog and Patched accumulation in Lipophorin RNAi discs can be reversed by adding purified Lipophorin particles to explanted discs. (PDF 1817 kb)

Supplementary Figure S7

Legend to accompany the above figure. (DOC 23 kb)

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Panáková, D., Sprong, H., Marois, E. et al. Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435, 58–65 (2005) doi:10.1038/nature03504

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