Advertisement

Caveolae pp 27-41 | Cite as

Analysis of Caveolin in Primary Cilia

  • Stine K. Morthorst
  • Johanne B. Mogensen
  • Søren T. Christensen
  • Lotte B. PedersenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2169)

Abstract

Recent evidence has indicated that caveolins are localized at the base of primary cilia, which are microtubule-based sensory organelles present on the cell surface, and that Caveolin-1 (CAV1) plays important roles in regulating ciliary membrane composition and function. Here we describe methods to analyze the localization and function of CAV1 in primary cilia of cultured mammalian cells. These include methods for culturing and transfecting mammalian cells with a CAV1-encoding plasmid or small interfering RNA (siRNA), analysis of mammalian cells by immunofluorescence microscopy (IFM) with antibodies against ciliary markers and CAV1, as well as methods for analyzing ciliary CAV1 function in siRNA-treated cells by IFM and cell-based signaling assays.

Key words

Mammalian cell culture Primary cilia Caveolin Immunofluorescence microscopy siRNA-mediated knockdown qPCR 

Notes

Acknowledgments

The authors acknowledge funding from the Independent Research Fund Denmark (6108-00457B and 8020-00162B), the Novo Nordisk Foundation (NNF14OC0011535, NNF15OC0016886 and NNF18OC0053024), Brødrene Hartmanns Fond (A31662), Kræftens Bekæmpelse (R146-A9590-16-S2), Carlsberg Foundation (CF18-0294), and the University of Copenhagen Excellence Programme for Interdisciplinary Research (2016 Funds). JBM and SKM were partially supported by PhD fellowships from the Department of Biology, University of Copenhagen.

References

  1. 1.
    Bangs F, Anderson KV (2017) Primary cilia and mammalian hedgehog signaling. Cold Spring Harb Perspect Biol 9:a028175CrossRefGoogle Scholar
  2. 2.
    Schou KB, Pedersen LB, Christensen ST (2015) Ins and outs of GPCR signaling in primary cilia. EMBO Rep 16:1099–1113CrossRefGoogle Scholar
  3. 3.
    Christensen ST, Morthorst SK, Mogensen JB, Pedersen LB (2017) Primary cilia and coordination of receptor tyrosine kinase (RTK) and transforming growth factor beta (TGF-beta) signaling. Cold Spring Harb Perspect Biol 9:a028167CrossRefGoogle Scholar
  4. 4.
    Reiter JF, Leroux MR (2017) Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol 18:533–547CrossRefGoogle Scholar
  5. 5.
    Garcia-Gonzalo FR, Phua SC, Roberson EC, Garcia G 3rd, Abedin M, Schurmans S, Inoue T, Reiter JF (2015) Phosphoinositides regulate ciliary protein trafficking to modulate hedgehog signaling. Dev Cell 34:400–409CrossRefGoogle Scholar
  6. 6.
    Chavez M, Ena S, Van Sande J, de Kerchove d’Exaerde A, Schurmans S, Schiffmann SN (2015) Modulation of ciliary phosphoinositide content regulates trafficking and sonic hedgehog signaling output. Dev Cell 34:338–350CrossRefGoogle Scholar
  7. 7.
    Garcia-Gonzalo FR, Reiter JF (2016) Open sesame: how transition fibers and the transition zone control ciliary composition. Cold Spring Harb Perspect Biol 9:a028134CrossRefGoogle Scholar
  8. 8.
    Taschner M, Lorentzen E (2016) The Intraflagellar transport machinery. Cold Spring Harb Perspect Biol 8:a028092CrossRefGoogle Scholar
  9. 9.
    Cao M, Ning J, Hernandez-Lara CI, Belzile O, Wang Q, Dutcher SK, Liu Y, Snell WJ (2015) Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding. eLife 4:e05242Google Scholar
  10. 10.
    Wood CR, Huang K, Diener DR, Rosenbaum JL (2013) The cilium secretes bioactive ectosomes. Curr Biol 23:906–911CrossRefGoogle Scholar
  11. 11.
    Wood CR, Rosenbaum JL (2015) Ciliary ectosomes: transmissions from the cell’s antenna. Trends Cell Biol 25:276–285CrossRefGoogle Scholar
  12. 12.
    Nager AR, Goldstein JS, Herranz-Perez V, Portran D, Ye F, Garcia-Verdugo JM, Nachury MV (2017) An actin network dispatches ciliary GPCRs into extracellular vesicles to modulate signaling. Cell 168:252–263CrossRefGoogle Scholar
  13. 13.
    Benmerah A (2013) The ciliary pocket. Curr Opin Cell Biol 25:78–84CrossRefGoogle Scholar
  14. 14.
    Clement CA, Ajbro KD, de Jesus MPR H, Koefoed K, Vestergaard ML, Veland IR, Pedersen LB, Benmerah A, Andersen CY, Larsen LA, Christensen ST (2013) Regulation of TGFβ signaling by endocytosis at the pocket region of the primary cilium. Cell Rep 3:1806–1814CrossRefGoogle Scholar
  15. 15.
    Pedersen LB, Mogensen JB, Christensen ST (2016) Endocytic control of cellular signaling at the primary cilium. Trends Biochem Sci 41:784–797CrossRefGoogle Scholar
  16. 16.
    Scheidel N, Kennedy J, Blacque OE (2018) Endosome maturation factors Rabenosyn-5/VPS45 and Caveolin-1 regulate ciliary membrane and Polycystin-2 homeostasis. EMBO J 37:e98248CrossRefGoogle Scholar
  17. 17.
    Schou KB, Mogensen JB, Nielsen BS, Morthorst SK, Aleliunaite A, Serra-Marques AMA, Saunier S, Bizet A, Veland IR, Akhmanova A, Christensen ST, Pedersen LB (2017) KIF13B establishes a CAV1-enriched microdomain at the ciliary transition zone to promote sonic hedgehog signaling. Nat Commun 8:14177CrossRefGoogle Scholar
  18. 18.
    Schreiber S, Fleischer J, Breer H, Boekhoff I (2000) A possible role for caveolin as a signaling organizer in olfactory sensory membranes. J Biol Chem 275:24115–24123CrossRefGoogle Scholar
  19. 19.
    Travis AJ, Merdiushev T, Vargas LA, Jones BH, Purdon MA, Nipper RW, Galatioto J, Moss SB, Hunnicutt GR, Kopf GS (2001) Expression and localization of caveolin-1, and the presence of membrane rafts, in mouse and Guinea pig spermatozoa. Dev Biol 240:599–610CrossRefGoogle Scholar
  20. 20.
    Trevino CL, Serrano CJ, Beltran C, Felix R, Darszon A (2001) Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm. FEBS Lett 509:119–125CrossRefGoogle Scholar
  21. 21.
    Schrøder JM, Larsen J, Komarova Y, Akhmanova A, Thorsteinsson RI, Grigoriev I, Manguso R, Christensen ST, Pedersen SF, Geimer S, Pedersen LB (2011) EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms. J Cell Sci 124:2539–2551CrossRefGoogle Scholar
  22. 22.
    Hayer A, Stoeber M, Ritz D, Engel S, Meyer HH, Helenius A (2010) Caveolin-1 is ubiquitinated and targeted to intralumenal vesicles in endolysosomes for degradation. J Cell Biol 191:615–629CrossRefGoogle Scholar
  23. 23.
    Zeng X, Goetz JA, Suber LM, Scott WJ Jr, Schreiner CM, Robbins DJ (2001) A freely diffusible form of sonic hedgehog mediates long-range signalling. Nature 411:716–720CrossRefGoogle Scholar
  24. 24.
    Millan J, Hewlett L, Glyn M, Toomre D, Clark P, Ridley AJ (2006) Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains. Nat Cell Biol 8:113–123CrossRefGoogle Scholar
  25. 25.
    Yue S, Tang LY, Tang Y, Tang Y, Shen QH, Ding J, Chen Y, Zhang Z, Yu TT, Zhang YE, Cheng SY (2014) Requirement of Smurf-mediated endocytosis of Patched1 in sonic hedgehog signal reception. eLife 3:e02555Google Scholar
  26. 26.
    Ghossoub R, Molla-Herman A, Bastin P, Benmerah A (2011) The ciliary pocket: a once-forgotten membrane domain at the base of cilia. Biol Cell 103:131–144CrossRefGoogle Scholar
  27. 27.
    Hirano T, Katoh Y, Nakayama K (2017) Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein-coupled receptors. Mol Biol Cell 28:429–439CrossRefGoogle Scholar
  28. 28.
    Vuolo L, Stevenson NL, Heesom KJ, Stephens DJ (2018) Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function. eLife 7:e39655Google Scholar
  29. 29.
    Rohatgi R, Milenkovic L, Scott MP (2007) Patched1 regulates hedgehog signaling at the primary cilium. Science 317:372–376CrossRefGoogle Scholar
  30. 30.
    Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L, Anderson KV (2003) Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426:83–87CrossRefGoogle Scholar
  31. 31.
    Eguether T, San Agustin JT, Keady BT, Jonassen JA, Liang Y, Francis R, Tobita K, Johnson CA, Abdelhamed ZA, Lo CW, Pazour GJ (2014) IFT27 links the BBSome to IFT for maintenance of the ciliary signaling compartment. Dev Cell 31:279–290CrossRefGoogle Scholar
  32. 32.
    Morthorst SK, Christensen ST, Pedersen LB (2018) Regulation of ciliary membrane protein trafficking and signalling by kinesin motor proteins. FEBS J 285:4535–4564CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Stine K. Morthorst
    • 1
    • 2
  • Johanne B. Mogensen
    • 1
    • 2
  • Søren T. Christensen
    • 1
  • Lotte B. Pedersen
    • 1
    Email author
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagen ØDenmark
  2. 2.Department of HematologyBiotech Research and Innovation CenterCopenhagen NDenmark

Personalised recommendations