Isolation of Primary Cilia by Shear Force

Kimberly A. P. Mitchell1

1 Department of Biology, Liberty University, Lynchburg, Virginia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.42
DOI:  10.1002/0471143030.cb0342s59
Online Posting Date:  June, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The cell's primary cilium is both a mechanical and chemical sensor involved in many signaling pathways. In order to ascertain protein enrichment in the primary cilium or study sub‐ciliary localization of various proteins, it is advantageous to remove the primary cilium from the cell body. The protocol described here gives detailed instructions on purifying primary cilia by separating them from the cell body using shear force. This simple technique avoids using harsh purification conditions that may affect signaling proteins in the cilium or cause the ciliary membrane to disintegrate. In addition, as the cell body remains mostly intact, contamination of the isolated cilia by proteins from the cell body is minimized. This protocol is ideally suited for isolating cilia from renal cell lines, as primary cilia in these cells grow to greater lengths than in other cell types (up to 50‐µm long in Xenopus A6 toad kidney cells as opposed to 1 to 5 µm in NIH3T3 fibroblast cells). Curr. Protoc. Cell Biol. 59:3.42.1‐3.42.9. © 2013 by John Wiley & Sons, Inc.

Keywords: primary cilia; isolation; purification; shear force

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolating Primary Cilia Using Shear Force
  • Basic Protocol 2: Visualization of Isolated Primary Cilia Fraction Using Electron Microscopy (EM)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Isolating Primary Cilia Using Shear Force

  • Cells such as Xenopus A6 cells
  • A6 tissue culture medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Resuspension buffer (see recipe)
  • 150‐mm tissue culture dishes
  • Humidified incubator with CO 2 supply
  • Vacuum aspirator
  • Rotary shaker
  • ∼50‐ml centrifuge tubes appropriate for chosen rotor
  • Micropipettor
  • Refrigerated centrifuge (Beckman J6 or equivalent) with either swinging‐bucket or fixed‐angle rotor capable of volumes of 45 ml or more
  • Felt tip pen
  • ∼24‐ml ultracentrifuge tubes appropriate for chosen rotor
  • Refrigerated ultracentrifuge with fixed‐angle rotor (Beckman 60Ti, or equivalent)

Basic Protocol 2: Visualization of Isolated Primary Cilia Fraction Using Electron Microscopy (EM)

  • Isolated primary cilia (see protocol 1)
  • Resuspension buffer (see recipe)
  • 2% uranyl acetate solution
  • Parafilm
  • Formvar‐coated carbon grids (200‐mesh; Ted Pella)
  • Fine forceps
  • Filter paper
  • Electron microscope such as Phillips CM12 or equivalent
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Anderson, R.G. 1974. Isolation of ciliated or unciliated basal bodies from the rabbit oviduct. J. Cell Biol 60:393–404 .
   Armour, E.A., Carson, R.P., and Ess, K.C. 2012. Cystogenesis and elongated primary cilia in Tsc1‐deficient distal convoluted tubules. Am. J. Physiol. Renal Physiol. 303:F584‐F592.
   Berbari, N.F., O'Connor, A.K., Haycraft, C.J., and Yoder, B.K. 2009. The primary cilium as a complex signaling center. Curr. Biol. 19:R526‐535.
   Chakravarthy, B., Gaudet, C., Menard, M., Brown, L., Atkinson, T., Laferla, F.M., Ito, S., Armato, U., Dal Pra, I., and Whitfield, J. 2012. Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD‐transgenic mice producing human Abeta(1‐42) and tau. Biochem. Biophys. Res. Commun. 427:218‐222.
   D'Angelo, A. and Franco, B. 2011. The primary cilium in different tissues‐lessons from patients and animal models. Pediatr. Nephrol. 26:655‐662.
   Davenport, J.R. and Yoder, B.K. 2005. An incredible decade for the primary cilium: A look at a once‐forgotten organelle. Am. J. Physiol. Renal Physiol. 289:F1159‐F1169.
   Deane, J.A. and Ricardo, S.D. 2012. Emerging roles for renal primary cilia in epithelial repair. Int. Rev. Cell. Mol. Biol. 293:169‐193.
   Doctor, R.B., Dahl, R., Fouassier, L., Kilic, G., and Fitz, J.G. 2002. Cholangiocytes exhibit dynamic, actin‐dependent apical membrane turnover. Am. J. Physiol. Cell. Physiol. 282:C1042‐C1052.
   Farnum, C.E. and Wilsman, N.J. 2011. Axonemal positioning and orientation in three‐dimensional space for primary cilia: What is known, what is assumed, and what needs clarification. Dev. Dyn. 240:2405‐2431.
   Gallagher, B.C. 1980. Primary cilia of the corneal endothelium. Am. J. Anat. 159:475‐484.
   Gerdes, J.M., Davis, E.E., and Katsanis, N. 2009. The vertebrate primary cilium in development, homeostasis, and disease. Cell 137:32‐45.
   Goetz, S.C. and Anderson, K.V. 2010. The primary cilium: A signalling centre during vertebrate development. Nat. Rev. Genet. 11:331‐344.
   Hastie, A.T., Dicker, D.T., Hingley, S.T., Kueppers, F., Higgins, M.L., and Weinbaum, G. 1986. Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms. Cell Motil. Cytoskeleton 6:25‐34.
   Hoey, D.A., Downs, M.E., and Jacobs, C.R. 2012. The mechanics of the primary cilium: An intricate structure with complex function. J Biomech. 45:17‐26.
   Hu, Q. and Nelson, W.J. 2011. Ciliary diffusion barrier: The gatekeeper for the primary cilium compartment. Cytoskeleton 68:313‐324.
   Huang, B.Q., Masyuk, T.V., Muff, M.A., Tietz, P.S., Masyuk, A.I., and Larusso, N.F. 2006. Isolation and characterization of cholangiocyte primary cilia. Am. J. Physiol. Gastrointest. Liver Physiol. 291:G500‐G509.
   Ishikawa, H., Thompson, J., Yates, J.R. 3rd, and Marshall, W.F. 2012. Proteomic analysis of mammalian primary cilia. Curr. Biol. 22:414‐419.
   Jones, T.J. and Nauli, S.M. 2012. Mechanosensory calcium signaling. Adv. Exp. Med. Biol. 740:1001‐1015.
   Lancaster, M.A. and Gleeson, J.G. 2009. The primary cilium as a cellular signaling center: Lessons from disease. Curr. Opin. Genet. Dev. 19:220‐229.
   Lienkamp, S., Ganner, A., and Walz, G. 2012. Inversin, Wnt signaling and primary cilia. Differentiation 83:S49‐S55.
   Linck, R.W. 1973. Comparative isolation of cilia and flagella from the lamellibranch mollusc, Aequipecten irradians. J. Cell Sci. 12:345‐367.
   Marley, A. and von Zastrow, M. 2012. A simple cell‐based assay reveals that diverse neuropsychiatric risk genes converge on primary cilia. PLoS One 7:e46647.
   Massinen, S., Hokkanen, M.E., Matsson, H., Tammimies, K., Tapia‐Paez, I., Dahlstrom‐Heuser, V., Kuja‐Panula, J., Burghoorn, J., Jeppsson, K.E., Swoboda, P., Peyrard‐Janvid, M., Toftgard, R., Castren, E., and Kere, J. 2011. Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons. PLoS One 6:e20580.
   McGlashan, S.R., Knight, M.M., Chowdhury, T.T., Joshi, P., Jensen, C.G., Kennedy, S., and Poole, C.A. 2010. Mechanical loading modulates chondrocyte primary cilia incidence and length. Cell Biol. Int. 34:441‐446.
   Mitchell, K.A., Gallagher, B.C., Szabo, G., and Otero Ade, S. 2004. NDP kinase moves into developing primary cilia. Cell Motil. Cytoskeleton 59:62‐73.
   Mitchell, K.A.P., Szabo, G., and Otero, A.d.S. 2009. Methods for the isolation of sensory and primary cilia: An overview. In Methods in Cell Biology, vol. 94 (D.S. Roger, ed.) pp. 87‐101. Academic Press.
   Miyoshi, K., Kasahara, K., Miyazaki, I., and Asanuma, M. 2011. Factors that influence primary cilium length. Acta Med. Okayama 65:279‐285.
   Narita, K., Kozuka‐Hata, H., Nonami, Y., Ao‐Kondo, H., Suzuki, T., Nakamura, H., Yamakawa, K., Oyama, M., Inoue, T., and Takeda, S. 2012. Proteomic analysis of multiple primary cilia reveals a novel mode of ciliary development in mammals. Biol. Open 1:815‐825.
   Pazour, G.J. and Witman, G.B. 2003. The vertebrate primary cilium is a sensory organelle. Curr. Opin. Cell Biol. 15:105‐110.
   Perez, J.B., Segura, J.M., Abankwa, D., Piguet, J., Martinez, K.L., and Vogel, H. 2006. Monitoring the diffusion of single heterotrimeric G proteins in supported cell‐membrane sheets reveals their partitioning into microdomains. J. Mol. Biol. 363:918‐930.
   Piperno, G., LeDizet, M., and Chang, X.J. 1987. Microtubules containing acetylated alpha‐tubulin in mammalian cells in culture. J. Cell Biol. 104:289‐302.
   Praetorius, H.A. and Spring, K.R. 2001. Bending the MDCK cell primary cilium increases intracellular calcium. J. Membr. Biol. 184:71‐79.
   Rich, D.R. and Clark, A.L. 2012. Chondrocyte primary cilia shorten in response to osmotic challenge and are sites for endocytosis. Osteoarthritis Cartilage 20:923‐930.
   Rondanino, C., Poland, P.A., Kinlough, C.L., Li, H., Rbaibi, Y., Myerburg, M.M., Al‐bataineh, M.M., Kashlan, O.B., Pastor‐Soler, N.M., Hallows, K.R., Weisz, O.A., Apodaca, G., and Hughey, R.P. 2011. Galectin‐7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells. Am. J. Physiol. Renal Physiol. 301:F622‐F633.
   Rosenbaum, J.L. and Child, F.M. 1967. Flagellar regeneration in protozoan flagellates. J. Cell Biol. 34:345‐364.
   Roth, K.E., Rieder, C.L., and Bowser, S.S. 1988. Flexible‐substratum technique for viewing cells from the side: some in vivo properties of primary (9+0) cilia in cultured kidney epithelia. J. Cell Sci. 89:457‐466.
   Saggese, T., Young, A., Huang, C., Braeckmans, K., and McGlashan, S. 2012. Development of a method for the measurement of primary cilia length in 3D. Cilia 1:1‐12.
   Sanan, D.A. and Anderson, R.G. 1991. Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells. J. Histochem. Cytochem. 39:1017‐1024.
   Satir, P., Pedersen, L.B., and Christensen, S.T. 2010. The primary cilium at a glance. J. Cell Sci. 123:499‐503.
   Sharma, N., Kosan, Z.A., Stallworth, J.E., Berbari, N.F., and Yoder, B.K. 2011. Soluble levels of cytosolic tubulin regulate ciliary length control. Mol. Biol. Cell 22:806‐816.
   Sloboda, R.D. 2009. Primary cilia: Methods in Cell Biology, Volume 94. Academic Press, San Diego, Calif.
   Veland, I.R., Awan, A., Pedersen, L.B., Yoder, B.K., and Christensen, S.T. 2009. Primary cilia and signaling pathways in mammalian development, health and disease. Nephron Physiol. 111:39‐53.
   Wann, A.K. and Knight, M.M. 2012. Primary cilia elongation in response to interleukin‐1 mediates the inflammatory response. Cell Mol. Life Sci. 69:2967‐2977.
   Watson, M.R. and Hopkins, J.M. 1962. Isolated cilia from Tetrahymena pyriformis. Exp. Cell Res. 28:280‐295.
   Wheatley, D.N. and Bowser, S.S. 2000. Length control of primary cilia: Analysis of monociliate and multiciliate PtK1 cells. Biol. Cell 92:573‐582.
   Wheatley, D.N., Wang, A.M., and Strugnell, G.E. 1996. Expression of primary cilia in mammalian cells. Cell Biol. Int. 20:73‐81.
   Zaghloul, N.A. and Brugmann, S.A. 2011. The emerging face of primary cilia. Genesis 49:231‐246.
   Zhang, Y.H., Ross, E.M., and Snell, W.J. 1991. ATP‐dependent regulation of flagellar adenylylcyclase in gametes of Chlamydomonas reinhardtii. J. Biol. Chem. 266:22954‐22959.
   Zimmerman, K. 1898. Beitrage zur Kenntniss einiger Drusen und epithelien. Arch. Mikrosk. Anat. 52:552‐706.
PDF or HTML at Wiley Online Library