Visualization of Live Primary Cilia Dynamics Using Fluorescence Microscopy

Carolyn Ott1, Jennifer Lippincott‐Schwartz1

1 Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.26
DOI:  10.1002/0471143030.cb0426s57
Online Posting Date:  December, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Methods useful for exploring the formation and functions of primary cilia in living cells are described here. First, multiple protocols for visualizing solitary cilia that extend away from the cell body are described. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The subsequent protocols describe how to quantify movement of fluorescent molecules within the cilium using kymographs, photobleaching, and photoconversion. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein‐mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary‐mediated signal perception and transmission. Curr. Protoc. Cell Biol. 57:4.26.1‐4.26.22. © 2012 by John Wiley & Sons, Inc.

Keywords: primary cilium; cilia; intraflagellar transport; kymograph

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Imaging Fluorescent Proteins in Live, Filter‐Grown, MDCK Cell Primary Cilia
  • Basic Protocol 2: Imaging Fluorescent Proteins in Primary Cilia of Live NIH3T3 Cells
  • Staining Ciliary Proteins or Ciliary Membranes with Exogenously Added Dyes
  • Alternate Protocol 1: Staining Primary Cilia with Fluorescently Labeled Lipids
  • Alternate Protocol 2: Staining Primary Cilia with BODIPY Cholesterol
  • Alternate Protocol 3: Lectin Staining of Primary Cilia
  • Basic Protocol 3: Visualizing and Quantifying Movement within Primary Cilia
  • Alternate Protocol 4: Improved Discrimination of Protein Movement Using Cilia Photobleaching
  • Alternate Protocol 5: Assessing Movement of Select Pools of Cilia Proteins Using Photoconversion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Imaging Fluorescent Proteins in Live, Filter‐Grown, MDCK Cell Primary Cilia

  Materials
  • MDCK II cells
  • MDCK cell growth medium (see recipe)
  • 1× PBS ( appendix 2A)
  • 0.25% trypsin/2.21 mM EDTA in Hank's buffered salt solution (HBSS)
  • Serum‐free medium or OptiMEM
  • DNA
  • Lipofectamine 2000
  • Imaging medium such as Liebovitz‐15 or CO 2‐independent medium with 4 mM glutamine (Invitrogen)
  • Centrifuge
  • Transwells contained in a multi‐well plate (Corning, cat. no. 3460 or 3470)
  • 37°C incubator
  • Inverted confocal microscope
  • Multi‐well plates
  • 1.5‐ml microcentrifuge tubes
  • 2‐well LabTek chambers (Nunc) or MatTek dishes or coverslip holders (e.g., Chamlide from Quarum Technologies)
  • Wire cutters and tweezers, optional
  • Additional reagents and equipment for cell counting (see unit 1.1)

Basic Protocol 2: Imaging Fluorescent Proteins in Primary Cilia of Live NIH3T3 Cells

  Materials
  • NIH3T3 cells
  • NIH3T3 growth medium (see recipe)
  • 1× PBS ( appendix 2A)
  • 0.05% Trypsin/0.53 mM EDTA in HBSS
  • NIH3T3 starvation medium (see recipe)
  • Serum‐free medium or Opti‐MEM (Invitrogen)
  • DNA
  • Lipofectamine 2000
  • Mineral oil, optional
  • Imaging medium such as Liebovitz‐15 or CO 2‐independent medium with 4 mM glutamine (Invitrogen)
  • Centrifuge
  • 10‐mm coverslip in a 24‐well dish or 4‐well coverslip‐bottom LabTek chamber
  • Inverted microscope and imaging equipment
  • Additional reagents and equipment for cell counting (see unit 1.1)

Alternate Protocol 1: Staining Primary Cilia with Fluorescently Labeled Lipids

  Materials
  • 1 mg/ml DOPE rhodamine (Avanti Polar Lipids, http://www.avantilipids.com) in ethanol
  • Imaging medium such as Liebovitz‐15 or CO 2‐independent medium with 4 mM glutamine
  • Cultured ciliated MDCK or NIH3T3 cells (see Basic Protocol protocol 11 and protocol 22)

Alternate Protocol 2: Staining Primary Cilia with BODIPY Cholesterol

  Materials
  • Imaging medium such as Liebovitz‐15 or CO 2‐independent medium with 4 mM glutamine
  • BODIPY cholesterol stock solution (see recipe)
  • Cultured ciliated MDCK or NIH3T3 cells (see Basic Protocols protocol 11 and protocol 22)
  • 37°C incubator

Alternate Protocol 3: Lectin Staining of Primary Cilia

  Materials
  • 1 mg/ml stock solution of WGA rhodamine (Invitrogen or EY Laboratories, http://eylabs.com/)
  • Imaging medium such as Liebovitz‐15 or CO 2‐independent medium with 4 mM glutamine
  • Cultured ciliated MDCK or NIH3T3 cells (see Basic Protocols protocol 11 and protocol 22)
  • 37°C incubator
  • Microscope

Basic Protocol 3: Visualizing and Quantifying Movement within Primary Cilia

  Materials
  • Cultured ciliated MDCK cells expressing fluorescent protein (see protocol 1)
  • Inverted confocal microscope
  • Image J software with kymograph plug‐ins (written by J. Rietdorf, FMI Basel, and A. Seitz, EMBL Heidelberg)

Alternate Protocol 4: Improved Discrimination of Protein Movement Using Cilia Photobleaching

  Materials
  • Cultured ciliated MDCK cells expressing fluorescent protein (see protocol 1)
  • Inverted confocal microscope capable of photobleaching a specific region of interest
  • Image J software with kymograph plug‐ins

Alternate Protocol 5: Assessing Movement of Select Pools of Cilia Proteins Using Photoconversion

  Materials
  • Cultured ciliated MDCK cells expressing fluorescent protein (see protocol 1)
  • Inverted confocal microscope capable of photoconverting a specific region of interest
  • Image J software with kymograph plug‐ins
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Literature Cited

   Berbari, N.F., Bishop, G.A., Askwith, C.C, Lewis, J.S, and Mykytyn, K. 2007. Hippocampal neurons possess primary cilia in culture. J. Neurosci. Res. 85:1095‐1100.
   Bloodgood, R.A. 2009. From central to rudimentary to primary: The history of an underappreciated organelle whose time has come. The primary cilium. Methods Cell Biol. 94:3‐52.
   Caspary, T., Larkins, C.E., and Anderson, K.V. 2007. The graded response to Sonic Hedgehog depends on cilia architecture. Dev. Cell 12:767‐778.
   Corbit, K.C., Aanstad, P., Singla, V., Norman, A.R., Stainier, D.Y., and Reiter, J.F. 2005. Vertebrate smoothened functions at the primary cilium. Nature 437:1018‐1021.
   Cuevas, P. and Gutierrez Diaz, J.A. 1985. Absence of filipin‐sterol complexes from the ciliary necklace of ependymal cells. Anat. Embryol. 172:97‐99.
   Follit, J.A., Tuft, R.A., Fogarty, K.E., and Pazour, G.J. 2006. The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly. Mol. Biol. Cell 17:3781‐3792.
   Goetz, S.C. and Anderson, K.V. 2010. The primary cilium: A signalling centre during vertebrate development. Nat. Rev. Genet. 11:331‐344.
   Handel, M., Schulz, S., Stanarius, A., Schreff, M., Erdtmann‐Vourliotis, M., Schmidt, H., Wolf, G., and Hollt, V. 1999. Selective targeting of somatostatin receptor 3 to neuronal cilia. Neuroscience 89:909‐926.
   Haycraft, C.J., Banizs, B., Aydin‐Son, Y., Zhang, Q., Michaud, E.J., and Yoder, B.K. 2005. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet. 1:e53.
   Holtta‐Vuori, M., Uronen, R.L., Repakova, J., Salonen, E., Vattulainen, I., Panula, P., Li, Z., Bittman, R., and Ikonen, E. 2008. BODIPY‐cholesterol: A new tool to visualize sterol trafficking in living cells and organisms. Traffic 9:1839‐1849.
   Kozminski, K.G., Johnson, K.A., Forscher, P., and Rosenbaum, J.L. 1993. A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc. Natl. Acad. Sci. U.S.A. 90:5519‐5523.
   Leppimaki, P., Mattinen, J., and Slotte, J.P. 2000. Sterol‐induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double‐bond position in the sterol tetracyclic ring structure. Eur. J. Biochem. 267:6385‐6394.
   Morgan, D., Eley, L., Sayer, J., Strachan, T., Yates, L.M, Craighead, A.S, and Goodship, J.A. 2002. Expression analyses and interaction with the anaphase promoting complex protein Apc2 suggest a role for inversin in primary cilia and involvement in the cell cycle. Hum. Mol. Genet. 11:3345‐3350.
   Nachury, M.V., Loktev, A.V., Zhang, Q., Westlake, C.J., Peranen, J., Merdes, A., Slusarski, D.C., Scheller, R.H., Bazan, J.F, Sheffield, V.C, and Jackson, P.K. 2007. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129:1201‐1213.
   Ott, C.M., Elia, N., Jeong, S.Y., Insinna, C., Sengupta, P., and Lippincott‐Schwartz, J. 2012. Primary cilia utilize glycoprotein‐dependent adhesion mechanisms to stabilize long‐lasting cilia‐cilia contacts. Cilia 1:3.
   Pedersen, L.B. and Rosenbaum, J.L. 2008. Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling. Curr. Top. Dev. Biol. 85:23‐61.
   Praetorius, H.A. and Spring, K.R. 2001. Bending the MDCK cell primary cilium increases intracellular calcium. J. Membr. Biol. 184:71‐79.
   Praetorius, H.A. and Spring, K.R. 2003. Removal of the MDCK cell primary cilium abolishes flow sensing. J. Membr. Biol. 191:69‐76.
   Praetorius, H.A., Frokiaer, J., Nielsen, S., and Spring, K.R. 2003. Bending the primary cilium opens Ca2+‐sensitive intermediate‐conductance K+ channels in MDCK cells. J. Membr. Biol. 191:193‐200.
   Praetorius, H.A., Praetorius, J., Nielsen, S., Frokiaer, J., and Spring, K.R. 2004. Beta1‐integrins in the primary cilium of MDCK cells potentiate fibronectin‐induced Ca2+ signaling. Am. J. Physiol. Renal. Physiol. 287:F969‐F978.
   Schneider, L., Clement, C.A., Teilmann, S.C., Pazour, G.J., Hoffmann, E.K, Satir, P., and Christensen, S.T. 2005. PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts. Curr. Biol. 15:1861‐1866.
   Sfakianos, J., Togawa, A., Maday, S., Hull, M., Pypaert, M., Cantley, L., Toomre, D., and Mellman, I. 2007. Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells. J. Cell Biol. 179:1133‐1140.
   Sharma, N., Berbari, N.F., and Yoder, B.K. 2008. Ciliary dysfunction in developmental abnormalities and diseases. Curr. Top. Dev. Biol. 85:371‐427.
   Smith, C.L. 2008. Basic confocal microscopy. Curr. Protoc. Mol. Biol. 81:14.11.1‐14.11.18.
   Snow, J.J., Ou, G., Gunnarson, A.L., Walker, M.R., Zhou, H.M, Brust‐Mascher, I., and Scholey, J.M. 2004. Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons. Nat. Cell Biol. 6:1109‐1113.
   Taipale, J., Chen, J.K., Cooper, M.K., Wang, B., Mann, R.K., Milenkovic, L., Scott, M.P., and Beachy, P.A. 2000. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406:1005‐1009.
   Taschner, M., Bhogaraju, S., and Lorentzen, E. 2011. Architecture and function of IFT complex proteins in ciliogenesis. Differentiation 83:S12‐S22.
   Taulman, P.D., Haycraft, C.J., Balkovetz, D.F., and Yoder, B.K. 2001. Polaris, a protein involved in left‐right axis patterning, localizes to basal bodies and cilia. Mol. Biol. Cell 12:589‐599.
   Wakabayashi, Y., Chua, J., Larkin, J.M, Lippincott‐Schwartz, J., and Arias, I.M. 2007. Four‐dimensional imaging of filter‐grown polarized epithelial cells. Histochem. Cell Biol. 127:463‐472.
   Yoshimura, S., Egerer, J., Fuchs, E., Haas, A.K, and Barr, F.A. 2007. Functional dissection of Rab GTPases involved in primary cilium formation. J. Cell Biol. 178:363‐369.
Internet Resources
  http://rsb.info.nih.gov/ij/
  Image J
  http://www.embl.de/eamnet/html/body_kymograph.html
  Image J kymograph plug‐ins
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library