我要登录|
免费注册
|
我的丁香通
- 企业机构：
- 成为企业机构
- 个人用户：
- 个人中心
移动端

大家都在搜

0 人通过求购买到了急需的产品

免费发布求购

发布求购

Visualization of Live Primary Cilia Dynamics Using Fluorescence Microscopy

互联网2013-12-31

1224

Abstract
Table of Contents
Materials
Figures
Videos
Literature Cited

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

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 CO₂ ‐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 CO₂ ‐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 CO₂ ‐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 CO₂ ‐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 CO₂ ‐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

Figure 4.26.1 Multiple fluorescent labels can be used to visualize primary cilia. (A and B ) MDCK cells stably expressing tubulinGFP. Without confocal imaging, the signal in the cilium can be swamped out by the cellular pool of tubulinGFP. A shows a side view of the cell (an x ‐ z slice). B shows a y ‐axis maximum intensity projection of a field of cells. (C ) The enrichment of SmoYFP in MDCK cilia is clear in this z ‐axis maximum intensity projection. (D ) An NIH3T3 cell expressing SmoYFP. In the top panel (this z ‐axis maximum intensity projection), the cilium appears to be a dot, but in the lower panel (an x ‐ z slice), it is clear that the cilium projects out of the cell. Levels were adjusted in D to make the cilium clearer. Scale bars = 5 µm.

View Image

Figure 4.26.2 Exogenously added fluorescent dyes can be used to visualize primary cilia. (A ) x ‐axis maximum intensity projection of MDCK cells stained with DOPE rhodamine. (B ) z ‐axis maximum intensity projection of NIH3T3 cells expressing inversinTdTomato (red) stained with BODIPY cholesterol (green). (C ) MDCK cell expressing tubulinGFP labeled with WGA rhodamine ( z ‐axis maximum intensity projection). Scale bars = 5 µm.

View Image

Figure 4.26.3 Generation of a kymograph to quantify cilia movement. After collecting a time series, a kymograph can be used to calculate the velocity of particle movements. This figure provides an overview of the steps required to generate a kymograph. Scale bars = 2 µm.

View Image

Figure 4.26.4 Using photobleaching and photoconversion to highlight moving proteins within the cilium. (A ) Kymographs generated from a time series where a region of a polaris‐GFP expressing cilium was photobleached during acquisition. The raw data was analyzed, and then processed with a walking average or median filter. Lines used for measurements are visible in the far right kymograph. (B ) SmoEos in the cilium of an MDCK cell was imaged with both 488‐ and 561‐nm light and then photoconverted. The movement of photoconverted particles is evident in the right panel (561 nm).

View Image

Videos

Movement within the cilium

Duration: 0:30

Play Video
Photoconversion highlights small pools of proteins

Duration: 0:23

Play Video

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