A protocol for generation and live-cell imaging analysis of primary cilia reporter cell lines

. 2022 Mar 18 ; 3 (1) : 101199. [epub] 20220302

Jazyk angličtina Země Spojené státy americké Médium electronic-ecollection

Typ dokumentu časopisecké články, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/pmid35257113
Odkazy

PubMed 35257113
PubMed Central PMC8897589
DOI 10.1016/j.xpro.2022.101199
PII: S2666-1667(22)00079-X
Knihovny.cz E-zdroje

Primary cilia are hair-like sensory organelles protruding from the surface of most human cells. As cilia are dynamic, several aspects of their biology can only be revealed by real-time analysis in living cells. Here we describe the generation of primary cilia reporter cell lines. Furthermore, we provide a detailed protocol of how to use the reporter cell lines for live-cell imaging microscopy analysis of primary cilia to study their growth as well as intraciliary transport. For complete details on the use and execution of this protocol, please refer to Bernatik et al. (2020) and Pejskova et al. (2020).

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Bansal R., Engle S.E., Kamba T.K., Brewer K.M., Lewis W.R., Berbari N.F. Artificial intelligence approaches to assessing primary cilia. J. Vis. Exp. 2021:e62521. doi: 10.3791/62521. PubMed DOI PMC

Bauer M., Cubizolles F., Schmidt A., Nigg E.A. Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging. EMBO J. 2016;35:2152–2166. doi: 10.15252/embj.201694462. PubMed DOI PMC

Bernatik O., Paclikova P., Kotrbova A., Bryja V., Cajanek L. Primary cilia formation does not rely on WNT/β-catenin signaling. Front. Cell Dev. Biol. 2021;9:623753. doi: 10.3389/fcell.2021.623753. PubMed DOI PMC

Bernatik O., Pejskova P., Vyslouzil D., Hanakova K., Zdrahal Z., Cajanek L. Phosphorylation of multiple proteins involved in ciliogenesis by Tau Tubulin kinase 2. Mol. Biol. Cell. 2020;31:1032–1046. doi: 10.1091/mbc.E19-06-0334. PubMed DOI PMC

Bhogaraju S., Cajanek L., Fort C., Blisnick T., Weber K., Taschner M., Mizuno N., Lamla S., Bastin P., Nigg E.A., Lorentzen E. Molecular basis of tubulin transport within the cilium by IFT74 and IFT81. Science. 2013;341:1009–1012. doi: 10.1126/science.1240985. PubMed DOI PMC

Caspary T., Larkins C.E., Anderson K.V. The graded response to Sonic Hedgehog depends on cilia architecture. Dev. Cell. 2007;12:767–778. doi: 10.1016/j.devcel.2007.03.004. PubMed DOI

Cho N.H., Cheveralls K.C., Brunner A.-D., Kim K., Michaelis A.C., Raghavan P., Kobayashi H., Savy L., Li J.Y., Canaj H., et al. OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization. 2021. PubMed DOI PMC

Goetz S.C., Liem K.F., Jr., Anderson K.V. The spinocerebellar ataxia-associated gene Tau tubulin kinase 2 controls the initiation of ciliogenesis. Cell. 2012;151:847–858. doi: 10.1016/j.cell.2012.10.010. PubMed DOI PMC

Hansen J.N., Rassmann S., Stüven B., Jurisch-Yaksi N., Wachten D. CiliaQ: a simple, open-source software for automated quantification of ciliary morphology and fluorescence in 2D, 3D, and 4D images. Eur. Phys. J. E Soft Matter. 2021;44:18. doi: 10.1140/epje/s10189-021-00031-y. PubMed DOI PMC

Ishikawa H., Marshall W.F. Efficient live fluorescence imaging of intraflagellar transport in mammalian primary cilia. Methods Cell Biol. 2015;127:189–201. doi: 10.1016/bs.mcb.2015.01.002. PubMed DOI PMC

Jakobs M.A., Dimitracopoulos A., Franze K. KymoButler, a deep learning software for automated kymograph analysis. eLife. 2019;8:e42288. doi: 10.7554/eLife.42288. PubMed DOI PMC

Kiesel P., Alvarez Viar G., Tsoy N., Maraspini R., Gorilak P., Varga V., Honigmann A., Pigino G. The molecular structure of mammalian primary cilia revealed by cryo-electron tomography. Nat. Struct. Mol. Biol. 2020;27:1115–1124. doi: 10.1038/s41594-020-0507-4. PubMed DOI PMC

Kim J., Lee J.E., Heynen-Genel S., Suyama E., Ono K., Lee K., Ideker T., Aza-Blanc P., Gleeson J.G. Functional genomic screen for modulators of ciliogenesis and cilium length. Nature. 2010;464:1048–1051. doi: 10.1038/nature08895. PubMed DOI PMC

Larkins C.E., Aviles G.D.G., East M.P., Kahn R.A., Caspary T. Arl13b regulates ciliogenesis and the dynamic localization of Shh signaling proteins. MBoC. 2011;22:4694–4703. doi: 10.1091/mbc.e10-12-0994. PubMed DOI PMC

Lauring M.C., Zhu T., Luo W., Wu W., Yu F., Toomre D. New software for automated cilia detection in cells (ACDC) Cilia. 2019;8:1. doi: 10.1186/s13630-019-0061-z. PubMed DOI PMC

Longo P.A., Kavran J.M., Kim M.-S., Leahy D.J. Transient mammalian cell transfection with polyethylenimine (PEI) Methods Enzymol. 2013;529:227–240. doi: 10.1016/B978-0-12-418687-3.00018-5. PubMed DOI PMC

Mangeol P., Prevo B., Peterman E.J.G. KymographClear and KymographDirect: two tools for the automated quantitative analysis of molecular and cellular dynamics using kymographs. MBoC. 2016;27:1948–1957. doi: 10.1091/mbc.e15-06-0404. PubMed DOI PMC

Martin-Fernandez M., Tynan C., Webb S. A ‘pocket guide’ to total internal reflection fluorescence. J. Microsc. 2013;252:16–22. doi: 10.1111/jmi.12070. PubMed DOI PMC

Mazo G., Soplop N., Wang W.J., Uryu K., Tsou M.B. Spatial control of primary ciliogenesis by subdistal appendages alters sensation-associated properties of cilia. Dev. Cell. 2016;39:424–437. doi: 10.1016/j.devcel.2016.10.006. PubMed DOI PMC

Mok H.P., Javed S., Lever A. Stable gene expression occurs from a minority of integrated HIV-1-based vectors: transcriptional silencing is present in the majority. Gene Ther. 2007;14:741–751. doi: 10.1038/sj.gt.3302923. PubMed DOI

Naviaux R.K., Costanzi E., Haas M., Verma I.M. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 1996;70:5701–5705. doi: 10.1128/JVI.70.8.5701-5705.1996. PubMed DOI PMC

Pejskova P., Reilly M.L., Bino L., Bernatik O., Dolanska L., Ganji R.S., Zdrahal Z., Benmerah A., Cajanek L. KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling. J. Cell Biol. 2020;219:e201904107. doi: 10.1083/jcb.201904107. PubMed DOI PMC

Prasai A., Schmidt Cernohorska M., Ruppova K., Niederlova V., Andelova M., Draber P., Stepanek O., Huranova M. The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells. J. Biol. Chem. 2020;295:14279–14290. doi: 10.1074/jbc.RA120.013905. PubMed DOI PMC

Rosenbaum J.L., Witman G.B. Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 2002;3:813–825. doi: 10.1038/nrm952. PubMed DOI

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Shaner N.C., Lambert G.G., Chammas A., Ni Y., Cranfill P.J., Baird M.A., Sell B.R., Allen J.R., Day R.N., Israelsson M., et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods. 2013;10:407–409. doi: 10.1038/nmeth.2413. PubMed DOI PMC

Sorokin S. Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J. Cell Biol. 1962;15:363–377. PubMed PMC

Sowa M.E., Bennett E.J., Gygi S.P., Harper J.W. Defining the human deubiquitinating enzyme interaction landscape. Cell. 2009;138:389–403. doi: 10.1016/j.cell.2009.04.042. PubMed DOI PMC

Swift S., Lorens J., Achacoso P., Nolan G.P. Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell–based systems. Curr. Protoc. Immunol. 1999;31:10.17.14–10.17.29. doi: 10.1002/0471142735.im1017cs31. PubMed DOI

Tokunaga M., Imamoto N., Sakata-Sogawa K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods. 2008;5:159–161. doi: 10.1038/nmeth1171. PubMed DOI

Ward R.J., Alvarez-Curto E., Milligan G. Using the Flp-InTM T-RexTM system to regulate GPCR expression. Methods Mol. Biol. 2011;746:21–37. doi: 10.1007/978-1-61779-126-0_2. PubMed DOI

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