Live-cell fluorescence imaging of ciliary dynamics

Quanlong Lü; Chuanmao Zhang; Christopher J. Westlake

doi:10.52601/bpr.2021.210005

Volume 7 Issue 2

Feb. 2021

Turn off MathJax

Article Contents

Article Navigation > Biophysics Reports > 2021 > 7(2): 101-110

Quanlong Lü, Chuanmao Zhang, Christopher J. Westlake. Live-cell fluorescence imaging of ciliary dynamics. Biophysics Reports, 2021, 7(2): 101-110. doi: 10.52601/bpr.2021.210005

Citation:

Quanlong Lü, Chuanmao Zhang, Christopher J. Westlake. Live-cell fluorescence imaging of ciliary dynamics. Biophysics Reports, 2021, 7(2): 101-110. doi: 10.52601/bpr.2021.210005

Quanlong Lü, Chuanmao Zhang, Christopher J. Westlake. Live-cell fluorescence imaging of ciliary dynamics. Biophysics Reports, 2021, 7(2): 101-110. doi: 10.52601/bpr.2021.210005

Citation:

Quanlong Lü, Chuanmao Zhang, Christopher J. Westlake. Live-cell fluorescence imaging of ciliary dynamics. Biophysics Reports, 2021, 7(2): 101-110. doi: 10.52601/bpr.2021.210005

PDF (1466 KB)

Live-cell fluorescence imaging of ciliary dynamics

doi: 10.52601/bpr.2021.210005

1.
Laboratory of Cellular and Developmental Signaling, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
2.
Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing 100871, China

Funds: We are grateful to Dr. Adrian Cuenca for the critical reading and editing of this manuscript. Q. Lü and C. J. Westlake are supported by federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. C. Zhang is supported by the National Natural Science Foundation of China (91854204) and the Ministry of Science and Technology of China (2016YFA0500201 and 2016YFA0100501).

More Information

Corresponding author: quanlong@outlook.com (Q. Lü); chris.westlake@nih.gov (C. J. Westlake)
Received Date: 09 February 2021
Accepted Date: 23 March 2021

Available Online: 17 May 2021

Publish Date: 28 February 2021

Abstract

Abstract

The cilium was one of the first organelles observed through a microscope. Motile cilia appear as oscillating cell appendages and have long been recognized to function in cell motility. In contrast, the far more widespread non-motile cilia, termed primary cilia, were thought to be vestigial and largely ignored following their initial description over a century ago. Only in the last two decades has the critical function of primary cilia been elucidated. Primary cilia play essential roles in signal transduction, chemical sensation, mechanosensation and light detection. Various microscopy approaches have been important for characterizing the structure, dynamics and function of the cilia. In this review, we discuss the application of live-cell imaging technologies and their contribution to our current understanding of ciliary processes.
- Cilia,
- Ciliogenesis,
- Ciliary dynamics,
- Live-cell fluorescence imaging

FullText(HTML)

References(59)

References

[1]	Bloodgood RA (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
[2]	Chadha A, Volland S, Baliaouri NV, Tran EM, Williams DS (2019) The route of the visual receptor rhodopsin along the cilium. J Cell Sci 132: jcs229526. https://doi.org/10.1242/jcs.229526
[3]	Crivat G, Taraska JW (2012) Imaging proteins inside cells with fluorescent tags. Trends Biotechnol 30(1): 8−16 doi: 10.1016/j.tibtech.2011.08.002
[4]	Diener D (2009) Analysis of cargo transport by IFT and GFP imaging of IFT in Chlamydomonas. Methods Cell Biol 93: 111−119
[5]	Emmer BT, Maric D, Engman DM (2010) Molecular mechanisms of protein and lipid targeting to ciliary membranes. J Cell Sci 123(Pt 4): 529−536
[6]	Flaherty J, Feng Z, Peng Z, Young YN, Resnick A (2020) Primary cilia have a length-dependent persistence length. Biomech Model Mechanobiol 19(2): 445−460 doi: 10.1007/s10237-019-01220-7
[7]	Follit JA, Tuft RA, Fogarty KE, Pazour GJ (2006) The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly. Mol Biol Cell 17(9): 3781−3792 doi: 10.1091/mbc.e06-02-0133
[8]	Ford MJ, Yeyati PL, Mali GR, Keighren MA, Waddell SH, Mjoseng HK, Douglas AT, Hall EA, Sakaue-Sawano A, Miyawaki A, Meehan RR, Boulter L, Jackson IJ, Mill P, Mort RL (2018) A cell/cilia cycle biosensor for single-cell kinetics reveals persistence of cilia after G1/S transition is a general property in cells and mice. Dev Cell 47(4): 509−523 e505 doi: 10.1016/j.devcel.2018.10.027
[9]	Fouquet C, Gilles JF, Heck N, Dos Santos M, Schwartzmann R, Cannaya V, Morel MP, Davidson RS, Trembleau A, Bolte S (2015) Improving axial resolution in confocal microscopy with new high refractive index mounting media. PLoS One 10(3): e0121096. https://doi.org/10.1371/journal.pone.0121096
[10]	Frigault MM, Lacoste J, Swift JL, Brown CM (2009) Live-cell microscopy -- tips and tools. J Cell Sci 122(Pt 6): 753−767
[11]	Fu W, Wang L, Kim S, Li J, Dynlacht BD (2016) Role for the IFT-A complex in selective transport to the primary cilium. Cell Rep 17(6): 1505−1517 doi: 10.1016/j.celrep.2016.10.018
[12]	Gautier A, Juillerat A, Heinis C, Correa IR, J r., Kindermann M, Beaufils F, Johnsson K (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15(2): 128−136 doi: 10.1016/j.chembiol.2008.01.007
[13]	Guadiana SM, Semple-Rowland S, Daroszewski D, Madorsky I, Breunig JJ, Mykytyn K, Sarkisian MR (2013) Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase. J Neurosci 33(6): 2626−2638 doi: 10.1523/JNEUROSCI.2906-12.2013
[14]	He X, Tan C, Wang F, Wang Y, Zhou R, Cui D, You W, Zhao H, Ren J, Feng B (2016) Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res 44(9): e85. https://doi.org/10.1093/nar/gkw064
[15]	Hu L, Wang B, Zhang Y (2017) Serotonin 5-HT6 receptors affect cognition in a mouse model of Alzheimer's disease by regulating cilia function. Alzheimers Res Ther 9(1): 76. https://doi.org/10.1186/s13195-017-0304-4
[16]	Hu Q, Milenkovic L, Jin H, Scott MP, Nachury MV, Spiliotis ET, Nelson WJ (2010) A septin diffusion barrier at the base of the primary cilium maintains ciliary membrane protein distribution. Science 329(5990): 436−439 doi: 10.1126/science.1191054
[17]	Insinna C, Lu Q, Teixeira I, Harned A, Semler EM, Stauffer J, Magidson V, Tiwari A, Kenworthy AK, Narayan K, Westlake CJ (2019a) Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport. Nat Commun 10(1): 428. https://doi.org/10.1038/s41467-018-08192-9
[18]	Insinna C, Lu Q, Teixeira I, Harned A, Semler EM, Stauffer J, Magidson V, Tiwari A, Kenworthy AK, Narayan K, Westlake CJ (2019b) Publisher correction: investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport. Nat Commun 10(1): 919. https://doi.org/10.1038/s41467-019-08872-0
[19]	Ishikawa H, Marshall WF (2015) Efficient live fluorescence imaging of intraflagellar transport in mammalian primary cilia. Methods Cell Biol 127: 189−201
[20]	Ishikawa H, Marshall WF (2017) Intraflagellar transport and ciliary dynamics. Cold Spring Harb Perspect Biol 9(3): a021998. https://doi.org/10.1101/cshperspect.a021998
[21]	Iwanaga T, Miki T, Takahashi-Iwanaga H (2011) Restricted expression of somatostatin receptor 3 to primary cilia in the pancreatic islets and adenohypophysis of mice. Biomed Res 32(1): 73−81 doi: 10.2220/biomedres.32.73
[22]	Jensen O, Ansari S, Gebauer L, Muller SF, Lowjaga K, Geyer J, Tzvetkov MV, Brockmoller J (2020) A double-Flp-in method for stable overexpression of two genes. Sci Rep 10(1): 14018. https://doi.org/10.1038/s41598-020-71051-5
[23]	Jiang YY, Lechtreck K, Gaertig J (2015) Total internal reflection fluorescence microscopy of intraflagellar transport in Tetrahymena thermophila. Methods Cell Biol 127: 445−456
[24]	Kozminski KG, Johnson KA, Forscher P, Rosenbaum JL (1993) A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad Sci USA 90(12): 5519−5523 doi: 10.1073/pnas.90.12.5519
[25]	Kukic I, Rivera-Molina F, Toomre D (2016) The IN/OUT assay: a new tool to study ciliogenesis. Cilia 5: 23. https://doi.org/10.1186/s13630-016-0044-2
[26]	Lechtreck KF (2013) In vivo imaging of IFT in Chlamydomonas flagella. Methods Enzymol 524: 265−284
[27]	Lechtreck KF (2016) Methods for studying movement of molecules within cilia. Methods Mol Biol 1454: 83−96
[28]	Lee N, Park J, Bae YC, Lee JH, Kim CH, Moon SJ (2018) Time-lapse live-cell imaging reveals dual function of Oseg4, Drosophila WDR35, in ciliary protein trafficking. Mol Cells 41(7): 676−683
[29]	Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, Wood MG, Learish R, Ohana RF, Urh M, Simpson D, Mendez J, Zimmerman K, Otto P, Vidugiris G, Zhu J, Darzins A, Klaubert DH, Bulleit RF, Wood KV (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6): 373−382 doi: 10.1021/cb800025k
[30]	Lu Q, Insinna C, Ott C, Stauffer J, Pintado PA, Rahajeng J, Baxa U, Walia V, Cuenca A, Hwang YS, Daar IO, Lopes S, Lippincott-Schwartz J, Jackson PK, Caplan S, Westlake CJ (2015a) Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat Cell Biol 17(4): 531. https://doi.org/10.1038/ncb3155
[31]	Lu Q, Insinna C, Ott C, Stauffer J, Pintado PA, Rahajeng J, Baxa U, Walia V, Cuenca A, Hwang YS, Daar IO, Lopes S, Lippincott-Schwartz J, Jackson PK, Caplan S, Westlake CJ (2015b) Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat Cell Biol 17(3): 228−240 doi: 10.1038/ncb3109
[32]	Mazo G, Soplop N, Wang WJ, Uryu K, Tsou MF (2016) Spatial control of primary ciliogenesis by subdistal appendages alters sensation-associated properties of cilia. Dev Cell 39(4): 424−437 doi: 10.1016/j.devcel.2016.10.006
[33]	Mirvis M, Siemers KA, Nelson WJ, Stearns TP (2019) Primary cilium loss in mammalian cells occurs predominantly by whole-cilium shedding. PLoS Biol 17(7): e3000381. https://doi.org/10.1371/journal.pbio.3000381
[34]	Murray JM (2011) Methods for imaging thick specimens: confocal microscopy, deconvolution, and structured illumination. Cold Spring Harb Protoc 2011(12): 1399−1437
[35]	Musgrave A, de Wildt P, van Etten I, Pijst H, Scholma C, Kooyman R, Homan W, van den Ende H (1986) Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonas eugametos gametes. Planta 167(4): 544−553 doi: 10.1007/BF00391231
[36]	Ocbina PJ, Anderson KV (2008) Intraflagellar transport, cilia, and mammalian Hedgehog signaling: analysis in mouse embryonic fibroblasts. Dev Dyn 237(8): 2030−2038 doi: 10.1002/dvdy.21551
[37]	Ott C, Lippincott-Schwartz J (2012) Visualization of live primary cilia dynamics using fluorescence microscopy. Curr Protoc Cell Biol Chapter 4: Unit 4.26. https://doi.org/10.1002/0471143030.cb0426s57
[38]	Paridaen JT, Wilsch-Brauninger M, Huttner WB (2013) Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division. Cell 155(2): 333−344 doi: 10.1016/j.cell.2013.08.060
[39]	Phua SC, Chiba S, Suzuki M, Su E, Roberson EC, Pusapati GV, Schurmans S, Setou M, Rohatgi R, Reiter JF, Ikegami K, Inoue T (2017) Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision. Cell 168(1-2): 264−279 doi: 10.1016/j.cell.2016.12.032
[40]	Piotrowska-Nitsche K, Caspary T (2012) Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response. Cilia 1(1): 6. https://doi.org/10.1186/2046-2530-1-6
[41]	Plotnikova OV, Pugacheva EN, Golemis EA (2009) Primary cilia and the cell cycle. Methods Cell Biol 94: 137−160
[42]	Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren BZ, Lahn BT (2010) Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 5(5): e10611. https://doi.org/10.1371/journal.pone.0010611
[43]	Rangel L, Bernabe-Rubio M, Fernandez-Barrera J, Casares-Arias J, Millan J, Alonso MA, Correas I (2019) Caveolin-1alpha regulates primary cilium length by controlling RhoA GTPase activity. Sci Rep 9(1): 1116. https://doi.org/10.1038/s41598-018-38020-5
[44]	Santos N, Reiter JF (2008) Building it up and taking it down: the regulation of vertebrate ciliogenesis. Dev Dyn 237(8): 1972−1981 doi: 10.1002/dvdy.21540
[45]	Satir P (1995) Landmarks in cilia research from Leeuwenhoek to us. Cell Motil Cytoskeleton 32(2): 90−94 doi: 10.1002/cm.970320203
[46]	Satir P, Christensen ST (2008) Structure and function of mammalian cilia. Histochem Cell Biol 129(6): 687−693 doi: 10.1007/s00418-008-0416-9
[47]	Shimada H, Lu Q, Insinna-Kettenhofen C, Nagashima K, English MA, Semler EM, Mahgerefteh J, Cideciyan AV, Li T, Brooks BP, Gunay-Aygun M, Jacobson SG, Cogliati T, Westlake CJ, Swaroop A (2017) In vitro modeling using ciliopathy-patient-derived cells reveals distinct cilia dysfunctions caused by CEP290 mutations. Cell Rep 20(2): 384−396 doi: 10.1016/j.celrep.2017.06.045
[48]	Sorokin SP (1968) Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J Cell Sci 3(2): 207−230
[49]	Stemmer A, Beck M, Fiolka R (2008) Widefield fluorescence microscopy with extended resolution. Histochem Cell Biol 130(5): 807−817 doi: 10.1007/s00418-008-0506-8
[50]	Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159(3): 635−646 doi: 10.1016/j.cell.2014.09.039
[51]	Toro-Tapia G, Das RM (2020) Primary cilium remodeling mediates a cell signaling switch in differentiating neurons. Sci Adv 6(21): eabb0601. https://doi.org/10.1126/sciadv.abb0601
[52]	Trivedi D, Colin E, Louie CM, Williams DS (2012) Live-cell imaging evidence for the ciliary transport of rod photoreceptor opsin by heterotrimeric kinesin-2. J Neurosci 32(31): 10587−10593 doi: 10.1523/JNEUROSCI.0015-12.2012
[53]	Tyler KM, Fridberg A, Toriello KM, Olson CL, Cieslak JA, Hazlett TL, Engman DM (2009) Flagellar membrane localization via association with lipid rafts. J Cell Sci 122(Pt 6): 859−866
[54]	Westlake CJ, Baye LM, Nachury MV, Wright KJ, Ervin KE, Phu L, Chalouni C, Beck JS, Kirkpatrick DS, Slusarski DC, Sheffield VC, Scheller RH, Jackson PK (2011) Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. Proc Natl Acad Sci USA 108(7): 2759−2764 doi: 10.1073/pnas.1018823108
[55]	Xie C, Li L, Li M, Shao W, Zuo Q, Huang X, Chen R, Li W, Brunnbauer M, Okten Z, Chen L, Ou G (2020) Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia. EMBO J 39(12): e103955. https://doi.org/10.15252/embj.2019103955
[56]	Ye F, Breslow DK, Koslover EF, Spakowitz AJ, Nelson WJ, Nachury MV (2013) Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors. Elife 2: e00654. https://doi.org/10.7554/eLife.00654
[57]	Zhang P, Kiseleva AA, Korobeynikov V, Liu H, Einarson MB, Golemis EA (2019) Microscopy-based automated live cell screening for small molecules that affect ciliation. Front Genet 10: 75. https://doi.org/10.3389/fgene.2019.00075
[58]	Zhou J (2009) Polycystins and primary cilia: primers for cell cycle progression. Annu Rev Physiol 71: 83−113 doi: 10.1146/annurev.physiol.70.113006.100621
[59]	Zimmermann KW (1898) Beiträge zur Kenntniss einiger Drüsen und Epithelien. Archiv für mikroskopische Anatomie 52(3): 552−706