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Cell Models Adapted to Real-Time Imaging of the Cytoskeleton Dynamics in Altered Gravity

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Abstract

Spatial and temporal regulation of cell phenotype by mechanical forces is a growing field of research in health sciences since these stimuli influence cellular functions, such as proliferation, migration, differentiation and gene expression. In the context of the Fluolive project selected by the European Space Agency and aiming at evaluating the impact of gravity alterations on the cell phenotype, we have developed new bone-derived cell lines adapted for live-cell imaging of the cytoskeleton. Osteoblastic cells derived from human osteosarcomas were used as experimental models. U2-OS and SaoS-2 cells stably expressing TagGFP2- β-actin and mCherry- α-tubulin were established and single-cell clonal cultures were characterized in terms of recombinant proteins production and localization, fluorescence intensity, cell proliferation and migration rates. Living fluorescently-tagged cell lines allow real-time fluorescence microscopy of the cytoskeleton dynamics without bleaching and without alteration of cell morphology. U2-OS and SaoS-2 TagGFP2- β-actin and mCherry- α-tubulin clones will be used to monitor the effect of mechanical forces in models of altered gravity on Earth and possibly on the ISS.

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References

  • Akhmanova, A., Stehbens, S.J., Yap, A.S.: Touch, grasp, deliver and control: Functional cross-talk between microtubules and cell adhesions. Traffic 10(3), 268–274 (2009). doi:10.1111/j.1600-0854.2008.00869.x

    Article  Google Scholar 

  • Ballestrem, C., Wehrle-Haller, B., Imhof, B.A.: Actin dynamics in living mammalian cells. J. Cell. Sci. 111(Pt 12), 1649–1658 (1998)

    Google Scholar 

  • Becker, J., Schuppan, D., Benzian, H., Bals, T., Hahn, E.G., Cantaluppi, C., Reichart, P.: Immunohistochemical distribution of collagens types IV, V, and VI and of pro-collagens types I and III in human alveolar bone and dentine. J. Histochem. Cytochem. 34(11), 1417–1429 (1986)

    Article  Google Scholar 

  • Chhabra, E.S., Higgs, H.N.: The many faces of actin: Matching assembly factors with cellular structures. Nat. Cell. Biol. 9(10), 1110–1121 (2007). doi:10.1038/ncb1007-1110

    Article  Google Scholar 

  • Chichester, C.O., Fernandez, M., Minguell, J.J.: Extracellular matrix gene expression by human bone marrow stroma and by marrow fibroblasts. Cell Adhes. Commun. 1(2), 93–99 (1993)

    Article  Google Scholar 

  • Chiquet, M., Gelman, L., Lutz, R., Maier, S.: From mechanotransduction to extracellular matrix gene expression in fibroblasts. Biochim. Biophys. Acta 1793(5), 911–920 (2009). doi:10.1016/j.bbamcr.2009.01.012

    Article  Google Scholar 

  • Cooper, J.A.: Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105(4), 1473–1478 (1987)

    Article  Google Scholar 

  • Czekanska, E.M., Stoddart, M.J., Richards, R.G., Hayes, J.S.: In search of an osteoblast cell model for in vitro research. Eur. Cell Mater. 24, 1–17 (2012)

    Google Scholar 

  • Discher, D.E., Janmey, P., Wang, Y.L.: Tissue cells feel and respond to the stiffness of their substrate. Science 310(5751), 1139–1143 (2005). doi:10.1126/science.1116995

    Article  Google Scholar 

  • Emi, N., Friedmann, T., Yee, J.K.: Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus. J. Virol. 65(3), 1202–1207 (1991)

    Google Scholar 

  • Etienne-Manneville, S.: Actin and microtubules in cell motility: Which one is in control? Traffic 5(7), 470–477 (2004). doi:10.1111/j.1600-0854.2004.00196.x

    Article  Google Scholar 

  • Etienne-Manneville, S.: Microtubules in cell migration. Annu. Rev. Cell Dev. Biol. 29, 471–499 (2013). doi:10.1146/annurev-cellbio-101011-155711

    Article  Google Scholar 

  • Even-Ram, S., Doyle, A.D., Conti, M.A., Matsumoto, K., Adelstein, R.S., Yamada, K.M.: Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk. Nat. Cell Biol. 9(3), 299–309 (2007). doi:10.1038/ncb1540

    Article  Google Scholar 

  • Fletcher, D.A., Mullins, R.D.: Cell mechanics and the cytoskeleton. Nature 463 (7280), 485–492 (2010)

    Article  Google Scholar 

  • Frantz, C., Stewart, K.M., Weaver, V.M.: The extracellular matrix at a glance. J. Cell Sci. 123(Pt 24), 4195–4200 (2010). doi:10.1242/jcs.023820

    Article  Google Scholar 

  • Geeraert, C., Ratier, A., Pfisterer, S.G., Perdiz, D., Cantaloube, I., Rouault, A., Pattingre, S., Proikas-Cezanne, T., Codogno, P., Pous, C.: Starvation-induced hyperacetylation of tubulin is required for the stimulation of autophagy by nutrient deprivation. J. Biol. Chem. 285(31), 24184–24194 (2010). doi:10.1074/jbc.M109.091553

    Article  Google Scholar 

  • Guignandon, A., Akhouayri, O., Laroche, N., Lafage-Proust, M.H., Alexandre, C., Vico, L.: Focal contacts organization in osteoblastic cells under microgravity and cyclic deformation conditions. Adv. Space Res. 32(8), 1561–1567 (2003). doi:10.1016/s0273-1177(03)90396-6

    Article  Google Scholar 

  • Guignandon, A., Faure, C., Neutelings, T., Rattner, A., Mineur, P., Linossier, M.T., Laroche, N., Lambert, C., Deroanne, C., Nusgens, B., Demets, R., Colige, A., Vico, L.: Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells. FASEB journal: Official publication of the Federation of American Societies for Experimental Biology doi:10.1096/fj.14-249714 (2014)

  • Halper, J., Kjaer, M.: Basic components of connective tissues and extracellular matrix: Elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins. Adv. Exp. Med. Biol. 802, 31–47 (2014). doi:10.1007/978-94-007-7893-1_3

    Article  Google Scholar 

  • Horgan, C.P., McCaffrey, M.W.: Rab GTPases and microtubule motors. Biochem. Soc. Trans. 39(5), 1202–1206 (2011). doi:10.1042/BST0391202

    Article  Google Scholar 

  • Ingber, D.E.: Mechanobiology and diseases of mechanotransduction. Ann. Med. 35(8), 564–577 (2003a)

    Article  Google Scholar 

  • Ingber, D.E.: Tensegrity I. cell structure and hierarchical systems biology. J. Cell Sci. 116(Pt 7), 1157–1173 (2003b)

    Article  Google Scholar 

  • Jaalouk, D.E., Lammerding, J.: Mechanotransduction gone awry. Nat. Rev. Mol. Cell Biol. 10(1), 63–73 (2009). doi:10.1038/nrm2597

    Article  Google Scholar 

  • Janmey, P.A., Miller, R.T.: Mechanisms of mechanical signaling in development and disease. J. Cell Sci. 124(Pt 1), 9–18 (2011). doi:10.1242/jcs.071001

    Article  Google Scholar 

  • Johnson, I.D.: Practical considerations in the selection and application of fluorescent probes. In: Handbook of Biological Confocal Microscopy, pp. 353-367. Springer (2006)

  • Johnson, R.B.: The bearable lightness of being: Bones, muscles, and spaceflight. Anat. Rec. 253(1), 24–27 (1998)

    Article  Google Scholar 

  • Kartsogiannis, V., Ng, K.W.: Cell lines and primary cell cultures in the study of bone cell biology. Mol. Cell Endocrinol. 228(1-2), 79–102 (2004). doi:10.1016/j.mce.2003.06.002

    Article  Google Scholar 

  • Kline-Smith, S.L., Walczak, C.E.: Mitotic spindle assembly and chromosome segregation: refocusing on microtubule dynamics. Mol. Cell 15(3), 317–327 (2004). doi:10.1016/j.molcel.2004.07.012

    Article  Google Scholar 

  • Lang, T., LeBlanc, A., Evans, H., Lu, Y., Genant, H., Yu, A.: Cortical and trabecular bone mineral loss from the spine and hip in long-duration spaceflight. J. Bone Miner. Res. Off. J. Amer. Soc. Bone Miner. Res. 19(6), 1006–1012 (2004). doi:10.1359/jbmr.040307

    Article  Google Scholar 

  • Lanyon, L.E.: Osteocytes, strain detection, bone modeling and remodeling. Calcif Tissue Int 53 Suppl 1, S102–106; discussion S106–107 (1993)

    Article  Google Scholar 

  • Mammoto, A., Mammoto, T., Ingber, D.E.: Mechanosensitive mechanisms in transcriptional regulation. J. Cell Sci. 125(Pt 13), 3061–3073 (2012). doi:10.1242/jcs.093005

    Article  Google Scholar 

  • Martin, R.B.: The importance of mechanical loading in bone biology and medicine. J. Musculoskelet Neuronal Interact 7(1), 48–53 (2007)

    Google Scholar 

  • Mitchison, T., Kirschner, M.: Dynamic instability of microtubule growth. Nature 312(5991), 237–242 (1984)

    Article  Google Scholar 

  • Mounier, N., Perriard, J.C., Gabbiani, G., Chaponnier, C.: Transfected muscle and non-muscle actins are differentially sorted by cultured smooth muscle and non-muscle cells. J. Cell Sci. 110(Pt 7), 839–846 (1997)

    Google Scholar 

  • Nabavi, N., Khandani, A., Camirand, A., Harrison, R.E.: Effects of microgravity on osteoclast bone resorption and osteoblast cytoskeletal organization and adhesion. Bone 49(5), 965–974 (2011). doi:10.1016/j.bone.2011.07.036

    Article  Google Scholar 

  • Pautke, C., Schieker, M., Tischer, T., Kolk, A., Neth, P., Mutschler, W., Milz, S.: Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts. Anticancer Res. 24(6), 3743–3748 (2004)

    Google Scholar 

  • Pollard, T.D., Borisy, G.G.: Cellular motility driven by assembly and disassembly of actin filaments. Cell 112(4), 453–465 (2003)

    Article  Google Scholar 

  • Ridley, A.J.: Life at the leading edge. Cell 145(7), 1012–1022 (2011). doi:10.1016/j.cell.2011.06.010

    Article  Google Scholar 

  • Roca-Cusachs, P., Iskratsch, T., Sheetz, M.P.: Finding the weakest link: Exploring integrin-mediated mechanical molecular pathways. J. Cell Sci. 125(Pt 13), 3025–3038 (2012). doi:10.1242/jcs.095794

    Article  Google Scholar 

  • Sadoshima, J., Jahn, L., Takahashi, T., Kulik, T.J., Izumo, S.: Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. An in vitro model of load-induced cardiac hypertrophy. J. Biol. Chem. 267(15), 10551–10560 (1992)

    Google Scholar 

  • Salbreux, G., Charras, G., Paluch, E.: Actin cortex mechanics and cellular morphogenesis. Trends Cell Biol. 22(10), 536–545 (2012) doi:10.1016/j.tcb.2012.07.001

    Article  Google Scholar 

  • Schambach, A., Zychlinski, D., Ehrnstroem, B., Baum, C.: Biosafety features of lentiviral vectors. Hum. Gene Ther. 24(2), 132–142 (2013). doi:10.1089/hum.2012.229

    Article  Google Scholar 

  • Schuh, M.: An actin-dependent mechanism for long-range vesicle transport. Nat. Cell Biol. 13(12), 1431–1436 (2011). doi:10.1038/ncb2353

    Article  Google Scholar 

  • Schwarz, U.S., Gardel, M.L.: United we stand: Integrating the actin cytoskeleton and cell-matrix adhesions in cellular mechanotransduction. J. Cell Sci. 125(Pt 13), 3051–3060 (2012). doi:10.1242/jcs.093716

    Article  Google Scholar 

  • Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., Tsien, R.Y.: Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22(12), 1567–1572 (2004). doi:10.1038/nbt1037

    Article  Google Scholar 

  • Sorger, P.K., Dobles, M., Tournebize, R., Hyman, A.A.: Coupling cell division and cell death to microtubule dynamics. Curr. Opin. Cell Biol. 9(6), 807–814 (1997)

    Article  Google Scholar 

  • Stehbens, S., Wittmann, T.: Targeting and transport: How microtubules control focal adhesion dynamics. J Cell Biol 198(4), 481–489 (2012). doi:10.1083/jcb.201206050

    Article  Google Scholar 

  • Stricker, J., Falzone, T., Gardel, M.L.: Mechanics of the F-actin cytoskeleton. J. Biomech. 43(1), 9–14 (2010). doi:10.1016/j.jbiomech.2009.09.003

    Article  Google Scholar 

  • Subach, O.M., Gundorov, I.S., Yoshimura, M., Subach, F.V., Zhang, J., Gruenwald, D., Souslova, E.A., Chudakov, D.M., Verkhusha, V.V.: Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 15(10), 1116–1124 (2008). doi:10.1016/j.chembiol.2008.08.006

    Article  Google Scholar 

  • Tojkander, S., Gateva, G., Lappalainen, P.: Actin stress fibers–assembly, dynamics and biological roles. J. Cell Sci. 125(Pt 8), 1855–1864 (2012). doi:10.1242/jcs.098087

    Article  Google Scholar 

  • Van Loon, J., Krause, J., Cunha, H., Goncalves, J., Almeida, H., Schiller, P.: The large diameter centrifuge, LDC, for life and physical sciences and technology. In: Proceedings of the ‘Life In Space For Life On Earth Symposium’, pp. 22–27. Angers (2008)

  • van Loon, J.J.: Some history and use of the random positioning machine, RPM, in gravity related research. Adv. Space Res. 39(7), 1161–1165 (2007)

    Article  Google Scholar 

  • Vassy, J., Portet, S., Beil, M., Millot, G., Fauvel-Lafeve, F., Gasset, G., Schoevaert, D.: Weightlessness acts on human breast cancer cell line MCF-7. Adv. Space Res. 32(8), 1595–1603 (2003)

    Article  Google Scholar 

  • Vicente-Manzanares, M., Ma, X., Adelstein, R.S., Horwitz, A.R.: Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat. Rev. Mol. Cell Biol. 10(11), 778–790 (2009). doi:10.1038/nrm2786

    Article  Google Scholar 

  • Wang, N., Tytell, J.D., Ingber, D.E.: Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus. Nat. Rev. Mol. Cell Biol. 10(1), 75–82 (2009). doi:10.1038/nrm2594

    Article  Google Scholar 

  • Wanisch, K., Yanez-Munoz, R.J.: Integration-deficient lentiviral vectors: a slow coming of age. Mol. Ther. 17(8), 1316–1332 (2009). doi:10.1038/mt.2009.122

    Article  Google Scholar 

  • Wittmann, T., Waterman-Storer, C.M.: Cell motility: Can Rho GTPases and microtubules point the way? J. Cell Sci. 114(Pt 21), 3795–3803 (2001)

    Google Scholar 

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Acknowledgments

The lentiviral strategy was developed thanks to E. Di Valentin (Viral Vectors Platform, GIGA, University of Liège, Belgium). We thank the GIGA-Imaging and Flow Cytometry Platform (University of Liège, Belgium) for FACS analyses. We thank Pr. Poüs and Dr. Tsien for sharing plasmids. A. Colige and C. Deroanne are supported by the Fond de la Recherche Scientifique-FNRS. J. Willems and N. Garbacki are supported by Belspo/Prodex.

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  1. Laboratory of Connective Tissues Biology, GIGA-Research, University of Liège, avenue de l’Hôpital, 3, 4000, Liège, Belgium

    Jérôme Willems, Christophe Deroanne, Alain Colige & Nancy Garbacki

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  1. Jérôme Willems
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  2. Christophe Deroanne
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Correspondence to Alain Colige.

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Willems, J., Deroanne, C., Colige, A. et al. Cell Models Adapted to Real-Time Imaging of the Cytoskeleton Dynamics in Altered Gravity. Microgravity Sci. Technol. 26, 257–270 (2014). https://doi.org/10.1007/s12217-014-9392-y

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