Hostname: page-component-cd9895bd7-mkpzs Total loading time: 0 Render date: 2024-12-26T16:52:41.210Z Has data issue: false hasContentIssue false
  • English
  • Français

Novel Electron Tomographic Methods to Study the Morphology of Keratin Filament Networks

Published online by Cambridge University Press:  02 July 2010

Michaela Sailer ,
Katharina Höhn ,
Sebastian Lück ,
Volker Schmidt ,
Michael Beil  and
Paul Walther
Michaela Sailer
Affiliation:
Electron Microscopy Facility, Ulm University, D-89069 Ulm, Germany
Katharina Höhn
Affiliation:
Electron Microscopy Facility, Ulm University, D-89069 Ulm, Germany
Sebastian Lück
Affiliation:
Institute of Stochastics, Ulm University, D-89069 Ulm, Germany
Volker Schmidt
Affiliation:
Institute of Stochastics, Ulm University, D-89069 Ulm, Germany
Michael Beil
Affiliation:
Department of Internal Medicine I, University Hospital Ulm, D-89070 Ulm, Germany
Paul Walther*
Affiliation:
Electron Microscopy Facility, Ulm University, D-89069 Ulm, Germany
*
Corresponding author. E-mail: [email protected]
Get access

Abstract

The three-dimensional (3D) keratin filament network of pancreatic carcinoma cells was investigated with different electron microscopical approaches. Semithin sections of high-pressure frozen and freeze substituted cells were analyzed with scanning transmission electron microscope (STEM) tomography. Preservation of subcellular structures was excellent, and keratin filaments could be observed; however, it was impossible to three-dimensionally track the individual filaments. To obtain a better signal-to-noise ratio in transmission mode, we observed ultrathin sections of high-pressure frozen and freeze substituted samples with low-voltage (30 kV) STEM. Contrast was improved compared to 300 kV, and individual filaments could be observed. The filament network of samples prepared by detergent extraction was imaged by high-resolution scanning electron microscopy (SEM) with very good signal-to-noise ratio using the secondary electron signal and the 3D structure could be elucidated by SEM tomography. In freeze-dried samples it was possible to discern between keratin filaments and actin filaments because the helical arrangement of actin subunits in the F-actin could be resolved. When comparing the network structures of the differently prepared samples, we found no obvious differences in filament length and branching, indicating that the intermediate filament network is less susceptible to preparation artifacts than the actin network.

Keywords

keratin (intermediate) filamentsSEM tomographySTEM tomographypancreaskeratinactinelectron microscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 16 , Issue 4 , August 2010 , pp. 462 - 471
Copyright
Copyright © Microscopy Society of America 2010

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Alberts, B., Johnson, A., Lewis, L., Raff, M., Roberts, K. & Walter, P. (2008). Molecular Biology of the Cell, 5th Ed.New York: Garland Publishing.Google Scholar
Baumeister, W. (2004). Mapping molecular landscapes inside cells. Biol Chem 385, 865–872.CrossRefGoogle ScholarPubMed
Beil, M., Braxmeier, H., Fleischer, F., Schmidt, V. & Walther, P. (2005). Quantitative analysis of keratin filament networks in scanning electron microscopy images of cancer cells. J Micros 220, 84–95.CrossRefGoogle ScholarPubMed
Beil, M., Eckel, S., Fleischer, F., Schmidt, H., Schmidt, V. & Walther, P. (2006). Fitting of random tessellation models to keratin filament networks. J Theor Biol 241, 62–72.CrossRefGoogle ScholarPubMed
Beil, M., Micoulet, A., von Wichert, G., Paschke, S., Walther, P., Omary, M.B., Van Veldhoven, P.P., Gern, U., Wolff-Hieber, E., Eggermann, J., Waltenberger, J., Adler, G., Spatz, J. & Seufferlein, T. (2003). Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells. Nat Cell Biol 5, 803–811.CrossRefGoogle ScholarPubMed
Buser, C. & Walther, P. (2008). Freeze-substitution: The addition of water to polar solvents enhances the retention of structure and acts at temperatures around −60 °C. J Mircrosc 230, 268–277.CrossRefGoogle Scholar
Buzug, T.M. (2008). Computed Tomography. Berlin: Springer.Google Scholar
Coulombe, P.A. & Omary, M.B. (2002). “Hard” and “soft” principles defining the structure, function and regulation of keratin intermediate filaments. Curr Opin Cell Biol 14, 110–122.CrossRefGoogle ScholarPubMed
Dubochet, J. (2007). The physics of rapid cooling and its implications for cryoimmobilization of cells. Methods Cell Biol 79, 7–21.CrossRefGoogle ScholarPubMed
Echlin, P. (1992). Low-Temperature Microscopy and Analysis. New York, London: Plenum Press.CrossRefGoogle Scholar
Fuchs, E. & Weber, K. (1994). Keratin (intermediate) filaments: Structure, dynamics, function and disease. Annu Rev Biochem 63, 345–382.CrossRefGoogle Scholar
Goldstein, J., Newbury, D., Joy, D., Lyman, C., Echlin, P., Lifshin, E., Sawyer, L. & Michael, J. (2007). Scanning Electron Microscopy and X-Ray Microanalysis, 3rd Ed.New York: Springer.Google Scholar
Hatzfeld, M. & Franke, W.W. (1985). Pair formation and promiscuity of cytokeratins: Formation in vitro of heterotypic complexes and keratin (intermediate)-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol 101, 1826–1841.CrossRefGoogle ScholarPubMed
Hawkes, P.W. (2005). The electron microscope as a structure projector. In Electron Tomography, 2nd Ed., Frank, J. (Ed.), pp. 83–107. New York: Springer.Google Scholar
Herrmann, H., Hesse, M., Reichenzeller, M., Aebi, U. & Magin, T.M. (2003). Functional complexity of keratin (intermediate) filament cytoskeletons: From structure to assembly to gene ablation. Int Rev Cytol 223, 83–175.CrossRefGoogle ScholarPubMed
Hohenberg, H.H., Müller-Reichert, T., Schwarz, H. & Zierold, K. (2003). Foreword, special issue on high pressure freezing. J Microsc 212, 1–2.CrossRefGoogle Scholar
Höhn, K., Pusapati, G.V., Seufferlein, T., Adler, G. & Walther, P. (2009). 3D reconstruction of Golgi stacks in Brefeldin A treated cells. First results using STEM tomography. In Proceedings of MC2009, 2 Life Science, Pabst, M.A. & Zellnig, G. (Eds.), pp. 43–44. Graz, Austria: Verlag der TU.Google Scholar
Hoppe, W., Gassmann, J., Hunsmann, N., Schramm, H.J. & Sturm, M. (1974). Three-dimensional reconstruction of individual negatively stained yeast fatty-acid synthetase molecules from tilt series in the electron microscope. Z Physiol Chem 355, 1483–1487.Google ScholarPubMed
Humbel, B.M. (2009). Freeze substitution. In Handbook of Cryo-Preparation Methods for Electron Microscopy, Cavalier, A., Spehner, D. & Humbel, B.M. (Eds.), pp. 319–341. Boca Raton, FL: CRC Press.Google Scholar
Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116, 71–76.CrossRefGoogle ScholarPubMed
Lautenschläger, F., Paschke, S., Schinkinger, S., Bruel, A., Beil, M. & Guck, J. (2009). The regulatory role of cell mechanics for migration of differentiating myeloid cells. Proc Natl Acad Sci USA 106, 15696–15701.CrossRefGoogle ScholarPubMed
Lück, S., Sailer, M., Schmidt, V. & Walther, P. (2010). Three-dimensional analysis of intermediate filament networks using SEM tomography. J Microsc 236, 1–16.CrossRefGoogle Scholar
Marti, O., Holzwarth, M. & Beil, M. (2008). Measuring the nanomechanical properties of cancer cells by digital pulsed force mode imaging. Nanotechnology 19, 384015.CrossRefGoogle ScholarPubMed
Midgley, P.A. & Dunin-Borkowski, R.E. (2009). Electron tomography and holography in materials science. Nat Mater 8, 271–280.CrossRefGoogle ScholarPubMed
Midgley, P.A., Weyland, M., Thomas, J.M. & Johnson, B.F.G. (2001). Z-contrast tomography: A technique in 3-dimensional nanostructural analysis based on Rutherford scattering. Chem Comm 18, 907–908.CrossRefGoogle Scholar
Pawley, J. (2008). LVSEM for biology. In Biological Low-Voltage Scanning Electron Microscopy, Schatten, H. & Pawley, J.B. (Eds.), pp. 27–106. New York: Springer.CrossRefGoogle Scholar
Radon, J. (1917). Über die Bestimmung von Funktionen längs gewisser Mannigfaltigkeiten. Berichte der math.-phys. Kl. Sächsischen Gesellschaft der Wissenschaften 59, 262–277.Google Scholar
Resch, G.P., Goldie, K.N., Krebs, A., Hoenger, A. & Small, J.V. (2002). Visualisation of the actin cytoskeleton by cryo-electron microscopy. J Cell Sci 115, 1877–1882.CrossRefGoogle ScholarPubMed
Ris, H. (1985). The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections. J Cell Biol 100, 1474–1487.CrossRefGoogle ScholarPubMed
Sailer, M., Lück, S., Schmidt, V., Beil, M., Adler, G. & Walther, P. (2008). Three-dimensional analysis of the keratin (intermediate) filament network using SEM-tomography. In Proceedings of EMC 2008, Aretz, A., Hermanns-Sachweh, B. & Mayer, J. (Eds.), pp. 91–92. Berlin, Heidelberg: Springer.Google Scholar
Sailer, M., Lück, S., Schmidt, V., Beil, M., Adler, G. & Walther, P. (2009). Three-dimensional analysis of the keratin (intermediate) filament network using SEM-tomography. In Proceedings of MC2009, Pabst, M.A. & Zellnig, G. (Eds.), pp. LI.P603–LI.P604. Graz, Austria: Verlag der TU.Google Scholar
Seiler, H. (1967). Einige aktuelle Probleme der Sekundärelektronenemission. Z Angew Phys 22, 249–263.Google Scholar
Svitkina, T.M. (2007). Electron microscopic analysis of the leading edge in migrating cells. Methods Cell Biol 79, 295–319.CrossRefGoogle Scholar
Svitkina, T.M. & Borisy, G.G. (1998). Correlative light and electron microscopy of the cytoskeleton of cultured cells. Method Enzymol 298, 570–592.CrossRefGoogle ScholarPubMed
Vignal, E. & Resch, G. (2003). Shedding light and electrons on the lamellipodium: Imaging the motor of crawling cells. Biotechniques 34, 780–784, 786, 788–789.CrossRefGoogle ScholarPubMed
Walther, P. (2003). The potential of high resolution cryo-SEM in life science. Hitachi Instrument News 40, 3–8.Google Scholar
Walther, P. (2008). High-resolution cryo-SEM allows direct identification of F-actin at the inner nuclear membrane of Xenopus oocytes by virtue of its structural features. J Microsc 232, 379–385.CrossRefGoogle ScholarPubMed
Walther, P. & Ziegler, A. (2002). Freeze substitution of high-pressure frozen samples: The visibility of biological membranes is improved when the substitution medium contains water. J Microsc 208, 3–10.CrossRefGoogle ScholarPubMed
Windoffer, R., Wöll, S., Strnad, P. & Leube, R.E. (2004). Identification of novel principles of keratin filament network turnover in living cells. Mol Biol Cell 5, 2436–2448.CrossRefGoogle Scholar
Yakushevska, A.E., Lebbink, M.N., Geerts, W.J., Spek, L., van Donselaar, E.G., Jansen, K.A., Humbel, B.M., Post, J.A., Verkleij, A.J. & Koster, A.J. (2007). STEM tomography in cell biology. J Struct Biol 159, 381–391.CrossRefGoogle ScholarPubMed