Hostname: page-component-586b7cd67f-2brh9 Total loading time: 0 Render date: 2024-11-24T12:07:46.475Z Has data issue: false hasContentIssue false
  • English
  • Français

Imaging of Xenopus laevis Oocyte Plasma Membrane in Physiological-Like Conditions by Atomic Force Microscopy

Published online by Cambridge University Press:  10 June 2013

Massimo Santacroce ,
Federica Daniele ,
Andrea Cremona ,
Diletta Scaccabarozzi ,
Michela Castagna  and
Francesco Orsini
Massimo Santacroce
Affiliation:
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134 Milano, Italy
Federica Daniele
Affiliation:
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134 Milano, Italy
Andrea Cremona
Affiliation:
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134 Milano, Italy
Diletta Scaccabarozzi
Affiliation:
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134 Milano, Italy
Michela Castagna
Affiliation:
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134 Milano, Italy
Francesco Orsini*
Affiliation:
Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, 20133 Milano, Italy
*
*Corresponding author. E-mail: [email protected]
Get access

Abstract

Xenopus laevis oocytes are an interesting model for the study of many developmental mechanisms because of their dimensions and the ease with which they can be manipulated. In addition, they are widely employed systems for the expression and functional study of heterologous proteins, which can be expressed with high efficiency on their plasma membrane. Here we applied atomic force microscopy (AFM) to the study of the plasma membrane of X. laevis oocytes. In particular, we developed and optimized a new sample preparation protocol, based on the purification of plasma membranes by ultracentrifugation on a sucrose gradient, to perform a high-resolution AFM imaging of X. laevis oocyte plasma membrane in physiological-like conditions. Reproducible AFM topographs allowed visualization and dimensional characterization of membrane patches, whose height corresponds to a single lipid bilayer, as well as the presence of nanometer structures embedded in the plasma membrane and identified as native membrane proteins. The described method appears to be an applicable tool for performing high-resolution AFM imaging of X. laevis oocyte plasma membrane in a physiological-like environment, thus opening promising perspectives for studying in situ cloned membrane proteins of relevant biomedical/pharmacological interest expressed in this biological system.

Keywords

atomic force microscopyXenopus laevis oocyteplasma membranemembrane proteinsbiological membranessample preparation protocol
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 5 , October 2013 , pp. 1358 - 1363
Copyright
Copyright © Microscopy Society of America 2013 

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

Bahatyrova, S., Frese, R.N., Siebert, C.A., Olsen, J.D., Van der Werf, K.O., Van Grondelle, R., Niederman, R.A., Bullough, P.A., Otto, C. & Hunter, C.N. (2004). The native architecture of a photosynthetic membrane. Nature 430, 1058–1062.CrossRefGoogle ScholarPubMed
Bartlett, G.R. (1959). Phosphorus assay in column chromatography. J Biol Chem 234, 466–468.CrossRefGoogle ScholarPubMed
Bligh, E.G. & Dyer, W.J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911–917.CrossRefGoogle ScholarPubMed
Cucu, D., Simaels, J., Jans, D. & Van Driessche, W. (2004). The transoocyte voltage clamp: A non-invasive technique for electrophysiological experiments with Xenopus laevis oocytes. Pflügers Arch 447, 934–942.CrossRefGoogle Scholar
Frederix, P.L.T.M., Bosshart, P.D. & Engel, A. (2009). Atomic force microscopy of biological membranes. Biophysical J 96, 329–338.CrossRefGoogle ScholarPubMed
Luria, A., Vegelyte-Avery, V., Stith, B., Tsvetkova, N.M., Wolkers, W.F., Crowe, J.H., Tablin, F. & Nuccitelli, R. (2002). Detergent-free domain isolated from Xenopus laevis egg plasma membrane with properties similar to those of detergent-resistant membranes. Biochemistry 41, 13189–13197.CrossRefGoogle ScholarPubMed
Mari, S.A., Soragna, A., Castagna, M., Santacroce, M., Perego, C., Bossi, E., Peres, A. & Sacchi, V.F. (2006). Role of the conserved glutamine 291 in the γ-aminobutyric acid transporter rGAT-1. Cell Mol Life Sci 63, 100–111.CrossRefGoogle ScholarPubMed
Müller, D.J. & Engel, A. (2008). Strategies to prepare and characterize native membrane proteins and protein membranes by AFM. Curr Opin Colloid Interface Sci 13, 338–350.CrossRefGoogle Scholar
Müller, D.J., Sapra, K.T., Scheuring, S., Kedrov, A., Frederix, P.L., Fotiadis, D. & Engel, A. (2006). Single-molecule studies of membrane proteins. Curr Opin Struc Biol 16, 489–495.Google Scholar
Nakamura, F., Goshima, Y., Strittmatter, S.M. & Kawamoto, S. (1999). Isolation of receptor clones by expression screening in Xenopus oocytes. Methods Mol Biol 128, 1–18.Google ScholarPubMed
Orsini, F., Cremona, A., Arosio, P., Corsetto, P.A., Montorfano, G., Lascialfari, A. & Rizzo, A.M. (2012). Atomic force microscopy imaging of lipid rafts of human breast cancer cells. Biochim Biophys Acta – Biomembranes 1818, 2943–2949.CrossRefGoogle ScholarPubMed
Orsini, F., Santacroce, M., Arosio, P., Castagna, M., Lenardi, C., Poletti, G. & Sacchi, V.F. (2009). Intermittent contact mode AFM investigation of native plasma membrane of Xenopus laevis oocyte. Eur Biophys J 38, 903–910.CrossRefGoogle ScholarPubMed
Orsini, F., Santacroce, M., Arosio, P. & Sacchi, V.F. (2010). Observing Xenopus laevis oocyte plasma membrane by atomic force microscopy. Methods 51, 106–113.CrossRefGoogle ScholarPubMed
Orsini, F., Santacroce, M., Perego, C., Lenardi, C., Castagna, M., Mari, S.A., Sacchi, V.F. & Poletti, G. (2006). Atomic force microscopy characterization of Xenopus laevis oocyte plasma membrane. Microsc Res Tech 69, 826–834.CrossRefGoogle ScholarPubMed
Santacroce, M., Orsini, F., Mari, S.A., Marinone, M., Lenardi, C., Bettè, S., Sacchi, V.F. & Poletti, G. (2008). Atomic force microscopy imaging of X. laevis oocyte plasma membrane purified by ultracentrifugation. Microsc Res Tech 71, 397–402.CrossRefGoogle ScholarPubMed
Scheuring, S., Goncalves, R.P., Prima, V. & Sturgis, J.N. (2006). The photosynthetic apparatus of Rhodopseudomonas palustris: Structures and organization. J Mol Biol 358, 83–96.CrossRefGoogle ScholarPubMed
Wagner, C.A., Friedrich, B., Setiawan, I., Lang, F. & Broer, S. (2000). The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 10, 1–12.CrossRefGoogle ScholarPubMed