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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]
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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 

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