title: Imaging of Xenopus laevis oocyte plasma membrane in physiological-like conditions by atomic force microscopy.
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.
authors.affiliation: Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134 Milano, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Santacroce
authors.forename: Federica
authors.initials: F
authors.lastname: Daniele
authors.forename: Andrea
authors.initials: A
authors.lastname: Cremona
authors.forename: Diletta
authors.initials: D
authors.lastname: Scaccabarozzi
authors.forename: Michela
authors.initials: M
authors.lastname: Castagna
authors.forename: Francesco
authors.initials: F
authors.lastname: Orsini
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force; D009865:Oocytes / Q000648:ultrastructure*; D014461:Ultracentrifugation; D014982:Xenopus laevis