title: Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.
abstract: Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes.
authors.affiliation: Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, 20133, Milano, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Orsini
authors.forename: Massimo
authors.initials: M
authors.lastname: Santacroce
authors.forename: Andrea
authors.initials: A
authors.lastname: Cremona
authors.forename: Nitya N
authors.initials: NN
authors.lastname: Gosvami
authors.forename: Alessandro
authors.initials: A
authors.lastname: Lascialfari
authors.forename: Bart W
authors.initials: BW
authors.lastname: Hoogenboom
mesh_terms: D000818:Animals; D051401:Aquaporin 4 / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D005260:Female; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*; D014982:Xenopus laevis / Q000254:growth & development / Q000378:metabolism*
chemical_list: C496030:AQP4 protein, human; D051401:Aquaporin 4
keywords: Xenopus laevis oocyte; aquaporin 4; atomic force microscopy; heterologous expression; membrane proteins