Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

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.

Imaging of Xenopus laevis oocyte plasma membrane in physiological-like conditions by atomic force microscopy.

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.

Passive water permeability of some wild type and mutagenized amino acid cotransporters of the SLC6/NSS family expressed in Xenopus laevis oocytes.

In this paper passive water movement across the cell membrane mediated by wild type and mutagenized cotransporters was investigated. We evaluated water movement and, in parallel, amino acid uptake induced by some members of the SLC6/NSS family belonging to different kingdoms, namely the rat GABA transporter GAT1, the insect amino acid transporters KAAT1 and CAATCH1 and the bacterial leucine transporter LeuT, whose structure was recently solved. We also tested whether mutated proteins in which the solute translocation mechanism is altered or even abolished were able to induce water movement across cell membrane. The proteins of interest were expressed in Xenopus laevis oocytes and osmotic water permeabilities were estimated from the rate of cell volume change induced by an osmotic gradient in the absence of cotransported solutes. Under osmotic stress all the studied wild type amino acid cotransporters increased the water permeability of the membrane. The GABA transport inhibitor SKF 89976A inhibited both GABA transport and water movement induced by the expression of GAT1. Interestingly, the capacity of mutant proteins to induce water movement was not predictable on the basis of their substrate transport ability. In particular the GAT1 mutant Q291N, void of any transport activity, induced a water permeability similar to that induced by the wt protein. The KAAT1 mutant T339C, which showed a higher transport activity, induced a water permeability not significantly different from the wild type transporter. Interestingly, the bacterial leucine cotransporter LeuT, whose binding site for leucine and Na(+) is void of water, induced water movement through the plasma membrane.

Observing Xenopus laevis oocyte plasma membrane by Atomic Force Microscopy.

This paper describes the use of Atomic Force Microscopy (AFM) to investigate the plasma membrane of Xenopus laevis oocyte. Different protocols of sample preparation to perform an AFM investigation of both external and intracellular sides of the oocyte plasma membrane are presented and discussed. Reproducible AFM images allowed visualization and dimensional characterization of protein complexes differently arranged on both sides of the oocyte plasma membrane. In particular, two different arrangements were visualized: (1) a heterogeneous and irregular distribution of the protein complexes and (2) in some cases a distribution of nanometer-sized membrane domains where protein complexes are densely packed and spatially arranged in an ordered hexagonal motif. In addition, a methodological approach based on the purification of oocyte plasma membrane by ultracentrifugation on sucrose gradient is also described in this work. The potential of AFM as a useful tool for the structural characterization of proteins in a native eukaryotic membrane was established and its relevance for describing the organization of protein complexes in native biological membranes was explored.

Atomic force microscopy imaging of Xenopus laevis oocyte plasma membrane purified by ultracentrifugation.

Atomic force microscopy (AFM) was used to investigate the native plasma membrane of Xenopus laevis (X. laevis) oocyte purified by means of ultracentrifugation on sucrose gradient and subsequently adsorbed on mica leaves through a physisorption process. Reproducible AFM topography images were collected, analyzed, and compared. AFM images showed the presence of large single or double bilayer membrane sheets covered with protein complexes. The lateral dimension and height of protein complexes imaged in air showed a normal distribution centred on 15.4 +/- 0.4 nm (mean +/- SE; n = 59) and 3.9 +/- 0.2 nm (mean +/- SE; n = 57), respectively. A density of about 270 protein complexes per square micron was calculated. Less frequently, ordered nanometer domains with densely packed protein complexes arranged in hexagonal patterns were also visualized in AFM images, confirming previously published data. Their lateral dimension and height showed a normal distribution centred on 23.0 +/- 0.4 nm (mean +/- SE; n = 42) and 1.5 +/- 0.6 nm (mean +/- SE; n = 90), respectively. A density of about 870 protein complexes per square micrometer was calculated. Advantages and drawbacks of this new sample preparation for AFM imaging are discussed.