Orsini, F;
Santacroce, M;
Cremona, A;
Gosvami, NN;
Lascialfari, A;
Hoogenboom, BW;
(2014)
Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.
Jornal of Molecular Recognition
, 27
(11)
669 - 675.
10.1002/jmr.2390.
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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. Copyright © 2014 John Wiley & Sons, Ltd.
| Type: | Article |
|---|---|
| Title: | Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4. |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1002/jmr.2390 |
| Publisher version: | http://dx.doi.org/10.1002/jmr.2390 |
| Language: | English |
| Additional information: | Under Wiley copyright, authors are permitted to self-archive the peer-reviewed (but not final) version of a contribution on the contributor's personal website, in the contributor's company/institutional repository or archive, and in certain not for profit subject-based repositories such as PubMed Central as listed at the following website (http://olabout.wiley.com/WileyCDA/Section/id-820227.html), subject to an embargo period of 12 months for scientific, technical, and medical (STM) journals and 24 months for social science and humanities (SSH) journals following publication of the final contribution. |
| Keywords: | Xenopus laevis oocyte, Aquaporin 4, Atomic force microscopy, Heterologous expression, Membrane proteins |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Physics and Astronomy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1450641 |
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