The activity of many membrane proteins is regulated by interactions with partners in the plasma membrane, and thus membrane protein interactions play a critical role in cell life and in disease 1,2
. Studies of membrane protein interactions need to be performed in a native-like membrane environment in order to give accurate results 3,4
. However, the membrane of live cells is not suitable for quantitative biophysical characterization due to its heterogeneity. On the other hand, model lipid systems containing purified membrane proteins are challenging to produce as the expression levels of membrane proteins are generally low and they often misfold during purification and reconstitution. Plasma membrane-derived vesicles, defined as micron sized, cytoplasm-filled spheres of membrane released by live cells, are an alternative model system for the studies of membrane proteins because they mimic the molecular structure and composition of the native cell membrane and exhibit a homogenous distribution of proteins due to the lack of a cytoskeleton 5-8
We have used such plasma membrane derived vesicles to study the interactions between receptor tyrosine kinases (RTKs), membrane proteins which conduct biochemical signals via lateral dimerization 9,10
. RTK dimerization in the vesicles is analyzed using Förster resonance energy transfer (FRET) and a methodology which yields quantitative information about dimerization propensities 11-13
. For the FRET experiments, RTKs are tagged with fluorescent proteins that act either as a donor or an acceptor. Images of vesicle cross-sections are obtained and then analyzed to determine the donor and acceptor concentrations and the FRET efficiency, which are needed to calculate association constants and association free energies 11,12
The vesicles used in our previous FRET experiments were produced by incubating cells with a vesiculation buffer containing formaldehyde and DTT, following the procedure of Scott et al
(see ). This vesiculation method is usually the method of choice for researchers, as it quickly produces many large vesicles (5 to 25 μm in size). However, the presence of formaldehyde and DTT in the system is undesirable: DTT is a reducing agent, and formaldehyde may induce membrane protein cross-linking, thereby affecting the measured level of interactions 14
Cohen et al.
developed a vesiculation method that uses a hypotonic wash, followed by incubation with a “salt cocktail” osmotic buffer with the composition shown in
. This osmotic buffer does not rupture the cells, but stresses them so they produce vesicles 15
. However, this method is only effective for the highly adherent cancer cell line A431. The A431 line is an epidermoid carcinoma cell line that expresses very high levels of Epidermal Growth Factor Receptor (EGFR). It is highly sensitive to mitogenic stimuli, and is usually avoided in studies of recombinant proteins. On the other hand, cells that are widely used for the expression of recombinant proteins, such as the Chinese hamster ovary (CHO) cell line, quickly detach in the osmotic salt cocktail buffer and the vesicles cannot be purified prior to imaging.
Here we describe a new vesiculation method that uses an osmotic buffer to produce vesicles from live CHO cells without causing cell detachment. This vesiculation method relies on chloride salts and eliminates the use of formaldehyde and DTT, works in a cell line that readily over-expresses proteins, and allows easy separation of the vesicles from cell debris.
To assess whether membrane protein interactions can be studied in the vesicles produced via the new method, we characterized the dimerization of the membrane protein Fibroblast Growth Factor Receptor 3 (FGFR3). FGFR3 is an RTK that is known to form dimers in the cell membrane and to play an important role in the development of the skeletal system 16-18
. Here, experiments were performed using a truncated form of the protein containing the extracellular and transmembrane domains linked to fluorescent proteins. The dimerization of this protein construct has been characterized in vesicles produced with formaldehyde and DTT 13
, such that the results can be directly compared.
Here, we i) describe the vesiculation method, ii) characterize the vesicles obtained, iii) investigate the interactions of FGFR3, and iv) compare vesicles produced using the new method with those produced using the established formaldehyde/DTT vesiculation method. The results presented here show that the newly developed method of producing cell-derived vesicles is a useful tool for studies of membrane protein interactions using FRET-based detection techniques.