Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies

Abstract

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly.

Keywords: ER and plasma membrane protein separation; Nicotinic receptor; OptiPrep; Photobleaching; Protein trafficking; Single molecule; Stoichiometry; Vesicles.

Figure 1.. Instrumental setup used for nitrogen cavitation.

A. Take the precooled nitrogen cavitation chamber, close the valve (red circle) and add the cell slurry into the chamber. B. Attach the vessel head and open valve B1 to flow nitrogen gas inside the chamber and close valve B1 once the pressure reaches 600 psi. Place this chamber into ice for 5 min. Take the chamber out from the ice and slowly open valve B2 to simultaneously release the pressure and to collect the cell lysate into a 15 ml centrifuge tube.

Figure 2.. Pictorial presentation of the collection steps for ER and plasma membrane vesicles.

A. A wine cork (A1) is cut into a slice of about 2 mm thickness (A2). This slice (A3) is cut along the circumference to discard the outer area (A4) and keep a small circular slice (A5) which can easily fit into an Ultra-Clear ultracentrifuge tube. A scale (A6) is placed on the image for comparison of sizes. B. The small slice of cork is inserted into the tube and floats on the solution. C. As additional solution (20%, and 10% OptiPrep) is added the cork rises to the top. After the addition of all layers, the cork floats atop the solution. D. The cork slice is removed using a forceps without disturbing the gradient. E. A Pasteur pipette is broken to obtain the narrow tube which is inserted in the inlet tubing of a peristaltic pump. F. The resultant inlet tubing of the pump is inserted at the bottom-center of the Ultra-Clear tube containing the gradient solution.

Figure 3.. Presence of vesicles between the interfaces of OptiPrep solutions and the relative positions of different fractions collected.

A. Three layers of vesicles are visible: on the interface of 0% and 10% OptiPrep, 10% and 20% OptiPrep and 20% and 30% OptiPrep. This image was taken with a mobile phone camera. B. By inserting the inlet tubing of a peristaltic pump in the bottom-center of the tube, collect fractions 1 and 2 in series, 1.0-ml each, then fractions 3-8 in a series, 1.5 ml each.

Figure 4.. Western blot of the different fractions collected from OptiPrep gradient to isolate single receptors into the ER and plasma membrane specific vesicles.

Anti-sodium potassium ATPase antibody (1:200 dilution) was used to bind with the plasma membrane marker, sodium potassium ATPase, in the OptiPrep gradient (upper panel). An anti-calnexin antibody (1:200 dilution) was employed to bind with the ER marker, calnexin, in the gradients (lower panel). An HRP conjugated mouse anti-Rabbit secondary antibody (1:5,000 dilution) and Clarity^TM Western ECL Substrate on a ChemiDoc imaging system (Bio-Rad) were utilized to locate the ER and Plasma membrane markers on the gels. The plasma membrane marker was found in the cell lysate and fractions 5 to 8 and the ER marker was located in the cell lysate and fractions 3 to 6. Therefore, fractions 3 and 4 contain only the ER vesicles; fractions 7 and 8 contain only the plasma membrane vesicles. We selected fraction 3 for ER vesicles and fraction 7 for the plasma membrane vesicles.