The overall goal of this nanotube pulling methodology is to quantitatively study the interactions between specific proteins of interest and highly curved membranes. This method can help answer key question in the biology of cell membranes, such as endocytosis and intracellular trafficking. This technique allow us to directly quantify how proteins bind to curved membranes and reciprocally deform them.
Initially, this method was used to measure the basic mechanical properties of lipid membranes. Recently, it has been applied to elucidate important mechanisms in protein membrane interactions, particularly protein-shaping membranes. Begin by collecting the giant unilamellar vesicles directly from the platinum wires of the electroformation chamber.
To construct the experimental chamber, place two rectangular glass cover slips one millimeter apart on a metallic base, leaving openings along the long edges large enough to allow a glass micropipette tip to reach to at least the center of the chamber. Fill both the experimental chamber and a glass micropipette with five milligrams per milliliter of a highly pure beta-casein solution, taking care to avoid bubbles. While the chamber is being passivated, mount it onto a light microscope centered above the objective, and insert the tip of the aspiration micropipette through the opening along the long edge until the tip is above the objective.
Adjust the water tank level so that the aspiration pressure is near zero, and fill an injection micropipette with the molecule of interest dissolved in experimental buffer at the appropriate experimental concentration. Mount the injection micropipette inside a micromanipulator, and insert the micropipette through the other side of the experimental chamber. At the end of the incubation, remove the beta-casein solution from the chamber, and rinse the chamber several times with experimental buffer.
Then, add a few microliters of vesicle solution to the chamber, followed by a few microliters of streptavidin-coated beads, to a final concentration of 0.1 times 10 to the negative 3%bead rate per volume or less. After the vesicles and beads have settled to the bottom of the chamber, allow the experimental buffer to evaporate for about 15 minutes, at which point the vesicles should visibly undulate under a light microscope. When the vesicles are appropriately floppy, aspirate individual vesicles into the micropipette until the lengths of the membranes within the pipette are equal to or larger than the pipette radius.
When an appropriately sized aspiration tongue has been identified, seal the open edges of the chamber with mineral oil. To set the zero position of the aspiration pressure, position the end of the aspiration pipette near a bead, and adjust the height of the water tank so that the bead is neither sucked in nor blown away by the pipette. Adjusting the height of the water is critical to control the membrane tension of the vesicle that is aspirated by the micropipette.
Aspirate a vesicle, and move the micropipette up and out of focus without breaking the micropipette. Using optical tweezers, trap a bead approximately 20 micrometers away from the bottom of the chamber. Bring the vesicle back into focus away from the bead and aligned with the optical trap, and record the movement of the bead for one to two minutes to measure the equilibrium position.
Reduce the pressure inside the micropipette as much as possible without losing the vesicle to decrease the membrane tension, and carefully bring the vesicle in contact with the bead for around a second to establish the streptavidin-biotin bonds. Then, using a piezo actuator, gently pull the vesicle back to create a nanotube. Increase the aspiration pressure to recreate the aspiration tongue and align the tube within the axis of the aspiration pipette.
Bring the tube and the vesicle fully into focus, and record the movement of the bead at a 30-hertz acquisition speed and the height of the water tank with respect to the zero position for a few minutes under bright-field microscopy. When all of the images have been obtained, record the movement of the bead again at different aspiration pressures and membrane tensions. To inject proteins or molecules of interest, bring the injection micropipette near the nanotube, taking care that the bead in the optical trap is not perturbed, and gently inject the proteins at one to two pascals of pressure.
After the protein binding has equilibrated, repeat the stepwise measurements in the presence of the added proteins, as demonstrated for the bare membrane. Force and radius measurements on nanotubes pulled from 100%DOPC vesicles are independent, as the force is measured from the displacement of the bead in the optical trap and the radius from fluorescence intensity, providing two ways of calculating the membrane bending rigidity. The relative enrichment of BAR or potassium channel proteins on membrane tubes over the underlying vesicles indicates that the proteins are more likely to bind to curved membranes.
Indeed, binding of the N-BAR protein endophilin A2 to the membrane gradually reduces the tube force to zero, indicating the formation of a three-dimensional scaffold that keeps the tube stable. Following this procedure, other techniques can be combined to answer additional questions, for instance, high-resolution microscopy to detect protein organization on membrane tubes or single particle tracking to measure protein mobility.
