Pulling Membrane Nanotubes from Giant Unilamellar Vesicles
December 7th, 2017
Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.
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.
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Overview
This study introduces a method for pulling membrane nanotubes from lipid vesicles to investigate the interactions between proteins and curved membranes in vitro. The approach allows for quantification of protein binding to these membranes and their mechanical properties, shedding light on fundamental processes like endocytosis and intracellular trafficking.
Key Study Components
Research Area
- Cell biology
- Membrane dynamics
- Protein-membrane interactions
Background
- Understanding how proteins influence membrane curvature is critical for cellular processes.
- Investigating curved membranes helps unveil mechanisms involved in endocytosis.
- Membrane mechanics play an essential role in intracellular trafficking.
Methods Used
- Nanotube pulling methodology for studying protein interactions with curved membranes.
- Giant unilamellar vesicles as model systems.
- Optical tweezers and microscopy for measuring membrane properties.
Main Results
- Quantified how proteins like endophilin A2 interact with and stabilize membrane nanotubes.
- Demonstrated mechanical properties of lipid membranes through various observed behaviors.
- Provided insights into the preferential binding of proteins to curved membranes.
Conclusions
- This study highlights the importance of membrane curvature in protein interactions.
- The method presented can further research in cellular processes and membrane biology.
Frequently Asked Questions
Executive Industry Relevance
This method enables quantitative analysis of protein-membrane curvature interactions, providing mechanistic insights into endocytosis and intracellular trafficking pathways. By measuring protein binding to highly curved membranes and their mechanical effects, it supports target validation and de-risks early-stage discovery of membrane-associated therapeutics. The approach delivers predictive confidence in protein function by linking molecular interactions to membrane remodeling phenotypes.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by quantifying protein binding to curved membranes and their reciprocal deformation.
- Operational Value: Enables functional target validation through direct measurement of protein-induced membrane stabilization, such as force reduction to zero by endophilin A2.
- Predictive Value: Supports portfolio triage by identifying proteins that preferentially bind high-curvature membranes, indicating mechanistic relevance to vesicle formation.
Screening & Assay Development
- Assay Readiness: Prepares validated protein-membrane systems for downstream screening by enabling concurrent measurement of membrane curvature, tension, and protein surface density.
- Quantitative Outputs: Generates force and radius measurements from optical trap displacement and fluorescence intensity, providing dual readouts for bending rigidity calculations.
- Reproducibility: Standardizes membrane tension control via micropipette aspiration and zero-position calibration, ensuring consistent nanotube formation across experiments.
Translational & Preclinical Research
- Disease Relevance: Models curved cellular structures like endocytic necks, linking protein-membrane interactions to trafficking pathways implicated in neurodegenerative and metabolic disorders.
- Translational Continuity: Bridges in vitro findings to cellular contexts by enabling follow-up with high-resolution microscopy and single-particle tracking to assess protein organization and mobility on nanotubes.
- Risk-Adjusted Advancement: Informs go/no-go decisions by quantifying how proteins alter membrane mechanics, reducing ambiguity in mechanistic de-risking.
Pipeline & Workflow Integration
Positioned at the discovery biology stage, this method supports hypothesis testing and pathway clarification before advancing to lead identification and preclinical validation.
- Discovery Biology: Tests how specific proteins sense or induce membrane curvature, clarifying roles in endocytosis and intracellular trafficking.
- Screening: Delivers assay-ready systems with standardized curvature and tension, enabling reliable compound or protein evaluation.
- Analytics: Provides force, radius, and fluorescence intensity readouts that quantify membrane bending rigidity and protein enrichment on nanotubes.
- Translational Research: Connects to preclinical work by allowing correlation of in vitro protein-membrane binding with cellular localization and dynamics.
- Enterprise Reuse: Establishes a reusable platform for studying diverse proteins of interest across multiple projects, reducing redundant method development.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity in protein-membrane interactions.
- Operational Value: Ensures reproducibility through standardized vesicle preparation, tension control, and dual-mode measurement of nanotube properties.
- Strategic Value: Improves capital efficiency by enabling early go/no-go decisions based on quantitative protein-curvature binding data.
- Portfolio Impact: Supports risk-adjusted prioritization of membrane-associated targets by linking binding behavior to functional membrane remodeling.
Implementation Considerations
- Requires expertise in micromanipulation, optical tweezers, and fluorescence microscopy for nanotube pulling and protein injection.
- Depends on specialized instrumentation including micropipette aspirators, piezo actuators, and confocal systems for concurrent tension, curvature, and protein density measurements.
- Necessitates cross-team standardization of buffer conditions, vesicle preparation protocols, and protein labeling strategies to ensure data comparability.
- Involves adaptation considerations when extending the method to different lipid compositions or protein systems, particularly regarding biotin-streptavidin binding efficiency.
- Includes practical limitations such as the need for highly pure beta-casein for chamber passivation and careful avoidance of bubbles during micropipette filling to maintain measurement integrity.
