Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

Summary

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.

Abstract

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Introduction

Many cellular processes, such as endocytosis, trafficking, the formation of filopodia, infection, etc., are accompanied by a dramatic change in the shape of cell membranes^1,2. In the cell, a number of proteins participate in these processes by binding to the membrane and altering their shape. The most notable examples are members of the Bin/Amphiphysin/Rvs (BAR) protein family, containing a characteristic intrinsically curved BAR domain^{3,4,5,6,7<....}

Protocol

1. Preparation of GUVs by Electroformation on Pt-wires

1. Clean the electroformation chamber (see Introduction and Figure 1A) and the Pt-wires with an organic solvent such as ethanol or acetone to wash away the lipids and with water to wash away the salts.
   NOTE: We suggest thoroughly wiping away the residue with ethanol-soaked tissue then sonicating in acetone, ethanol, then water, each for 5 min.
2. Prepare a lipid mix of a desired lipid composition at 1 mg/mL in chloroform. The mix should contain ~ 0.05% molar fraction of a lipid conjugated with biotin (e.g., 1,2-distearoyl-sn-glycero-3-pho....

Results

The tube-pulling experiment can give vital mechanical information about the membrane. In the absence of proteins or other molecules that couple with membrane curvature, the membrane force and tube radius can be related with membrane tension by applying the Canham-Helfrich Hamiltonian equation to a tube pulled from a GUV^50,51

Discussion

The method of pulling tubes from GUVs gives rich information on the membrane-protein system, as it is not only the means to measure the fundamental mechanical properties of the membrane, but it helps to shed light on the coupling between proteins and membrane curvature. As discussed in the Introduction, other techniques exist to measure the effects of membrane-curving proteins, either by incubating the proteins with sub-micron liposomes tethered to a passivated surface^18,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)	Avanti	850375	Example lipid used in data for Figure 3
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] [DSPE-PEG(2000)-biotin]	Avanti	880129	biotinylated lipid
BODIPY-TR-C5-ceramide	Molecular Probes (ThermoFisher)	D7540	fluorescent lipid
BODIPY- FLC5-hexadecanoyl phosphatidylcholine (HPC*)	Molecular Probes (ThermoFisher)		fluorescent lipid for protein density calibration
egg L-α-phosphatidylcholine (eggPC)	Avanti	840051	used for calibrating the tube radius constant
β-casein from bovine milk (>99%)	Sigma Aldrich	C6905	used for passivating the micropipette and the experimental chamber
Sucrose	Sigma Aldrich	S7903
D(+) glucose	Sigma Aldrich	G7021
NaCl	Sigma Aldrich	S7653
Tris	Sigma Aldrich	10708976001
osmometer	Loser	n/a
Pt-wires, 0.5 mm diameter, 99.99% pure	Goodfellow USA	PT005139	used for GUV electroformation
function generator		n/a	used to create current for GUV electroformation
putty sealant	Vitrex (from CML France)	CRIT 140013	used to seal the electroformation chamber
bath sonicator		n/a	useful to clean the electroformation chamber, but not crucial
Nikon TE2000 inverted microscope, eC1 confocal system (Nikon), with two laser lines (λ = 488 nm and 543 nm); optical tweezers induced by a 5 W ytterbium fiber continuous wave laser (λ > 1070 nm; IPG GmBH Germany)			an example of a confocal microscopy system equipped with optical tweezers
micromanipulators		n/a
borosilicate capillaries (with internal and external radii of 0.78 mm and 1 mm, respectively)	Harvard apparatus	30-0036
micropipette puller	Sutter Instrument	P-2100
microforge	Narishige	MF-800
piezoelectric actuator	Physik Instrumente	n/a
polystyrene streptavidin coated beads (diameter 3.2 µm)	Spherotech	SVP-30-5