In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

Summary

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

Abstract

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

Introduction

The actin cytoskeleton plays a fundamental role in constructing the intracellular architecture of the cell by coordinating molecular-level contractility and force generation^1,2,3. As a result, it mediates numerous essential cellular activities, including cell deformation^4,5, division⁶, migration^7,8, and adhesion⁹. The in vitro reconstitution of actin networks has gained tremendous attention in recent yea....

Protocol

1. Preparation of buffers and protein solutions

1. Prepare the aqueous Inner Non-Polymerization (INP) Buffer in a total volume of 5 mL by mixing 0.1 mM CaCl₂, 10 mM HEPES (pH 7.5), 1 mM DTT, 0.5 mM Dabco, 320 mM sucrose, and 0.2 mM ATP.
2. Prepare the Protein Mix (PM) by adding proteins to the INP buffer at 4 °C with the following concentrations: 11.2 µM non-fluorescent G-actin, 2.8 µM fluorescently labeled actin, and 0.24 µM Arp2/3 (Table of Materials). To form an F-actin layer, add 100 nM gelsolin, 4 µM cofilin, and 2.2 µM VCA-His to the PM. As a control experiment, replace PM....

Results

The preparation of liposomes based on the inverted emulsion technique is illustrated graphically and schematically in Figure 1.

First, empty (bare) liposomes (~5-50 µm in diameter) that were composed of phospholipid (EPC) and fluorescent lipid (DHPE) were prepared. A bright, far-red fluorescent dye was encapsulated within bare liposomes as a control experiment. Whether a lipid monolayer has successfully formed in the peripheral of the droplet could be determined b.......

Discussion

Several key steps determine the success of a high yield of liposomes during the preparation process. To completely dissolve the lipid film in the oil, the sample must be sonicated until the lipid film at the bottom of the glass vial disappears completely. After the sonication, the lipid-oil mixture must be stored overnight at room temperature under dark conditions for the lipid molecules to disperse further²⁹. The mixture can be stored at 4 °C for up to a week. When preparing an FB/oil emulsi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-dioleoyl-sn-glycero-3-{[n(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DOGS-NTA-Ni)	Avanti Polar Lipids Inc.	231615773	Nickel Lipid
1,4-Diazabicyclo[2.2.2]octane	Sigma	D27802-25G	DABCO
Actin protein (>99% pure): rabbit skeletal muscle	Cytoskeleton, Inc	AKL99-D	non-fluorescent G-actin
Actin protein (rhodamine): rabbit skeletal muscle	Cytoskeleton, Inc	AR05	fluorescently labeled actin
Adenosine 5′-triphosphate disodium salt hydrate	Sigma	A2383-10G	ATP
Alexa Fluor 647 dye	ThermoFisher		fluorescent dye
Andor iQ3	Andor Technologies		control and acquisition software for confocal microscope
Arp2/3 Protein Complex: Porcine Brain	Cytoskeleton, Inc	RP01P-A	Arp 2/3
Calcium chloride dihydrate	Sigma	10035048	CaCl2
Chamlide Chambers (4-well for 12 mm round coverslip)	Quorum Technologies		incubation chamber
Cofilin protein: human recombinant	Cytoskeleton, Inc	CF01-C	cofilin
Confocal Microscope (63× oil-immersion objective)	Andor Technologies	LEICA DMi8
D-(+)-GLUCOSE BIOXTRA	Sigma	G7528	glucose
Dithiothreitol	DOT Scientific	DSD11000-10	DTT
Gelsolin Protein: Homo Sapiens Recombinant	Cytoskeleton, Inc	HPG6	gelsolin
Hamilton 1750 Gastight Syringe, 500 µL, cemented needle, 22 G, 2" conical tip	Cole-Parmer	UX-07940-53	glass syringe
HEPES	AmericanBio	7365-45-9
ImageJ/Fiji			https://imagej.net/tutorials/
L-alpha-Phosphatidylcholine	Avanti Polar Lipids Inc.	97281442	EPC
Magnesium chloride	Sigma	7786303	MgCl2
Mineral oil, BioReagent, for molecular biology, light oil	Sigma	8042475	mineral oil
N-WASP fragment WWA (aa400–501, VCA-His)			VCA-His is purified using lab protocol. The protocol can be provided upon reasonable requests
Oregon Green 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (Oregon Green 488 DHPE)	Thermo Fisher	O12650	DHPE
Potassium chloride	Sigma	7447407	KCl
Sucrose	Sigma	57-50-1	sucrose
β-Casein from bovine milk	Sigma	C6905-250MG