Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

Summary

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

Abstract

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

Introduction

Lipid bilayer compartments are used as model synthetic cells for studying enclosed organic reactions and membrane-based processes or as carrier modules in drug delivery applications^1,2. Bottom-up biology with purified components requires minimal experimental systems to explore properties and interactions between biomolecules, such as proteins and lipids^3,4. However, with the advancement of the field, there is an increased need for more complex experimental systems that better imitate the conditions in biological cells. Encapsulation in GUVs is a practi....

Protocol

1. Preparation of oil-lipid-mixture

NOTE: The step needs to be performed in a fume hood following all the safety guidelines for handling chloroform.

1. Take 0.5 mL of chloroform in a 15 mL glass vial. Add 88 µL of 25 mg/mL dioleoyl-phosphocholine (DOPC), 9.3 µL of 50 mg/mL cholesterol, and 5 µL of 1 mg/mL dioleoyl-phosphoethanolamine-lissamine rhodamine B (rhodamine PE) (see Table of Materials) into the 15 ml glass vial.
   NOTE: Th.......

Discussion

Different methods of generating GUVs have been explored for the creation of synthetic cells However, the complexity of the procedures, extended time to attain encapsulation, restriction of lipid types and molecular composition of the encapsulant, need for non-physiological chemicals to facilitate encapsulation, low GUV yield, and inconsistencies in encapsulation efficiency have continued to challenge researchers in this field. Considering the wide range of potential studies that can be embarked in bottom-up syntheti.......

Materials

Name	Company	Catalog Number	Comments
18:1 Liss Rhod PE lipid in chloroform	Avanti Polar Lipids	810150C
96 Well Optical Btm Pit PolymerBase	ThermoFisher Scientific	165305
Actin from rabbit skeletal muscle	Cytoskeleton	AKL99-A
ATTO 488-actin from rabbit skeletal muscle	Hypermol	8153-01
Axygen microtubes (200 µL)	Fisher Scientific	14-222-262	for handling ABPs
Black resin	Formlabs	RS-F2-GPBK-04
Cholesterol (powder)	Avanti Polar Lipids	700100P
Choloroform	Sigma Aldrich	67-66-3
Clear resin	Formlabs	RS-F2-GPCL-04
CSU-X1 Confocal Scanner Unit	YOKOGAWA	CSU-X1
Density gradient medium (Optiprep)	Sigma-Aldrich	D1556
DOPC lipid in chloroform	Avanti Polar Lipids	850375C
Fascin	homemade	N/A
F-buffer	homemade	N/A
Fisherbrand microtubes (1.5 mL)	Fisher Scientific	05-408-129
FS02 Sonicator	Fischer Scientific	FS20
G-buffer	homemade	N/A
Glucose	Sigma-Aldrich	158968
iXon X3 camera	Andor	DU-897E-CS0
Mineral oil	Acros Organics	8042-47-5
Olympus IX81 Inverted Microscope	Olympus	IX21
Olympus PlanApo N 60x Oil Microscope Objective	Olumpus	1-U2B933
Silicone oil	Sigma-Aldrich	317667