Artificial cells and microcompartmentation

IRTG Lecture on

„Microcompartments, Membranes and Cellular Communication - part II: Methods“

H. Merzendorfer, 25.7.2012

The basic unit of life:

The cell

Noireaux et al. (2011) PNAS 108, 3473-3480

Cells observed by Robert Hooke and Theodor Schwann

Robert Hooke (1635-1702) Matthias Jacob Schleiden (1804-1881) Theodor Schwann (1810-1882) Rudolf Virchow (1821-1902) Keith R. Porter (1912-1997)

Porter et al. (1945) J. Exp. Med. 81, 233

Membranes organize cells into functionally distinct compartments

• Each type of membrane has a unique function and unique protein and lipid components

• The interior (lumen) of each compartment has a unique chemical composition

• Membranes control the composition of the compartments by controlling movement of molecules across the membrane

Two working directions in cell biology

Modern living cell

Minimal cell (minimal life)

Simple molecules

Bottom-up approach

Top-down approach Incorporation of genes and enzymes into liposomes

“Construction of a minimal cell starting from scratch“

Membranes are made of amphipathic lipids

Cholin Inositol Ethanolamin Serine etc.

(Sphingosin in sphingomyelins)

The basic structural unit of biological membranes is a lipid bilayer

Bilayers abhor free ends

• Pure phospholipid bilayers spontaneously seal to form closed structures

SUV: small unilammelar vesicle Ø ~ 50 nm (sonication)

GUV: giant unilammelar vesicle Ø ~ 10-100 µm (hydration)

1-palmytoyl-2-oleoyl-sn- glycero-3-phosphocholin

Artificial vesicles of different sizes

LUV: large unilammelar vesicle Ø ~ 100 nm (porous membranes)

MLV: multilamellar vesicles Ø ~ 0.5 – 10 µm (agigation)

OVV: oligivesicular vesicles Ø ~ 0.5 – 10 µm (agigation)

GUVs, as well as LUVs and SUVs are aggregates that are usually not at a “true” thermodynamic equilibrium, but rather in a kinetically trapped state, as energy barrier for interconversion is very high. Therefore they are stable for hours, days or even weeks.

Giant vesicles

• resembles the basic compartment

structure of all biological cells (artificial cells)

• Mimics the self-closed lipid matrix of the plasma membrane

• Easy to investigate by light and fluorescence microscopy, because they usually have a diameter of about 20 µm

How to generate giant vesicles?

Simple geometric considerations

50 m

4 mm

Note, that a 10 fold increase in diameter results in a 1000 fold increase in volume

4 nm

Spontaneous swelling, natural swelling or gentle hydration and electroformation

GH, EF

WOS

WOWE

L-WO S-WO

PLB

SUV LUV

WMOS MS

Walde et al. (2010) ChemBioChem 11, 848 – 865

2 L

Gentle hydration method • Originally reported by Reeves and Dowben (1969)

• Drying by passing N2

• Initial hydration by passing watersaturated N2

• Addition of ~ 20 ml aquous solution

• Swelling under N2 for 2 hrs

• Even slight agitation diminishes yield due to MLV formation

• Harvesting the vesicles by density centrifugation

5 µM egg yolk phosphatidylcholines in 0.5 ml of 1:2 chloroform-methanol

GUVs from dried lipids on solid surfaces

N2

Gentle hydration method

Aqueous solution runs in between the lamellae

Detachment and rounding up into vesicles

Lamellae of dried phospholipids

Swelling in a moist atmosphere leads to separation from one another

Electroformation of GUVs

multilayer stack of lipids

1.5 V and 10 Hz of an AC electric field

Shimanouchi et al. (2009) Langmuir, 25, 4835–4840

curvature fluctuations

no curvature fluctuations

Expansion model

Swelling model

Electroformation of GUVs

Membrane composition and fluidity are the main determinants!

Shimanouchi et al. (2009) Langmuir, 25, 4835–4840

Electroformation yield symetric GUVs

GUVs from water oil emulsions

• Size of GUV depends on size of water droplet • Allows engineering of asymmetric vesicles

oil

water

Water droplet in emulsion

w/o emulsion transfer method (L-WO)

Lipid of the inner leaflet

Lipid of the outer leaflet

POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine NBD-PC, 1-palmitoyl-2-{6-[(7-nitro2–1,3-benzoxadiazol-4-yl) amino caproyl]-sn-glycero-3-phosphocholine} NBD-PS, 1-palmitoyl-2-{6-[(7-nitro2–1,3-benzoxadiazol-4-yl) amino] caproyl}-sn-glycero-3-(phospho-L-serine) Quencher: sodium hydrosulfite

Pautot et al. (2003) PNAS 100, 10718-10721

POPS

POPC

Quencher TritonX-100

Engineering of assymetric vesicles

Inside POPS Outside POPC

Inside POPC Outside POPS

Hybrid vesicles of a inner diblock copolymer and a outer egg-PC labelled with rhodamine

Vesicles form even under energitally unfavourable conditions

polystyrene-polyacrylic-acid diblock copolymer

GUVs from water oil emulsions

surfactant-stabilized w/o emulsion method (S-WO)

• Not always unilammellar, but high entrapment yield • Inside and outsite aequous solutions may be different

Surfactant stabilized water droplet in emulsion liquid oil

frozen water droplets

Replacement of surfactant with the desired lipid at -10°C

Surfactant (Span 80, dodecylamine)

replacement of the oil with an aqueous suspension of small lipid vesicles (hydration)

hexane

Formation of W/O Emulsions by Microchannel (MC) Emulsification

Sugiura et al. (2008) Langmuir 24, 4581-4588

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Number of publications using GUVs

• Lipid domain formation and lateral lipid heterogeneity in biological membranes (GH)(EF)

• Lipid membrane dynamics, lipid order and membrane fluidity (EF)

• Interaction of virus-like particles with lipid bilayers (EF)

• Protein (peptide) lipid bilayer interactions (GH)(EF)

• Investigation of membrane proteins reconstituted within vesicle (GH) (EF)

• Study of giant vesicles as micro-reactors and minimal cells (WO)

• Giant vesicles with a reconstituted cytoskeleton (EF)

• Membrane fusion, budding, fission and scission of vesicles as biomembrane model system (GH) (EF)

Reconstitution of membrane proteins in GUVs

sarcoplasmic-reticulum Ca2+-ATPase

Step 1 Detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1–0.2 µm in size.

Step 2 Proteoliposomes were partially dried under controlled humidity followed by electro-swelling of the partially dried film to give GUVs (EF).

Reconstitution of membrane proteins in GUVs

Girard et al. (2004) Biophys. J. 87, 419–429

Activity of Ca2+-ATPase in GUVs

ATPase activity measured by an enzyme-linked assay

Fluo-5N Girard et al. (2004) Biophys. J. 87, 419–429

Unilamellarity of reconstituted GUVs using elastic bending measurements

Girard et al. (2004) Biophys. J. 87, 419–429

GUVs as a bioreactors: one step towards an artificial cell assembly

Giant unilamellar vesicles containing bacterial extracts for protein biosynthesis are produced in an oil-extract emulsion. GUVs are added to a feeding solution containing ribonucleotides and amino acids. Expression of eGFP inside the vesicle. To overcome limitations due to low membrane Permeabilities, hemolysin pore protein was Expressed.

Noireaux and Libchaber (2004) PNAS 101, 17669–17674

a-hemolysin-eGFP Expressed inside GUV

Expression of eGFP Inside GUV

50% extract

100% extract

Expression of eGFP in bacterial extracts

GUVs as a bioreactors: eGFP production

a-hemolysin-eGFP

Noireaux and Libchaber (2004) PNAS 101, 17669–17674

Self-reproduction of supramolecular giant vesicles with encapsulated DNA

Kurihara et al. (2011) Nature Chem. 3, 475-79

Self-reproduction of supramolecular giant vesicles with encapsulated DNA

Kurihara et al. (2011) Nature Chem. 3, 475-79

Inside: 0.12 mM Arp2/3 50 nM gelsolin 2 mM ADF-cofilin 1 mM profilin 6.5 mM G-actin (20% labeled) 0.64 mM VVCA-His.0

Outside: 10 mM HEPES (pH 7.5) 2 mM MgCl2, 0.2 mM CaCl2

2 mM ATP, 6 mM DTT 0.13 mM Dabco 275 mM glucose 0.5 mg/mL casein

Polymerization: 150 mM KCl, 2 mM CaCl2, 5 mM HEPES (pH 7.5), 2 mM ATP, 6 mM DTT, 0.13 mM Dabco.

Reconstitution of cytoskeletal elements in/on GUVs

Pontani et al. (2009) Biophys. J. 96, 192-198

Reconstitution of cytoskeletal elements in/on GUVs

Pontani et al (2009) Biophys. J. 96, 192-1968

Actin Alexa Fluor 488 Actin rhodamine phalloidin

Reconstitution of cytoskeletal elements in/on GUVs

+ cholesterol - cholesterol

minus N-WASP minus N-WASP, ARP2/3 plus latrunculin

minus N-WASP minus N-WASP, ARP2/3

Pontani et al (2009) Biophys. J. 96, 192-1968

GUVs with bulged domains below their miscibility transition temperature (phase separation)

Scale bars: 20 µm

Mixtures of saturated lipids, unsaturated lipids, and cholesterol

Veatch and Keller (2003) Biophys. J. 85, 3074–3083

PIP2 lipids N-WASP ARP2/3

Assembly of actin networks on phase-separated GUVs

Liu and Fletcher (2006) Biophys. J. 91, 4064–4070

Actin Polymerization Serves as a Membrane Domain Switch in Model Lipid Bilayers

Liu and Fletcher (2006) Biophys. J. 91, 4064–4070 Tmisc: miscibility transition temperature

GUVs to analyze MVB formation

Hurley and Odorizzi (2012) Nature Cell Biol. 14, 654–655

Escrt III

Wollert et al. (2009) Nature 458, 172-177

Membrane Scission by the ESCRT-III Complex

Wollert et al. (2009) Nature 458, 172-177

Summary

• GUVs can be prepared in various ways

• Electroformation and water/oil emulsion systems are most frequently used

• GUVs serve as biomimetic models

– Membrane protein reconstitution

– Membrane fusion, fission and scission

– Function of membrane domains

– Contruction of bioreactors and „minimal cells“