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“
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