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
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)
Noireaux et al. (2011) PNAS 108, 3473-3480
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
Incorporation of genes and
enzymes into liposomes
Top-down approach
Minimal cell
(minimal life)
“Construction of a minimal
cell starting from scratch“
Bottom-up approach
Simple molecules
Membranes are made of amphipathic lipids
(Sphingosin in sphingomyelins)
Cholin
Inositol
Ethanolamin
Serine
etc.
The basic structural unit of biological membranes is
a lipid bilayer
Bilayers abhor free ends
• Pure phospholipid bilayers spontaneously
seal to form closed structures
Artificial vesicles of different sizes
SUV: small unilammelar vesicle
Ø ~ 50 nm (sonication)
LUV: large unilammelar vesicle
Ø ~ 100 nm (porous membranes)
GUVs, as well as LUVs and SUVs are aggregates that are usually not at a “true”
GUV: giant
unilammelar
thermodynamic equilibrium, but rather in a kinetically trapped
state,
as energy vesicle
Ø ~ stable
10-100for
µmhours,
(hydration)
barrier for interconversion is very high. Therefore they are
days or
even weeks.
MLV: multilamellar vesicles
Ø ~ 0.5 – 10 µm (agigation)
OVV: oligivesicular vesicles
Ø ~ 0.5 – 10 µm (agigation)
1-palmytoyl-2-oleoyl-snglycero-3-phosphocholin
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
4 nm
50 m
4 mm
Note, that a 10 fold increase in diameter results in a 1000 fold increase in volume
WOWE
L-WO
S-WO
PLB
WOS
WMOS
MS
SUV
LUV
Spontaneous swelling, natural swelling
or gentle hydration and electroformation
GH, EF
Walde et al. (2010) ChemBioChem 11, 848 – 865
GUVs from dried lipids on solid surfaces
Gentle hydration method
• Originally reported by Reeves and Dowben (1969)
N2
• Drying by passing N2
2L
• 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
5 µM egg yolk phosphatidylcholines
in 0.5 ml of 1:2 chloroform-methanol
• Harvesting the vesicles by density centrifugation
Gentle hydration method
Lamellae of dried
phospholipids
Aqueous solution runs in
between the lamellae
Swelling in a moist atmosphere
leads to separation from one another
Detachment and rounding
up into vesicles
Electroformation of GUVs
1.5 V and 10 Hz of an AC electric field
Expansion model
curvature fluctuations
Swelling model
multilayer stack
of lipids
no curvature fluctuations
Shimanouchi et al. (2009) Langmuir, 25, 4835–4840
Electroformation of GUVs
Shimanouchi et al. (2009) Langmuir, 25, 4835–4840
Membrane composition and fluidity
are the main determinants!
Electroformation yield symetric GUVs
GUVs from water oil emulsions
w/o emulsion transfer method (L-WO)
Water droplet
in emulsion
oil
Lipid of the
inner leaflet
Lipid of the
outer leaflet
water
• Size of GUV depends on size of water droplet
• Allows engineering of asymmetric vesicles
Engineering of assymetric vesicles
Quencher
TritonX-100
POPS
POPC
Pautot et al. (2003) PNAS 100, 10718-10721
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
Inside POPS
Outside POPC
Vesicles form even under
energitally unfavourable conditions
Hybrid vesicles of a inner diblock copolymer
and a outer egg-PC labelled with rhodamine
Inside POPC
Outside POPS
polystyrene-polyacrylic-acid diblock copolymer
GUVs from water oil emulsions
surfactant-stabilized w/o emulsion method (S-WO)
hexane
Surfactant
(Span 80, dodecylamine)
Surfactant stabilized
water droplet
in emulsion
Replacement of
surfactant with the
desired lipid at -10°C
liquid oil
frozen water droplets
replacement of the oil with an aqueous
suspension of small lipid vesicles (hydration)
• Not always unilammellar, but high entrapment yield
• Inside and outsite aequous solutions may be different
Formation of W/O Emulsions by Microchannel
(MC) Emulsification
Sugiura et al. (2008) Langmuir 24, 4581-4588
Number of publications using GUVs
120
100
80
60
2012
0
2010
2008
2006
2004
2002
2000
1998
1996
1994
1992
20
1990
1988
1986
1984
1982
1980
1978
1974
1976
1972
1970
40
• 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
Reconstitution of membrane proteins in GUVs
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 electroswelling of the partially dried film to give GUVs
(EF).
Girard et al. (2004) Biophys. J. 87, 419–429
Activity of Ca2+-ATPase in GUVs
ATPase activity measured by an enzyme-linked assay
Girard et al. (2004) Biophys. J. 87, 419–429
Fluo-5N
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
GUVs as a bioreactors: eGFP production
a-hemolysin-eGFP
100% extract
50% extract
Expression of eGFP
in bacterial extracts
Expression of eGFP
Inside GUV
Noireaux and Libchaber (2004) PNAS 101, 17669–17674
a-hemolysin-eGFP
Expressed inside
GUV
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
Reconstitution of cytoskeletal elements in/on GUVs
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
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
Polymerization: 150 mM KCl, 2 mM CaCl2, 5 mM HEPES (pH 7.5), 2
mM ATP, 6 mM DTT, 0.13 mM Dabco.
Pontani et al. (2009) Biophys. J. 96, 192-198
Reconstitution of cytoskeletal elements in/on GUVs
Actin Alexa Fluor 488
Actin rhodamine phalloidin
Pontani et al (2009) Biophys. J. 96, 192-1968
Reconstitution of cytoskeletal elements in/on GUVs
- cholesterol
minus N-WASP
minus N-WASP, ARP2/3
plus latrunculin
Pontani et al (2009) Biophys. J. 96, 192-1968
+ cholesterol
minus N-WASP
minus N-WASP, ARP2/3
GUVs with bulged domains below their miscibility
transition temperature (phase separation)
Mixtures of saturated lipids, unsaturated lipids, and cholesterol
Veatch and Keller (2003) Biophys. J. 85, 3074–3083
Scale bars: 20 µm
Assembly of actin networks on phase-separated GUVs
PIP2 lipids N-WASP ARP2/3
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
Escrt III
Wollert et al. (2009) Nature 458, 172-177
Hurley and Odorizzi (2012) Nature Cell Biol. 14, 654–655
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“