Presentation given as part of the Emerging Topics module, awarded 72.5%.

1. Positioning Lipid Membrane
Domains in Giant Vesicles by
Micro-organisation of Aqueous
Cytoplasm Mimic
A. Cans, M. Andes-Koback, C. Keating
Emerging Topics – Dr Beales
J. Ingram – 200743868
University of Leeds

2. Cell Membrane and Interior
Heterogeneity
In addition to
heterogeneity of
organelles in cells,
there is localised
membrane and
cytoplasm
heterogeneity. The
origin and
consequence of this is
not fully understood.

3. Cytoplasm Mimic – ATPS
Dextran Polyethylene
Glycol (PEG)
• Homogenous mixture at 37°C, but forms a
heterogeneous aqueous two phase system
(ATPS) at 4°C.
– “Cell” composition can be manipulated by
initial ratios of PEG: Dextran.

4. Cytoplasm Mimic –
Fluorescence Markers
FITC – Dextran Alexa647 – PEG
GREEN BLUE
To allow identification of the contents of
the giant vesicles, fluorescent versions of
PEG and Dextran were alternatively used.

5. Membrane Composition
Structural Preferential Phospholipids
NonPEGylated PolyPEGylated
Liquid Disordered (Ld) Liquid Ordered (Lo)
DOPC DOPE-mPEG
DPPC DSPE-mPEG
Fluorescent Preferential Phospholipids
DOPE-rhodamine DSPE-mPEG-FITC
RED GREEN

6. Giant Vesicle (GV) Formation
1. Lipid solution placed in
Non-PEGylated test tube.
lipids
2. Dried with argon, solvent
removed in vacuo.
3. 37°C homogeneous mix of
dextran and PEG added.
4. Left at 37°C for 2-3 days,
giant vesicles formed.
5. Cooled to 5°C, monolayer
~5μm diameter, two
aqueous phase GVs
PolyPEGylated lipids
selected.

7. Giant Vesicle Observations
Transmitted light Rhodamine-DOPE Alexa647-PEG
DCC CF-PEG-DSPE

8. Giant Vesicle Observations
Hypertonic sucrose 1. Reduced volume  surplus membrane
addition induces  distortion of vesicle = “budding”
osmotic stress. 2. Increased [PEG]+[Dextran]  Increased
interfacial energy  Increased solute
partitioning
3. Increased interfacial energy + surplus
membrane  expulsion of dextran from
inside the PEG.

9. Giant Vesicle Observations
• Budding • Redistribution • Increased
partitioning

10. What Decides Localisation?
1. Ld can curve more, so
encases the smaller
volume.
2. Lipid headgroup –
interior interactions, i.e.
the contents decide
membrane localisation.
3. Expulsion of dextran
from the centre causes a
more flexible lipid to
encase it

11. Proving Theory 2
For Theories 1 and 3 For Theory 2
• Ld is more flexible • Membrane overlaps
than the Lo due to PEG liquid phases
groups • Inverting Lo and Ld so Ld
is PEGylated does not
change distribution

12. Proving Theory 2
For Theories 1 and 3 For Theory 2
• Ld is more flexible • Membrane overlaps
than the Lo due to PEG liquid phases
groups • Inverting Lo and Ld so Ld
is PEGylated does not
change distribution

13. Concordant Results
Localised Cold • Heating caused mixing
of lipids but not
cytoplasm
• Cooling causes the
membrane to reform
around the cytoplasm.
Mixed
Hot
– Lipid domain position is
not random, nor does it
rely on Ld / Lo
mechanical properties,
but is decided by the
aqueous phase position.
Localised Cold

14. Conclusions
• Found a correlation between
internal and external
organisation
– Due to nonspecific interactions
• Created a basic model of a cell,
the first make:
– Model cells with chemically
asymmetric cytoplasm and
membrane.

15. Future Endeavour
• Asymmetric division of artificial cells.
• Local control over enzymatic reactions to
control mineralization.
• New routes to controlled encapsulation
of macromolecular solutes within
artificial cells.

16. Questions?