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Adhesion and phase separation in mixed-lipid membranes: steps toward a better
experimental modelVernita D. Gordon, University of Texas at Austin
Membranes are important for:
• Biophysics– Interface of cell and environment
• Physics– Rich model systems for interactions and transitions– Novel couplings of statistical mechanics & elasticity
• soft to perturbations caused by kBT
• Biotechnology– Controlled encapsulation and delivery – Artificial cells created by synthetic biology
Michael Edidin (2003)
Model systems reduce rich lipid compositions
Phospholipids
Structure from LIPIDAT
Michael Edidin (2003) Nature Reviews Molecular Cell Biology 4, 414-418
1000s of different lipid species
Lipid names: xxPy
xx == hydrophobic tail saturation and length
y == hydrophilic headgroup
Lipid amphiphilicity + aqueous solution self-assembled structures
membrane vesicle
waterwater
hyd
rop
ho
bichydrophilic hydrophilic
~10 m = Giant Unilamellar Vesicle (GUV)
bilayer
P′ II
Lipids in simple model bilayers form a variety of solid-like phases
L
tem
per
atu
re
L
L′
P′
and others
bend ~10 stiffer
L
tem
per
atu
re
LdL
L′P′ and others
Lo
In model bilayers containing cholesterol, lipids form different liquid phases
bend ~2 stiffer
= cholesterol
Each image = projection of upper or lower hemisphere
Models: Giant Unilamellar Vesicles (GUVs) containing preferentially-partitioning fluorescent dyes
BODIPYRh-DPPE or DiI-C-18
(Dyes are ~0.5 mol% of system composition.)
Most ordered phases exclude dyes as impurities:
For P′, dyes partition complementarily:
Membrane adhesion essential in biology
cells adhere to the extracellular environment
nutrients and pathogens interact with and enter cells
rafts and caveolae.
http://publications.nigms.nih.gov/insidethecell/chapter2.html
Lo
Adhesion favors demixing and localizes ordered
phases
P′ II hexagonal domains
(in 3 different lipid mixtures with the same headgroup)
Fluid-ordered domains
P′ “red” domains
VDG, M Deserno, et al, 2008 Europhysics Letters 84:48003
Why we think this happens:
Undulations favour mixing
f )/ln( 2ABBA
0ln
)/ln( BB TkTkF bendq4
ABBA21 )( ffff
ABAAB
Treat a membrane as a collection of classical oscillators, each with spring constant and free energy
Toy Case: For a membrane with 2 components, A:B 1:1, complete demixing changes the undulation contribution to the free energy of demixing by
Integrating over all oscillator modes gives
If disordered (soft) AB mixture demixes into disordered A and ordered B, moduli are
Undulations favour mixing
Fluid-ordered ~ 2Solid-like ~ 10
Suppressing undulations favours demixing
f
Systems demix when this reduces their free energy (U – TS)
221 mh
mq 4
f
Approximate adhesion as a confining, harmonic potential
Classical oscillators comprising the membrane have new spring constants
Confining the membrane suppresses fluctuations
)/1arctan(2)( 2/1xxxA
)()/(1
ln mAmAm
m Previous Toy Case: completely-demixed AB membrane with confinement has a change in the free energy of demixing
where
For ~ 10, at room temperature, effect of confinement ~ 1% or 3K
VDG, M Deserno, et al, 2008 Europhys Letts, 84:48003
Implications for biological & biotechnological structures
Raft localization, growth, stabilization
Functional vesicles
Unbound, fluctuating, fluid-phase membrane
Specifically adhering, fluctuations suppressed, solid-phase membrane
vesicle
Membrane binder
Molecular target
Steps toward this vision:
Unbound, fluctuating, fluid-phase membrane
Specifically adhering, fluctuations suppressed, solid-phase membrane
vesicle
Membrane binder
Molecular target
Scheme for specifically adhering membranes
Figure from Fenz, S.F., R. Merkel and K. Sengupta. Langmuir, 2009. 25: p. 1074-1085.
Specific adhesion in our lab
• Non-adhering vesicles drift.
• Adhering vesicles do not drift.
Specific adhesion in our lab
t=0 t=10 minutes
Plan of action:• Measure effect of adhesion on phase separation
– Area fraction of ordered phase– Transition temperature
• Measure effect of adhesion on fluctuations
• Correlate
• Vary: – Stiffness of ordered phase– Binder properties
Strategy for measuring effect of adhesion on phase separation
• Work from known phase diagrams, very near the demixing boundary– Binary system: DOPC-DPPC
• Solid-like ordered phase– Ternary system: DOPC-DPPC-cholesterol
• Fluid-like ordered phase• Incorporate trace amounts of binders, PEG, and
fluorescent dye• Measure area fractions of ordered phase
– Specifically-adhering vs non-adhering vesicles• Measure transition temperature
– Specifically-adhering vs non-adhering vesicles
Steps toward this
• Vesicles that incorporate binders, PEG, and dye show the right phase separation
• Good yields of unilamellar, isolated vesicles
• Good supported bilayers to provide targets for binding
TRACK 1: MEASURE FLUCTUATIONS
Strategy for measuring effect of adhesion on fluctuations
• Measure fluctuations in membranes– Specifically-adhering vs non-adhering
• Begin with non-phase-separating, fluid membranes
• Advance to phase-separating membranes
Microscopy techniques to study adhesion and fluctuations
Reflection interference (can be developed into reflection interference contrast)
Total internal reflection fluorescence
Calibrating TIRF measurements
Thanks to Prof. George Shubeita (UT Austin) and his group!
d=λo/4π(n22sin2θ-n1
2)-1/2
d=Iz/e length for evanescent wave (penetration depth)λo= excitation wavelength (532 nm for the setup)n2 = index of refraction of coverslip (~1.52)n1 = index of refraction of buffer (~1.34)θcritical= sin-1(n1/n2)= 1.08 radθ= angle of incidence
Binder concentration may make a difference
High concentration of neutravidin
Low concentration of neutravidin
Image processing and analysis
Correct for:Lateral driftPhotobleaching/z-driftBackground noise
Correcting for lateral drift
• Center of mass should stay in the same place
Correcting for photobleaching/z-drift
Remove trends in pixel brightness
Correcting for background noise
Measure noise for SLB alone, no vesicles
Final corrected image
Instead of
Measuring membrane fluctuations
Specifically-adhering membrane
h(x,y,t) = h(x,y,t) - <h(x,y)>
RMS displacement measured: ~13nm
13.198nm for a large region
13.283nm for a smaller region
TRACK 2: MEASURE PHASE SEPARATION
DOPC:DPPC + cholesterol• Phase behavior characterized by S. Keller
and S. Veatch, U. Washington, Seattle– Standing on the shoulders of giants– Transition temperatures and phase diagram
• At sufficient cholesterol concentrations, this system has fluid-fluid phase separation
– DOPC:DPPC 1:1 + 42mol% or 45mol% cholesterol
Experimental strategy
• Prepare a sample of DOPC:DPPC:cholesterol + trace amounts of biotin, PEG, fluorophores
Measure area fraction of ordered phase in specifically-adhering versus non-adhering membranes
Early experimental images
Most membranes show no phase separation
If we’re careful about how we load the sample, a few
membranes do show phase separation right at the
adhering bottom
Adhesion decreases the fraction of membranes that phase separate
42 mol% cholesterol:28% of specifically-adhering membranes phase separate42% of non-adhering
membranes phase separate~40 membranes/sample
For those membranes with ordered phase at the adhering area, area fraction of ordered phase at the adhering area:
specifically-adhering, 0.59non-adhering, 0.68~10 membranes/sample
This is the opposite of what we expected. Are we crazy?
New working hypothesis• We are not completely crazy.
• Adhesion can do more than one thing:– Suppress fluctuations– Tense the membrane
• If tension stretches the membrane enough to dilate area/headgroup, could that suppress phase separation?– Could be 2 regimes of phase separation
interacting with adhesion
Plan from here:
• Test the new working hypothesis:– If the adhering area is low:
• membrane fluctuations suppressed • ordered phase promoted
– If the adhering area is high:• membrane tension dilates area/headgroup• Ordered phase suppressed
Another richness that could arise:
• Preference of the binder for one phase over another
Summary Suppressing fluctuations alters demixing behavior
We want to use this to understand the cell membrane and to make functional membranes that combine targeting and triggering.
Thank you• You • (UT Austin) Matthew Leroux, Matthew Preble,
Nabiha Saklayen; Jeanne Stachowiak (BME); George Shubeita (Physics)
• (Edinburgh) Paul Beales, Markus Deserno, Wilson Poon, Stefan Egelhaaf
• EPSRC
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• Postdoc to work on a bacteria experiment: how does spatial structure develop in biofilms, and how does this impact cooperation? – This 4-investigator collaboration is funded by
the Human Frontiers Science Project and is a great opportunity to train across disciplines.