adhesion and phase separation in mixed- lipid membranes: steps toward a better experimental model...
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![Page 1: Adhesion and phase separation in mixed- lipid membranes: steps toward a better experimental model Vernita D. Gordon, University of Texas at Austin](https://reader030.vdocuments.us/reader030/viewer/2022032805/56649eec5503460f94bfdc2f/html5/thumbnails/1.jpg)
Adhesion and phase separation in mixed-lipid membranes: steps toward a better
experimental modelVernita D. Gordon, University of Texas at Austin
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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)
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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
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Lipid amphiphilicity + aqueous solution self-assembled structures
membrane vesicle
waterwater
hyd
rop
ho
bichydrophilic hydrophilic
~10 m = Giant Unilamellar Vesicle (GUV)
bilayer
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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
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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
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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:
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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
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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
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Why we think this happens:
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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)
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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
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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
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Steps toward this vision:
Unbound, fluctuating, fluid-phase membrane
Specifically adhering, fluctuations suppressed, solid-phase membrane
vesicle
Membrane binder
Molecular target
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Scheme for specifically adhering membranes
Figure from Fenz, S.F., R. Merkel and K. Sengupta. Langmuir, 2009. 25: p. 1074-1085.
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Specific adhesion in our lab
• Non-adhering vesicles drift.
• Adhering vesicles do not drift.
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Specific adhesion in our lab
t=0 t=10 minutes
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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
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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
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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
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TRACK 1: MEASURE FLUCTUATIONS
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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
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Microscopy techniques to study adhesion and fluctuations
Reflection interference (can be developed into reflection interference contrast)
Total internal reflection fluorescence
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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
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Binder concentration may make a difference
High concentration of neutravidin
Low concentration of neutravidin
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Image processing and analysis
Correct for:Lateral driftPhotobleaching/z-driftBackground noise
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Correcting for lateral drift
• Center of mass should stay in the same place
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Correcting for photobleaching/z-drift
Remove trends in pixel brightness
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Correcting for background noise
Measure noise for SLB alone, no vesicles
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Final corrected image
Instead of
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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
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TRACK 2: MEASURE PHASE SEPARATION
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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
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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
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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
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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?
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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
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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
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Another richness that could arise:
• Preference of the binder for one phase over another
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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.
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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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