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.

• gordon@chaos.utexas.edu