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Adhesion and phase separation in mixed-lipid membranes: steps toward a better experimental model

Adhesion and phase separation in mixed-lipid membranes: steps toward a better experimental model. Vernita 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

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Adhesion and phase separation in mixed-lipid membranes: steps toward a better experimental model

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  1. Adhesion and phase separation in mixed-lipid membranes: steps toward a better experimental model Vernita D. Gordon, University of Texas at Austin

  2. 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)

  3. Model systems reduce rich lipid compositions 1000s of different lipid species Michael Edidin (2003) Nature Reviews Molecular Cell Biology 4, 414-418 Phospholipids Structure from LIPIDAT xx == hydrophobic tail saturation and length y == hydrophilic headgroup Lipid names: xxPy

  4. Lipid amphiphilicity + aqueous solution  self-assembled structures bilayer water water membrane vesicle hydrophilic hydrophilic ~10 mm = Giant Unilamellar Vesicle (GUV) hydrophobic

  5. Lipids in simple model bilayers form a variety of solid-like phases La P′ II bend ~10 stiffer temperature L P′ L′ and others

  6. In model bilayers containing cholesterol, lipids form different liquid phases Ld La = cholesterol temperature Lo bend ~2 stiffer L′ L and others P′

  7. Models: Giant Unilamellar Vesicles (GUVs) containing preferentially-partitioning fluorescent dyes Each image = projection of upper or lower hemisphere Most ordered phases exclude dyes as impurities: For Pb′, dyes partition complementarily: Rh-DPPE or DiI-C-18 BODIPY (Dyes are ~0.5 mol% of system composition.)

  8. cells adhere to the extracellular environment nutrients and pathogens interact with and enter cells Membrane adhesion essential in biology rafts and caveolae. http://publications.nigms.nih.gov/insidethecell/chapter2.html Lo

  9. Adhesion favors demixing and localizes ordered phases Fluid-ordered domains Pb′ II hexagonaldomains (in 3 different lipid mixtures with the same headgroup) Pb′ “red” domains VDG, M Deserno, et al, 2008 Europhysics Letters 84:48003

  10. Why we think this happens:

  11. Undulations favour mixing Systems demix when this reduces their free energy (U – TS) Treat a membrane as a collection of classical oscillators, each with spring constant and free energy kbendq4 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 Fluid-ordered a ~ 2 Solid-like a ~ 10  Undulations favour mixing Suppressing undulations favours demixing

  12. Confining the membrane suppresses fluctuations Approximate adhesion as a confining, harmonic potential Classical oscillators comprising the membrane have new spring constants Previous Toy Case: completely-demixed AB membrane with confinement has a change in the free energy of demixing  where For a ~ 10, at room temperature, effect of confinement ~ 1% or 3K VDG, M Deserno, et al, 2008 Europhys Letts, 84:48003

  13. 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

  14. Steps toward this vision: Unbound, fluctuating, fluid-phase membrane Specifically adhering, fluctuations suppressed, solid-phase membrane vesicle Membrane binder Molecular target

  15. Scheme for specifically adhering membranes Figure from Fenz, S.F., R. Merkel and K. Sengupta. Langmuir, 2009. 25: p. 1074-1085.

  16. Specific adhesion in our lab • Non-adhering vesicles drift. • Adhering vesicles do not drift.

  17. Specific adhesion in our lab t=0 t=10 minutes

  18. 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

  19. 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

  20. 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

  21. Track 1: Measure fluctuations

  22. 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

  23. Microscopy techniques to study adhesion and fluctuations Total internal reflection fluorescence Reflection interference (can be developed into reflection interference contrast)

  24. Calibrating TIRF measurements d=λo/4π(n22sin2θ-n12)-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 rad θ= angle of incidence Thanks to Prof. George Shubeita (UT Austin) and his group!

  25. Binder concentration may make a difference Low concentration of neutravidin High concentration of neutravidin

  26. Image processing and analysis Correct for: Lateral drift Photobleaching/z-drift Background noise

  27. Correcting for lateral drift • Center of mass should stay in the same place

  28. Correcting for photobleaching/z-drift Remove trends in pixel brightness

  29. Correcting for background noise Measure noise for SLB alone, no vesicles

  30. Final corrected image Instead of

  31. Measuring membrane fluctuations Specifically-adhering membrane Dh(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

  32. Track 2: measure phase separation

  33. 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

  34. 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

  35. 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

  36. Adhesion decreases the fraction of membranes that phase separate 42 mol% cholesterol: 28% of specifically-adhering membranes phase separate 42% 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, 0.59 non-adhering, 0.68 ~10 membranes/sample This is the opposite of what we expected. Are we crazy?

  37. 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

  38. 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

  39. Another richness that could arise: • Preference of the binder for one phase over another

  40. 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.

  41. 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

  42. Advertisment • 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

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