Research Papers

Atomic Force Microscopy of Phase Separation on Ruptured, Giant Unilamellar Vesicles, and a Mechanical Pathway for the Co-Existence of Lipid Gel Phases

[+] Author and Article Information
Yanfei Jiang

Department of Biochemistry and
Molecular Biophysics,
School of Medicine,
Washington University,
St. Louis, MO 63110

Kenneth M. Pryse, Srikanth Singamaneni

Department of Mechanical Engineering
and Materials Science,
Washington University,
St. Louis, MO 63110

Guy M. Genin

Department of Mechanical Engineering
and Materials Science,
Washington University,
St. Louis, MO 63110;
NSF Science and Technology,
Center for Engineering Mechanobiology,
Washington University,
St. Louis, MO 63110
e-mail: genin@wustl.edu

Elliot L. Elson

Department of Biochemistry and
Molecular Biophysics,
School of Medicine,
Washington University,
St. Louis, MO 63110
e-mail: elson@wustl.edu

1Corresponding authors.

Manuscript received December 10, 2018; final manuscript received May 26, 2019; published online June 13, 2019. Assoc. Editor: Victor H. Barocas.

J Biomech Eng 141(7), 071003 (Jun 13, 2019) (7 pages) Paper No: BIO-18-1528; doi: 10.1115/1.4043871 History: Received December 10, 2018; Revised May 26, 2019

Phase separation of lipid species is believed to underlie formation of lipid rafts that enable the concentration of certain surface receptors. However, the dynamics and stabilization of the resulting surface domains are unclear. We developed a methodology for collapsing giant unilamellar vesicles (GUVs) into supported bilayers in a way that keeps membrane nanodomains stable and enables their imaging. We used a combination of fluorescence and atomic force microscopy (AFM) of this system to uncover how a surprising phase separation occurs on lipid vesicles, in which two different gel phases of the same lipid co-exist. This unusual phase behavior was evident in binary GUVs containing 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The approach showed that one of the phases is stabilized by lipid patches that become ejected from the membrane, thereby enabling the stabilization of what would otherwise be a thermodynamically impossible coexistence. These results show the utility of AFM on collapsed GUVs, and suggest a possible mechanical mechanism for stabilization of lipid domains.

Copyright © 2019 by ASME
Your Session has timed out. Please sign back in to continue.


Elson, E. L. , Fried, E. , Dolbow, J. E. , and Genin, G. M. , 2010, “ Phase Separation in Biological Membranes: Integration of Theory and Experiment,” Annu. Rev. Biophys., 39(1), pp. 207–226. [CrossRef] [PubMed]
Pike, L. J. , 2006, “ Rafts Defined: A Report on the Keystone Symposium on Lipid Rafts and Cell Function,” J. Lipid Res., 47(7), pp. 1597–1598. [CrossRef] [PubMed]
Hinz, H. J. , and Sturtevant, J. M. , 1972, “ Calorimetric Studies of Dilute Aqueous Suspensions of Bilayers Formed From Synthetic L-α-Lecithins,” J. Biol. Chem., 247(19), pp. 6071–6075. [PubMed]
Melchior, D. L. , Morowitz, H. J. , Sturtevant, J. M. , and Tsong, T. Y. , 1970, “ Characterization of the Plasma Membrane of Mycoplasma Laidlawii—VII: Phase Transitions of Membrane Lipids,” Biochim. Biophys. Acta, 219(1), pp. 114–122. [CrossRef] [PubMed]
Harder, T. , and Sangani, D. , 2009, “ Plasma Membrane Rafts Engaged in T Cell Signalling: New Developments in an Old Concept,” Cell Commun. Signal. CCS, 7, p. 21. [CrossRef]
Simons, K. , and Vaz, W. L. C. , 2004, “ Model Systems, Lipid Rafts, and Cell Membranes,” Annu. Rev. Biophys. Biomol. Struct., 33, pp. 269–295. [CrossRef] [PubMed]
Mouritsen, O. G. , 1991, “ Theoretical Models of Phospholipid Phase Transitions,” Chem. Phys. Lipids, 57(2–3), pp. 179–194. [CrossRef] [PubMed]
Korlach, J. , Schwille, P. , Webb, W. W. , and Feigenson, G. W. , 1999, “ Characterization of Lipid Bilayer Phases by Confocal Microscopy and Fluorescence Correlation Spectroscopy,” Proc. Natl. Acad. Sci., 96(15), pp. 8461–8466. [CrossRef]
Feigenson, G. W. , and Buboltz, J. T. , 2001, “ Ternary Phase Diagram of Dipalmitoyl-PC/Dilauroyl-PC/Cholesterol: Nanoscopic Domain Formation Driven by Cholesterol,” Biophys. J., 80(6), pp. 2775–2788. [CrossRef] [PubMed]
Morales-Penningston, N. F. , Wu, J. , Farkas, E. R. , Goh, S. L. , Konyakhina, T. M. , Zheng, J. Y. , Webb, W. W. , and Feigenson, G. W. ,. 2010, “ GUV Preparation and Imaging: Minimizing Artifacts,” Biochim. Biophys. Acta, 1798(7), pp. 1324–1332. [CrossRef] [PubMed]
Li, L. , and Cheng, J.-X. , 2006, “ Coexisting Stripe- and Patch-Shaped Domains in Giant Unilamellar Vesicles,” Biochemistry, 45, pp. 11819–11826. [CrossRef] [PubMed]
Embar, A. , Dolbow, J. , and Fried, E. , 2013, “ Microdomain Evolution on Giant Unilamellar Vesicles,” Biomech. Model. Mechanobiol., 12(3), pp. 597–615. [CrossRef] [PubMed]
Maleki, M. , and Fried, E. , 2013, “ Multidomain and Ground State Configurations of Two-Phase Vesicles,” J. R. Soc. Interface R. Soc., 10(83), p. 20130112. [CrossRef]
Brewster, R. , and Safran, S. A. , 2010, “ Line Active Hybrid Lipids Determine Domain Size in Phase Separation of Saturated and Unsaturated Lipids,” Biophys. J., 98(6), pp. L21–L23. [CrossRef] [PubMed]
Palmieri, B. , and Safran, S. A. , 2013, “ Hybrid Lipids Increase the Probability of Fluctuating Nanodomains in Mixed Membranes,” Langmuir ACS J. Surf. Colloids, 29(17), pp. 5246–5261. [CrossRef]
Frolov, V. A. J. , Chizmadzhev, Y. A. , Cohen, F. S. , and Zimmerberg, J. , 2006, “ Entropic Traps' in the Kinetics of Phase Separation in Multicomponent Membranes Stabilize Nanodomains,” Biophys. J., 91(1), pp. 189–205. [CrossRef] [PubMed]
Ursell, T. S. , Klug, W. S. , and Phillips, R. , 2009, “ Morphology and Interaction Between Lipid Domains,” Proc. Natl. Acad. Sci., 106(32), pp. 13301–13306. [CrossRef]
Ulrich, A. S. , 2002, “ Biophysical Aspects of Using Liposomes as Delivery Vehicles,” Biosci. Rep., 22(2), pp. 129–150. [CrossRef] [PubMed]
Tokumasu, F. , Jin, A. J. , Feigenson, G. W. , and Dvorak, J. A. , 2003, “ Nanoscopic Lipid Domain Dynamics Revealed by Atomic Force Microscopy,” Biophys. J., 84(4), pp. 2609–2618. [CrossRef] [PubMed]
Lin, W.-C. , Blanchette, C. D. , Ratto, T. V. , and Longo, M. L. , 2006, “ Lipid Asymmetry in DLPC/DSPC-Supported Lipid Bilayers: A Combined AFM and Fluorescence Microscopy Study,” Biophys. J., 90(1), pp. 228–237. [CrossRef] [PubMed]
Bernchou, U. , Midtiby, H. , Ipsen, J. H. , and Simonsen, A. C. , 2011, “ Correlation Between the Ripple Phase and Stripe Domains in Membranes,” Biochim. Biophys. Acta, 1808(12), pp. 2849–2858. [CrossRef] [PubMed]
Loura, L. M. , Fedorov, A. , and Prieto, M. , 2000, “ Partition of Membrane Probes in a Gel/Fluid Two-Component Lipid System: A Fluorescence Resonance Energy Transfer Study,” Biochim. Biophys. Acta, 1467(1), pp. 101–112. [CrossRef] [PubMed]
Pryse, K. M. , Rong, X. , Whisler, J. A. , McConnaughey, W. B. , Jiang, Y.-F. , Melnykov, A. V. , Elson, E. L. , and Genin, G. M. ,. 2012, “ Confidence Intervals for Concentration and Brightness From Fluorescence Fluctuation Measurements,” Biophys. J., 103(5), pp. 898–906. [CrossRef] [PubMed]
Tsukruk, V. V. , and Singamaneni, S. , 2011, Scanning Probe Microscopy of Soft Matter, Wiley-VCH, Hoboken, NJ.
Gibbs, J. W. , 1961, The Scientific Papers, Vol. 1, Dover Publications, Mineola, NY.
Tsukruk, V. V. , and Singamaneni, S. , 2012, Scanning Probe Microscopy of Soft Matter: Fundamentals and Practices, Wiley, New York.
McConney, M. E. , Singamaneni, S. , and Tsukruk, V. V. , 2010, “ Probing Soft Matter With the Atomic Force Microscopies: Imaging and Force Spectroscopy,” Polym. Rev., 50(3), pp. 235–286. [CrossRef]
Singamaneni, S. , McConney, M. E. , and Tsukruk, V. V. , 2010, “ Swelling-Induced Folding in Confined Nanoscale Responsive Polymer Gels,” ACS Nano, 4(4), pp. 2327–2337. [CrossRef] [PubMed]
Tian, L. , Fei, M. , Kattumenu, R. , Abbas, A. , and Singamaneni, S. , 2012, “ Gold Nanorods as Nanotransducers to Monitor the Growth and Swelling of Ultrathin Polymer Films,” Nanotechnology, 23(25), p. 255502. [CrossRef] [PubMed]
Tian, L. , Chen, E. , Gandra, N. , Abbas, A. , and Singamaneni, S. , 2012, “ Gold Nanorods as Plasmonic Nanotransducers: Distance-Dependent Refractive Index Sensitivity,” Langmuir ACS J. Surf. Colloids, 28(50), pp. 17435–17442. [CrossRef]
Richter, R. P. , and Brisson, A. R. , 2005, “ Following the Formation of Supported Lipid Bilayers on Mica: A Study Combining AFM, QCM-D, and Ellipsometry,” Biophys. J., 88(5), pp. 3422–3433. [CrossRef] [PubMed]
Axelrod, D. , Koppel, D. E. , Schlessinger, J. , Elson, E. , and Webb, W. W. , 1976, “ Mobility Measurement by Analysis of Fluorescence Photobleaching Recovery Kinetics,” Biophys. J., 16(9), pp. 1055–1069. [CrossRef] [PubMed]
Leidy, C. , Kaasgaard, T. , Crowe, J. H. , Mouritsen, O. G. , and Jørgensen, K. , 2002, “ Ripples and the Formation of Anisotropic Lipid Domains: Imaging Two-Component Supported Double Bilayers by Atomic Force Microscopy,” Biophys. J., 83(5), pp. 2625–2633. [CrossRef] [PubMed]
Simonsen, A. C. , and Bagatolli, L. A. , 2004, “ Structure of Spin-Coated Lipid Films and Domain Formation in Supported Membranes Formed by Hydration,” Langmuir ACS J. Surf. Colloids, 20(22), pp. 9720–9728. [CrossRef]
Kollmitzer, B. , Heftberger, P. , Rappolt, M. , and Pabst, G. , 2013, Monolayer Spontaneous Curvature of Raft-Forming Membrane Lipids, Soft Matter, 9(45), pp. 10877–10884.
Melcher, J. , Carrasco, C. , Xu, X. , Carrascosa, J. L. , Gomez-Herrero, J. , Jose de Pablo, P. , and Raman, A. , 2009, “ Origins of Phase Contrast in the Atomic Force Microscope in Liquids,” Proc. Natl. Acad. Sci., 106(33), pp. 13655–13660. [CrossRef]


Grahic Jump Location
Fig. 1

Confocal images of a binary DLPC/DSPC GUV and ruptured GUV on a cover slip. GUVs were labeled with Bodipy-HPC (dark gray; green online) and DiI-C20 (light gray; red online). The pictured GUVs, made of 30% DSPC and 70% DLPC, show unusual phase behavior: although the GUVs have only two components, three phases are visible in this image, which is an apparent violation of the Gibbs phase rule.

Grahic Jump Location
Fig. 2

AFM measurements on ruptured GUVs: (a) fluorescence image of domains on which AFM scanning was performed. Images were taken using a standard fluorescence microscope instead of the confocal microscope used for Fig. 1, resulting in a lower resolution; (b) height profiles from the domains shown in panel A; (c) height profiles along the four lines shown in panel B. The origins of the curves (left) correspond to the numbered ends of lines shown in panel B. Cartoons of colored lipid molecules correspond to the hypothesized stacking of lipid layers (lightest gray (orange online): DLPC monolayer; second lightest gray (pink online): DLPC bilayer; second darkest gray (red online): DSPC bilayer, dark domain; darkest gray (blue online): DSPC bilayer, bright domain). (d) Magnified image from panel B, with contrast changed to better illustrate the topography. (e) Color rendering of the topography in panel D. Colors match those of panel C.

Grahic Jump Location
Fig. 3

Bright domains on DLPC/DSPC collapsed GUVs could be knocked off by an AFM tip. (a) Height images from a continuous scanning. The scale bar is for the first four images. For the last two images, showing the domain that appears on the left side in the first four images, the scale bar indicates 6 μm. Note that these figures have been modified using interpolation tools in the software package Gwyddion (Department of Nanometrology, Czech Metrology Institute); unmodified images can be found in Fig. S4 available in the Supplemental Materials on the ASME Digital Collection. (b) Fluorescence images (DiI-C20) of the domains before and after the AFM scanning. The scanning area is indicated by the black square, which is 15 μm by 15 μm.

Grahic Jump Location
Fig. 4

Height image of a dark domain on a ruptured DLPC/DSPC GUV membrane. The GUV was labeled only with Bodipy-HPC. The image shows that the second gel phase was not dependent upon DiI-C20.

Grahic Jump Location
Fig. 5

AFM of ruptured DLPC/DPPC GUVs: (a) fluorescence image, (b) AFM height image, (c) height profile along the red line in figure B, revealing that the DLPC/DPPC membrane shows no height difference between the dark gel domain and the bright gel domain surrounding it, and showed no evidence of the bright layer residing atop other membrane components



Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging and repositioning the boxes below.

Related Journal Articles
Related eBook Content
Topic Collections

Sorry! You do not have access to this content. For assistance or to subscribe, please contact us:

  • TELEPHONE: 1-800-843-2763 (Toll-free in the USA)
  • EMAIL: asmedigitalcollection@asme.org
Sign In