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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.

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Figures

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

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