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CAS: 138026-69-4
MF: C46BN3O8F2P
MW: 802.2891
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Robert C. Dunn

University of Kansas
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Krzysztof Kuczera

University of Kansas
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Kei Murakoshi

Hokkaido University
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Single-molecule observations for determining the orientation and diffusivity of dye molecules in lipid bilayers
Co-reporter: Toshinori Motegi, Hideki Nabika and Kei Murakoshi  
pp: 12895-12902
Publication Date(Web):28 Jun 2013
DOI: 10.1039/C3CP51585K
The molecular orientation and diffusion of dye molecules in artificial lipid bilayers were observed using total internal reflection fluorescence microscopy. An artificial lipid bilayer composed of a ternary lipid mixture of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and cholesterol was used. The molecular orientation, which was obtained through defocused imaging, clarified the microscopic features, including cholesterol-induced changes in the local packing structure. Diffusion analysis gave insights into the macroscopic aspects of phase distribution in the heterogeneous bilayer system. Combining these two independent investigations, we revealed the effect of cholesterol addition on microscopic local packing and macroscopic phase structures. Our observations showed a transition from a DLPC-network-like structure to a DPPC-network-like structure upon the addition of cholesterol, which was not evident from previous domain shape observations. The present single-molecule observations yielded the actual phase structure that controls the motion of molecules in the membrane. The results imply that the orientation and diffusivity of molecules offer useful information regarding the phase distribution, which may be hindered by the apparent phase structure in a heterogeneous lipid bilayer that contains cholesterol.
Single-molecule observations for determining the orientation and diffusivity of dye molecules in lipid bilayers
Co-reporter: Toshinori Motegi, Hideki Nabika and Kei Murakoshi
pp: NaN12902-12902
Publication Date(Web):2013/06/28
DOI: 10.1039/C3CP51585K
The molecular orientation and diffusion of dye molecules in artificial lipid bilayers were observed using total internal reflection fluorescence microscopy. An artificial lipid bilayer composed of a ternary lipid mixture of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and cholesterol was used. The molecular orientation, which was obtained through defocused imaging, clarified the microscopic features, including cholesterol-induced changes in the local packing structure. Diffusion analysis gave insights into the macroscopic aspects of phase distribution in the heterogeneous bilayer system. Combining these two independent investigations, we revealed the effect of cholesterol addition on microscopic local packing and macroscopic phase structures. Our observations showed a transition from a DLPC-network-like structure to a DPPC-network-like structure upon the addition of cholesterol, which was not evident from previous domain shape observations. The present single-molecule observations yielded the actual phase structure that controls the motion of molecules in the membrane. The results imply that the orientation and diffusivity of molecules offer useful information regarding the phase distribution, which may be hindered by the apparent phase structure in a heterogeneous lipid bilayer that contains cholesterol.

Claudia Steinem

Institute of Organic and Biomolecular Chemistry
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Creating and Modulating Microdomains in Pore-Spanning Membranes
Co-reporter: Alexer Orth;Dr. Ludger Johannes; Dr. Winfried Römer; Dr. Claudia Steinem
pp: 108-114
Publication Date(Web):
DOI: 10.1002/cphc.201100644

Abstract

The architecture of the plasma membrane is not only determined by the lipid and protein composition, but is also influenced by its attachment to the underlying cytoskeleton. Herein, we show that microscopic phase separation of “raft-like” lipid mixtures in pore-spanning bilayers is strongly determined by the underlying highly ordered porous substrate. In detail, lipid membranes composed of DOPC/sphingomyelin/cholesterol/Gb3 were prepared on ordered pore arrays in silicon with pore diameters of 0.8, 1.2 and 2 μm, respectively, by spreading and fusion of giant unilamellar vesicles. The upper part of the silicon substrate was first coated with gold and then functionalized with a thiol-bearing cholesterol derivative rendering the surface hydrophobic, which is prerequisite for membrane formation. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liquid-ordered- (lo) and liquid-disordered (ld) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb3 (40:35:20:5) membranes. The size of the lo-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temperature, and the cholesterol content in the membrane, which was modulated by the addition of methyl-β-cyclodextrin. Binding of Shiga toxin B-pentamers to the Gb3-doped membranes increased the lo-phase considerably and even induced lo-phase domains in non-phase separated bilayers composed of DOPC/sphingomyelin/cholesterol/Gb3 (65:10:20:5).