GUV preparation and imaging: Minimizing artifacts |
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Authors: | Nelson F. Morales-Penningston Elaine R. Farkas Tatyana M. Konyakhina Watt W. Webb |
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Affiliation: | a Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA b Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA c School of Applied and Engineering Physics, Cornell University, Ithaca, NY, 14853, USA |
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Abstract: | The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases. Any such phase separation would result in preferential partition of some proteins and lipids between the coexisting phases, and thus would influence which proteins could be in contact, and whether a protein could find its target. Phase separation in a plasma membrane would also influence the binding of molecules from outside the cell to the membrane, including recognition proteins on viruses, bacteria, and other cells. The concept of these and other events associated with membrane phase separation are sometimes grouped together as the “raft model” of biological membranes. Several types of experiments are aimed at detecting and characterizing membrane phase separation. Visualizing phase separation has special value, both because the immiscibility is so decisively determined, and also because the type of phase can often be identified. The fluorescence microscope has proven uniquely useful for yielding images of separated phases, both in certain cell preparations, and especially in models of cell membranes. Here we discuss ways to prepare useful model membranes for image studies, and how to avoid some of the artifacts that can plague these studies. |
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Keywords: | GUV, giant unilamellar vesicle PG, phosphatidylglycerol PS, phosphatidylserine PC, phosphatidylcholine DSPC, 1,2-Distearoyl-sn-Glycero-3-Phosphocholine SM, sphingomyelin POPC, 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine EDTA, ethylenediaminetetraacetic acid ITO, indium tin oxide DOPC, 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine Ld, liquid-disordered Lo, liquid-ordered SOPC, 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine chol, cholesterol TLC, Thin layer chromatography C12:0-DiI, 1,1'-didodecanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate C18:0-DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate C20:0-DiI, 1,1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (16:0,Bodipy-PC), 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl -sn-glycero-3-phosphocholine TR-DHPE, Texas Red 1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine PAH, polycyclic aromatic hydrocarbons LR-DPPE, Lissamine Rhodamine 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine NPG, n-propyl gallate b-SM, brain sphingomyelin |
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