Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York.
Joint Institute for Biological Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee; Large Scale Structures Group, Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, Tennessee.
Biophys J. 2018 Apr 24;114(8):1921-1935. doi: 10.1016/j.bpj.2018.03.014.
Biomembranes with as few as three lipid components can form coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. In the coexistence region of Ld and Lo phases, the lipid mixtures 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/chol or brain sphingomyelin (bSM)/DOPC/chol form micron-scale domains that are easily visualized with light microscopy. Although large domains are not observed in the mixtures DSPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/chol and bSM/POPC/chol, lateral heterogeneity is nevertheless detected using techniques with nanometer-scale spatial resolution. We propose a simple and accessible method to measure domain sizes below optical resolution (∼200 nm). We measured nanodomain size for the latter two mixtures by combining experimental Förster resonance energy transfer data with a Monte-Carlo-based analysis. We found a domain radius of 7.5-10 nm for DSPC/POPC/chol, similar to values obtained previously by neutron scattering, and ∼5 nm for bSM/POPC/chol, slightly smaller than measurable by neutron scattering. These analyses also detect the domain-size transition that is observed by fluorescence microscopy in the four-component lipid mixture bSM/DOPC/POPC/chol. Accurate measurements of fluorescent-probe partition coefficients are especially important for the analysis; therefore, we exploit three different methods to measure the partition coefficient of fluorescent molecules between Ld and Lo phases.
生物膜只需三种脂质成分即可形成共存的无序液体（Ld）和有序液体（Lo）相。在 Ld 和 Lo 相共存区域，脂质混合物 1,2-二硬脂酰-sn-甘油-3-磷酸胆碱（DSPC）/1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC）/胆固醇或脑鞘磷脂（bSM）/DOPC/胆固醇形成可通过相差显微镜轻松观察到的微米级域。尽管在混合物 DSPC/1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱（POPC）/胆固醇和 bSM/POPC/胆固醇中未观察到大域，但使用具有纳米级空间分辨率的技术仍检测到侧向异质性。我们提出了一种简单易行的方法来测量低于光学分辨率（约 200nm）的域大小。我们通过将实验Förster 共振能量转移数据与基于蒙特卡罗的分析相结合，测量了后两种混合物的纳米域大小。我们发现 DSPC/POPC/胆固醇的域半径为 7.5-10nm，与先前通过中子散射获得的值相似，而 bSM/POPC/胆固醇的域半径约为 5nm，略小于中子散射可测值。这些分析还检测到在四组分脂质混合物 bSM/DOPC/POPC/胆固醇中通过荧光显微镜观察到的域尺寸转变。荧光探针分配系数的准确测量对于分析特别重要；因此，我们利用三种不同的方法来测量荧光分子在 Ld 和 Lo 相之间的分配系数。
