Department of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen 37077, Germany.
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, UK.
Nat Commun. 2014 Nov 20;5:5412. doi: 10.1038/ncomms6412.
The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.
脂质和蛋白质的相互作用在质膜生物活性中起着重要作用，并且可以从它们的扩散特性中学到很多东西。在这里，我们将超分辨率 STED 显微镜与扫描荧光相关光谱（扫描 STED-FCS，sSTED-FCS）相结合，以表征脂质相互作用的空间和时间异质性。sSTED-FCS 揭示了荧光神经鞘脂类似物的瞬时分子相互作用热点。相互作用位点的直径小于 80nm，并且脂质在这些区域中被暂时捕获数毫秒。相比之下，具有改进的相分离特性的新型荧光磷脂和胆固醇类似物显示出更均匀的扩散，而与对有序或无序膜环境的偏好无关。我们的结果不支持在我们培养的非刺激细胞的基底膜中存在基于脂质相分离的纳米域，并且表明替代相互作用负责我们的神经鞘脂类似物的强局部捕获。
