Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California 94720, USA.
J Am Chem Soc. 2012 Jul 4;134(26):10833-42. doi: 10.1021/ja300374c. Epub 2012 Jun 14.
This study examines the dynamic co-localization of lipid-anchored fluorescent proteins in living cells using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and fluorescence lifetime analysis. Specifically, we look at the pairwise co-localization of anchors from lymphocyte cell kinase (LCK: myristoyl, palmitoyl, palmitoyl), RhoA (geranylgeranyl), and K-Ras (farnesyl) proteins in different cell types. In Jurkat cells, a density-dependent increase in cross-correlation among RhoA anchors is observed, while LCK anchors exhibit a more moderate increase and broader distribution. No correlation was detected among K-Ras anchors or between any of the different anchor types studied. Fluorescence lifetime data reveal no significant Förster resonance energy transfer in any of the data. In COS 7 cells, minimal correlation was detected among LCK or RhoA anchors. Taken together, these observations suggest that some lipid anchors take part in anchor-specific co-clustering with other existing clusters of native proteins and lipids in the membrane. Importantly, these observations do not support a simple interpretation of lipid anchor-mediated organization driven by partitioning based on binary lipid phase separation.
本研究使用脉冲交错激发荧光相关光谱（PIE-FCCS）和荧光寿命分析，研究了活细胞中脂锚定荧光蛋白的动态共定位。具体来说，我们观察了淋巴细胞激酶（LCK：豆蔻酰、棕榈酰、棕榈酰）、RhoA（香叶基）和 K-Ras（法呢基）蛋白的锚定物在不同细胞类型中的成对共定位。在 Jurkat 细胞中，观察到 RhoA 锚的交叉相关密度依赖性增加，而 LCK 锚则呈现出更适度的增加和更广泛的分布。在 K-Ras 锚或研究的任何不同锚定类型之间均未检测到相关性。荧光寿命数据显示，在任何数据中均未检测到明显的Förster 共振能量转移。在 COS 7 细胞中，仅检测到 LCK 或 RhoA 锚的最小相关性。总之，这些观察结果表明，一些脂锚定物与膜中存在的其他天然蛋白质和脂质簇的特定共聚类有关。重要的是，这些观察结果不支持基于二元脂质相分离的基于分区的简单脂质锚介导组织的解释。
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