Sharma Pranav, Varma Rajat, Sarasij R C, Gousset Karine, Krishnamoorthy G, Rao Madan, Mayor Satyajit
National Centre for Biological Science (TIFR), UAS-GKVK Campus, GKVK PO, Bangalore 560 065, India.
Cell. 2004 Feb 20;116(4):577-89. doi: 10.1016/s0092-8674(04)00167-9.
Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosyl-phosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments, combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.
长期以来，富含胆固醇和鞘脂的“脂筏”被认为是用于分选特定膜成分（包括糖基磷脂酰肌醇锚定蛋白，GPI-APs）的平台，然而，它们的存在和物理性质一直存在争议。在这里，我们使用基于同源和异源荧光共振能量转移（FRET）的实验，并结合理论建模，研究活细胞中GPI-APs脂质依赖性组织的大小。这些研究揭示了一种意想不到的组织形式，其中细胞表面的GPI-APs以单体形式存在，较小一部分（20%-40%）以纳米级（<5 nm）胆固醇敏感簇的形式存在。这些簇最多由四个分子组成，容纳多种GPI-AP种类；交联GPI-APs将它们与预先存在的GPI-AP簇分离，并通过GPI-AP选择性胞饮途径阻止交联物种的内吞作用。结合对簇的统计分布分析，这些观察结果提示了一种GPI-APs功能脂质依赖性聚集的机制。
