Molecular Dissection of the Organization and the Dynamics of Intracellular Lipid Domains

细胞色素c是一种促凋亡因子，它优先与线粒体脂质心磷脂(CL)结合，而不是与心磷脂氢过氧化氢(CL-OOH)结合。我们利用线粒体磷脂过氧化氢谷胱甘肽过氧化物酶(PHGPx)过表达的研究表明，线粒体CL-OHO的产生可能是触发细胞色素c.II.晚期内体中溶酶双磷脂酸结构域的特征。晚期内体在细胞器内腔内积累内膜。这些内膜富含特殊的脂质--异二磷脂酸(LBPA)。利用LBPA特异性的单抗和荧光相关光谱，我们已经确定LBPA结构域仅定位于晚期内体的管腔。III.小窝和糖基磷脂酰肌醇结构域。大多数哺乳动物细胞的膜上至少有两种类型的脂质m…更多的微区、非内陷的脂筏和小窝。糖基磷脂酰肌醇(GPI)锚定的蛋白质组成了一类蛋白质，在木筏中富含，但在稳定状态下不存在小窝。通过使用GPI缺陷细胞和高表达小窝蛋白-1的细胞，我们发现GPI锚定蛋白的表达与小窝蛋白-1呈负相关。IV.鞘磷脂特异性蛋白Lysenin是从赤子爱胜子体腔液中获得的一种新的4 kDa的鞘磷脂结合蛋白。我们研制了重组赖氨酸，并在电子显微镜下观察了神经鞘蛋白的分布。神经鞘磷脂在血管内皮细胞的小窝中积聚。鞘磷脂也分布在内体和跨高尔基体网络中，但不包括高尔基体。我们还检测了Niemann-Pick A型(NPA)成纤维细胞中的鞘磷脂，神经鞘磷脂在NPA的晚期内体溶酶体中积累。有趣的是，其他脂筏成分，如神经节苷脂GM1和胆固醇也积累在相同的细胞器中。我们的结果提示NPA中发生了脂筏的重新分布。较少

I. Involvement of mitochondrial cardiolipin domains in apoptosis.Cytochrome c is a proapoptotic factor that binds preferentially to cardiolipin (CL), a mitochondrial lipid, but not to cardiolipin hydroperoxide (Cl-OOH). Our studies using overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx) suggests that the generation of CL-OOH in mitochondria might be a primary event that triggers the release of cytochrome c.II. Characterization of lysobisphosphatidic acid domain in late endosomes.Late endosomes accumulates internal membranes within the lumen of the organelle. These intemal membranes are enriched in the specific lipid, Iysobisphosphatidic acid (LBPA). Using LBPA-specific monoclonal antibody and fluorescence correlation spectroscopy, we have determined that LBPA domain is exclusively localized in the lumen of late endosomes.III. Caveolae and glycosylphosphatidylinositol domain.Most mammalian cells have in their membrane at least two types of lipid m … More icrodomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. By using GPI-deficient cells and the cells overexpressing caveolin-1, we showed there is an inverse correlation between the expression of GPI-anchored proteins and caveolin-1.IV. Sphingomyelin-specific proteinLysenin is a novel 4lkDa sphingomyelin-binding protein obtained from coelomic fluid of the earthworrn Eisenia foetida. We have developed recombinant lysenin and examined the distribution of sphingomyelin under electron microscope. Sphingomyelin was accumulated in caveolae in endothelial cells. Sphingomyelin was also distributed in endosomes and the trans-Golgi network but excluded from the Golgi cistemae. We also examined sphingomyelin in Niemann-Pick type A (NPA) fibroblpsts, which accumulate sphingomyelin intracellularly Sphingomyelin is accumuiated in late endosomes lysosomes in NPA. Interestingly, other lipid raft components such as ganglioside GM1, and cholesterol were aiso accumulated in the same organelle. Our results suggest that re-distribution of lipid raft occurs in NPA. Less