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The role of transbilayer asymmetry of phospholipids in exocytosis

磷脂跨双层不对称性在胞吐作用中的作用

基本信息

批准号：
14572037
负责人：
HIRASHIMA Naohide
金额：
$ 1.86万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572037/
关键词：
exocytosis lipid scramblase transbilayer asymmetry of phospholipids flip-flop mast cell PC12 cholesterol flotillin PC12

项目摘要

In this study, we investigated the role of membrane components other than membrane protein such as phospholipids and cholesterol in exocytotic release.1.The role of transbilayer asymmetric distribution of phospholipids in exocytosisIn order to disrupt the transbilayer asymmetry of phospholipids, lipid scramblase, which catalyze hi-directional movement of phospholipids between two membrane layers, was expressed in mast cells or PC12 cells. We found that exocytosis was significantly inhibited in these cells. This result suggests that transbilayer asymmetry of phospholipids plays an important role in exocytotic release.2.The role of cholesterol in the plasma membrane in exocytotic processThe effects of cholesterol depletion from the plasma membrane with methyl-β-cyclodextrin (MβCD) on exocytotic processes were investigated in mast cells. Pretreatment with MβCD inhibited antigen-evoked exocytosis. The inhibition of exocytosis by MβCD was observed even under stimulation with phorbol ester a … More nd calcium ionophore. Therefore, MβCD affected membrane fusion between granule and the plasma membrane. Intracellular Ca^<2+> measurements revealed that MβCD inhibited Ca^<2+> influx from the extracellular medium through the store-operated calcium channel (SOC) without affecting Ca^<2+> from Ca^<2+> store. These results suggest that cholesterol depletion inhibits degranulation mainly by impairing SOC and exocytotic membrane fusion between granules and the plasma membrane.3.Raft protein flotillin regulates IgE receptor mediated signalingWe investigated the role of flotillin, which is localized in lipid rafts, in the early exocytotic process. We obtained flotillin-1 knockdown RBL-2H3 cells. In these cells, we observed a significant decrease in Ca^<2+> tyrosine phosphorylation of IgE-R, and exocytosis. We also found that flotillin is associated with Lyn in lipid rafts. Antigen stimulation causes augmentation of flotillin binding to Lyn, resulting in increased activity of Lyn. These results suggest that flotillin-1 is an essential molecule in IgE-R mediated activation, regulating the kinase activity of Lyn. Less

本研究探讨了膜蛋白以外的磷脂、胆固醇等膜成分在胞吐释放中的作用。1.磷脂跨双层不对称分布在胞吐作用中的作用为了破坏磷脂的跨双层不对称性，脂质扰乱酶催化磷脂的双向运动。两膜层之间的磷脂在肥大细胞或PC12细胞中表达。我们发现这些细胞的胞吐作用被显着抑制。这一结果表明磷脂的跨双层不对称性在胞吐释放中起着重要作用。2.质膜中胆固醇在胞吐过程中的作用在肥大细胞中研究了甲基-β-环糊精(MβCD)从质膜上去除胆固醇对胞吐过程的影响。 MβCD 预处理可抑制抗原诱发的胞吐作用。即使在佛波酯和钙离子载体的刺激下，也观察到 MβCD 对胞吐作用的抑制。因此，MβCD影响颗粒与质膜之间的膜融合。细胞内Ca ^ 2+ 测量表明，MβCD抑制Ca ^ 2+ 从细胞外介质通过钙库操纵的钙通道(SOC)流入，而不影响来自Ca ^ 2+ 库的Ca ^ 2+ 。这些结果表明，胆固醇消耗主要通过损害SOC和颗粒与质膜之间的胞吐膜融合来抑制脱颗粒。3.筏蛋白flotillin调节IgE受体介导的信号传导我们研究了位于脂筏中的flotillin在早期胞吐过程中的作用。我们获得了flotillin-1敲低的RBL-2H3细胞。在这些细胞中，我们观察到IgE-R的Ca 2+ 酪氨酸磷酸化和胞吐作用显着减少。我们还发现 flotillin 与脂筏中的 Lyn 相关。抗原刺激导致 flotillin 与 Lyn 的结合增强，从而导致 Lyn 活性增加。这些结果表明，flotillin-1 是 IgE-R 介导的激活中的重要分子，调节 Lyn 的激酶活性。较少的