The role of transbilayer asymmetry of phospholipids in exocytosis

在这项研究中，我们研究了膜蛋白以外的膜组分如磷脂和胆固醇在胞外释放中的作用。为了破坏磷脂的跨层不对称分布，肥大细胞或PC12细胞中表达了催化磷脂在两层膜间高定向运动的脂质超燃酶（脂质超燃酶）。我们发现这些细胞的胞吐明显受到抑制。这表明磷脂的跨双分子层不对称在胞外释放中起重要作用。以肥大细胞为研究对象，研究了甲基β-环糊精（m -β- cd）从质膜中去除胆固醇对肥大细胞胞外过程的影响。m - β cd预处理可抑制抗原诱导的胞吐。m - β cd对胞外分泌的抑制作用在磷酯和钙离子载体的刺激下也能观察到。因此，MβCD影响颗粒与质膜之间的膜融合。细胞内Ca^<2+>测量显示，MβCD抑制Ca^<2+>从细胞外介质通过存储操作的钙通道（SOC）内流，而不影响Ca^<2+>从Ca^<2+>存储的Ca^<2+>。这些结果表明，胆固醇消耗主要通过损害颗粒与质膜之间的SOC和胞外膜融合来抑制脱颗粒。筏蛋白flotillin调控IgE受体介导的信号传导我们研究了定位于脂筏中的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显著降低。我们还发现漂浮素与脂筏中的Lyn有关。抗原刺激导致flotillin与Lyn结合增强，导致Lyn活性增加。这些结果表明，flotilin -1是IgE-R介导的活化的重要分子，调节Lyn的激酶活性。少

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