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成分，如磷脂和胆固醇在胞外释放中的作用。1.磷脂跨双层不对称分布在胞外的作用为了破坏磷脂的跨双层不对称性，在肥大细胞或PC12细胞中表达了催化磷脂在两层膜之间双向移动的脂质扰乱酶。我们发现这些细胞的胞吐作用受到显著抑制。2.膜胆固醇在胞吐过程中的作用研究了甲基-β-环糊精(M-β-CD)去除质膜胆固醇对肥大细胞胞吐过程的影响。MβCD可抑制抗原诱导的胞吐作用。M-β-CD在佛波醇酯-α-…刺激下仍能抑制胞吐作用更多的钙离子载体。因此，M-β-CD影响了颗粒与质膜的膜融合。细胞内Ca~(2+)测定显示，M-β-CD抑制细胞外钙离子通过钙离子通道的内流，而不影响细胞外钙离子的内流。这些结果表明，胆固醇耗竭主要通过损害SOC和颗粒与质膜之间的胞吐膜融合来抑制颗粒脱颗粒。3.Raft蛋白Flotillin调节IgE受体介导的信号。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基因敲除的RBL-2H3细胞。在这些细胞中，我们观察到IgE-R的Ca^&lt；2+&Gt；酪氨酸磷酸化和胞吐作用显著降低。我们还发现，在脂筏中，Flotillin与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，Flotillin-1在IgE-R介导的激活过程中是一个必不可少的分子，调节Lyn的激酶活性。较少

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