"Mechanism of extracellular vesicle budding in C. elegans embryos".

描述(申请人提供)：细胞释放细胞外小泡(ECV)，可作为信号平台调节免疫反应的强度，影响发育过程中的分化，促进血小板凝集，或诱导肿瘤细胞转移。许多ECV是由质膜向外萌发形成的，但调节外部囊泡萌发的分子机制尚不清楚。已经观察到ECV膜双层的外叶含有通常被隔离到质膜内叶的脂类，这导致了一种假设，即脂类不对称性的破坏可能在ECV的形成中起作用。然而，尚未发现脂质不对称和ECV萌发之间的分子联系。在初步数据中，我们通过证明保守的脂质翻转酶TAT-5的缺失导致线虫胚胎中ECV的大规模萌发，确定了脂质不对称与ECV萌发之间的潜在分子联系。Flipase通过将特定的脂类从膜双层的外叶转移到内叶来维持脂质的不对称性。TAT-5的缺失导致细胞表面脂质磷脂酰乙醇胺(PE)的过量暴露，从而使PE不对称性的丧失与囊泡萌发的增加相关。我们还表明，已知的病毒出芽调节因子是由TAT-5缺失诱导的ECV出芽所必需的，这表明出芽是通过保守机制发生的。这项建议的目的是确定哪些因素与TAT-5相反地作用于诱导囊泡萌发，并检验PE外化导致ECV萌发的假说。在初步数据中，我们表明已知的调节病毒出芽的蛋白质(RAB-11和ESCRT复合体)也调节线虫胚胎中的ECV出芽。在目标1中，我们测试RAB-11和ESCRT复合体是如何调控ECV出芽的。这一目标将扩大我们对RAB-11和ESCRT复合体如何被招募到质膜形成和释放芽的理解。在目标2中，我们测试是否任何破坏脂质不对称的脂质扰乱也可以诱导ECV萌发。扰乱酶的作用与翻转酶相反，通过使双层之间的脂类定位随机化来破坏脂类的不对称性。调控ECV产生的扰乱酶的发现将进一步支持脂质不对称性的丧失导致ECV萌发的假说。最后，在目标3中，我们改变PE水平(通过破坏脂质生物合成途径)来验证PE调节ECV萌发的假设。总之，这项拟议的研究将测试TAT-5是否直接将PE的不对称性与质膜上囊泡的动态萌发联系起来。这项工作为未来的研究奠定了基础，以确定哺乳动物细胞是否使用相同的分子机制来调节血液凝固、免疫反应和转移过程中ECV的萌发。由于这些机制也可能被病毒从宿主质膜上萌发时所选择，我们的研究也可能揭示新的抗病毒靶点。

DESCRIPTION (provided by applicant): Cells release extracellular vesicles (ECVs) that can act as signaling platforms to modulate the strength of an immune response, influence differentiation during development, promote the coagulation of platelets, or induce the metastasis of tumor cells. Many ECVs form by budding outwards from the plasma membrane, but the molecular mechanisms that regulate external vesicle budding are unknown. It has been observed that the outer leaflet of the membrane bilayer of ECVs contains lipids that are normally sequestered to the inner leaflet of the plasma membrane, leading to the hypothesis that disruption of lipid asymmetry might have a role in ECV formation. However, a molecular link between lipid asymmetry and ECV budding has not been found. In preliminary data, we identify a potential molecular link between lipid asymmetry and ECV budding by showing that loss of the conserved lipid flippase TAT-5 causes the large-scale budding of ECVs in C. elegans embryos. Flippases maintain lipid asymmetry by transferring specific lipids from the outer leaflet of the membrane bilayer to the inner leaflet. Loss of TAT-5 results in excess exposure of the lipid phosphatidylethanolamine (PE) on cell surfaces, thereby correlating loss of PE asymmetry with an increase in vesicle budding. We also show that known regulators of viral budding are required for ECV budding induced by loss of TAT-5, suggesting that budding occurs via conserved mechanisms. The goals of this proposal are to determine what factors work in opposition to TAT-5 to induce vesicle budding, and to test the hypothesis that PE externalization causes ECV budding. In preliminary data, we show that proteins known to regulate viral budding (RAB-11 and the ESCRT complex) also regulate ECV budding in C. elegans embryos. In Aim 1, we test how RAB-11 and the ESCRT complex regulate ECV budding. This aim will expand our understanding of how RAB-11 and the ESCRT complex are recruited to the plasma membrane to form and release buds. In Aim 2, we test whether any lipid scramblases that disrupt lipid asymmetry also induce ECV budding. Scramblases act in opposition to flippases and disrupt lipid asymmetry by randomizing lipid localization between the bilayers. The identification of a scramblase that regulates ECV production would provide further support for the hypothesis that loss of lipid asymmetry induces ECV budding. Finally, in Aim 3, we alter PE levels (via disruption of lipid biosynthetic pathways) to test the hypothesis that PE regulates ECV budding. In summary, the proposed research will test whether TAT-5 directly links PE asymmetry to the dynamic budding of vesicles from the plasma membrane. This work provides the groundwork for future studies determining whether mammalian cells use the same molecular mechanisms to regulate ECV budding during blood clotting, the immune response, and metastasis. Because these mechanisms may also be co-opted by viruses as they bud from the host plasma membrane, our studies may also reveal new antiviral targets.