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

描述（由申请人提供）：细胞释放细胞外囊泡（ECV），其可作为信号平台调节免疫应答的强度，影响发育期间的分化，促进血小板凝固或诱导肿瘤细胞转移。许多ECV通过从质膜向外出芽而形成，但调节外部囊泡出芽的分子机制尚不清楚。已经观察到，ECV的膜双层的外小叶含有通常被隔离到质膜的内小叶的脂质，导致脂质不对称性的破坏可能在ECV形成中起作用的假设。然而，脂质不对称性和ECV出芽之间的分子联系还没有被发现。在初步的数据中，我们通过显示保守的脂质翻转达特-5的缺失导致C.线虫胚胎翻转酶通过将特定脂质从膜双层的外小叶转移到内小叶来维持脂质不对称性。达特-5的缺失导致脂质磷脂酰乙醇胺（PE）在细胞表面上的过度暴露，从而将PE不对称性的缺失与囊泡出芽的增加相关联。我们还表明，已知的病毒出芽的监管机构所需的损失达特-5诱导的ECV出芽，这表明出芽发生通过保守的机制。这项提议的目的是确定哪些因素与达特-5相反，以诱导囊泡出芽，并测试PE外化导致ECV出芽的假设。 在初步数据中，我们发现已知调节病毒出芽的蛋白质（RAB-11和ESCRT复合物）也调节C.线虫胚胎在目标1中，我们测试RAB-11和ESCRT复合物如何调节ECV出芽。这一目标将扩大我们对RAB-11和ESCRT复合物如何被招募到质膜以形成和释放芽的理解。在目标2中，我们测试了是否任何破坏脂质不对称性的脂质乱序酶也诱导ECV出芽。乱序酶与翻转酶作用相反，通过使双层之间的脂质定位随机化来破坏脂质不对称性。鉴定调控ECV产生的乱序酶将进一步支持脂质不对称性的丧失诱导ECV出芽的假说。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）来测试PE调节ECV出芽的假设。 总之，拟议的研究将测试达特-5是否直接将PE不对称性与质膜上囊泡的动态出芽联系起来。这项工作为未来的研究提供了基础，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的ECV出芽。由于这些机制也可能被病毒从宿主质膜出芽时所吸收，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.