Role of phospholipids in membrane traffic at the synapse

磷脂在突触膜交通中的作用

• 批准号: 7460777
• 负责人: Gilbert Di Paolo
• 金额: $ 41.29万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7460777
• 关键词: Ablation; Address; Affect; Animal Model; Biochemical; Calcium; Catalysis; Cell membrane; Chromosome Pairing; Clathrin; DNA; Data; Defect; Endocytosis; Ensure; Enzymes; Genetic; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Hydrolysis; Life; Link; Lipids; Mediating; Membrane; Membrane Protein Traffic; Metabolism; Molecular; Monomeric GTP-Binding Proteins; Mus; Nerve; Neurons; Pathway interactions; Phosphatidic Acid; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase D; Phospholipids; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiology; Process; Property; Protein Dephosphorylation; Proteins; RNA Interference; Recycling; Regulation; Research; Retrieval; Role; Series; Signal Transduction; Site; Source; Staging; Subfamily lentivirinae; Synapses; Synaptic Vesicles; Testing; Transport Vesicles; Vesicle; base; cofactor; fluorescence imaging; interest; neurotransmission; research study; sensor; small hairpin RNA; synaptojanin; trafficking

DESCRIPTION (provided by applicant): Neurotransmission relies on the proper trafficking of synaptic vesicles in nerve terminals. Recently, a series of genetic studies have unmasked a fundamental role for phosphoinositides (i.e. phosphorylated derivatives of phosphatidylinositol, Ptdlns) and other lipids in this process. In particular, ablation of the two main enzymes regulating the levels of PIP2 at the synapse, PIPK1 gamma and the phosphoinositides phosphatase synaptojanin 1, was shown to produce defects at multiple stages in the synaptic vesicle cycle, thereby reflecting the multifaceted role of PIP2 in signaling at the synapse. Although a role for PIP2 in membrane traffic is now well established, several fundamental questions have emerged concerning how distinct pools of this lipid may exert specialized functions in such compartments as the plasma membrane. First, what are the molecular mechanisms ensuring the local regulation of PIP2 synthesis? Based on growing evidence showing that the small monomeric GTPase Arf6 is a major regulator of PIPK1 gamma and vesicular transport to and from the plasma membrane, we will explore its potential role in synaptic vesicle trafficking. Second, what are the mechanisms controlling the spatial restriction of PIP2 dephosphorylation at the membrane? An interesting hypothesis is that the PIP2 hydrolysis machinery may utilize membrane curvature sensors, such as BAR proteins, to eliminate this lipid preferentially from curved (i.e. in the endocytic bud), rather than flat, membranes. This hypothesis is strongly supported by studies showing the tight partnership between the membrane curvature sensor, endophilin, and synaptojanin 1, as well as by our preliminary biochemical data. Third, what are the main effectors for PIP2 at the synapse? In addition to components of the exo-endocytic machinery, major phospholipid-metabolizing enzymes, such as phospholipase D (PLD), have a strong requirement for PIP2 as a cofactor for their function. Since PLD enzymes are primary sources for the signaling lipid, phosphatidic acid, the metabolism of PIP2 is tightly linked to that of this phospholipid. Therefore, we will investigate the significance of this crosstalk as well as the role of PLD-derived phosphatidic acid for membrane traffic at the synapse. To test our hypotheses, we will utilize mouse genetics as well as lentivirus-mediated delivery of short hairpin RNAs and dominant interfering DNA constructs into cultured neurons. Effects of these manipulations will be analyzed using biochemical, ultrastructural and live fluorescence imaging approaches. Altogether, we anticipate that our research will significantly advance our understanding of how phospholipids regulate membrane traffic at the synapse.

描述（由申请人提供）：神经传递依赖于神经末梢突触小泡的正确运输。最近，一系列遗传学研究揭示了磷酸肌醇（即磷脂酰肌醇的磷酸化衍生物，Ptdlns）和其他脂质在此过程中的基本作用。特别是，调节突触 PIP2 水平的两种主要酶 PIPK1 gamma 和磷酸肌醇磷酸酶 synaptojanin 1 的消融被证明会在突触小泡周期的多个阶段产生缺陷，从而反映了 PIP2 在突触信号传导中的多方面作用。尽管 PIP2 在膜运输中的作用现已得到充分证实，但一些基本问题已经出现，涉及这种脂质的不同池如何在质膜等区室中发挥专门的功能。首先，确保 PIP2 合成局部调控的分子机制是什么？越来越多的证据表明，小单体 GTP 酶 Arf6 是 PIPK1 γ 和囊泡往返质膜运输的主要调节因子，我们将探讨其在突触囊泡运输中的潜在作用。其次，控制 PIP2 去磷酸化在膜上的空间限制的机制是什么？一个有趣的假设是，PIP2 水解机制可能利用膜曲率传感器（例如 BAR 蛋白）优先从弯曲膜（即内吞芽中）而非平坦膜中消除这种脂质。这一假设得到了显示膜曲率传感器、endophilin 和 synaptojanin 1 之间紧密合作关系的研究以及我们的初步生化数据的有力支持。第三，PIP2在突触处的主要效应器是什么？除了外吞机制的组成部分之外，主要的磷脂代谢酶，例如磷脂酶 D (PLD)，也强烈需要 PIP2 作为其功能的辅助因子。由于 PLD 酶是信号脂质磷脂酸的主要来源，因此 PIP2 的代谢与这种磷脂的代谢紧密相关。因此，我们将研究这种串扰的重要性以及 PLD 衍生的磷脂酸对突触膜交通的作用。为了检验我们的假设，我们将利用小鼠遗传学以及慢病毒介导的短发夹 RNA 和显性干扰 DNA 构建体递送到培养的神经元中。将使用生化、超微结构和实时荧光成像方法来分析这些操作的效果。总而言之，我们预计我们的研究将显着增进我们对磷脂如何调节突触膜交通的理解。