Self-organized membrane polarity of a phosphatidylinositol-based signaling system

基于磷脂酰肌醇的信号系统的自组织膜极性

• 批准号: 8718516
• 负责人: Scott David Hansen
• 金额: $ 5.15万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718516
• 关键词: Actins; Affinity; Algorithms; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Assay; Cell membrane; Cell physiology; Cells; Computer Simulation; Cues; Cytoskeleton; Detection; Disease; Elements; Engineering; Enzymes; Evolution; Feedback; Fluorescence; Fluorescence Spectroscopy; Future; Generations; Genetic; Goals; In Vitro; Individual; Intracellular Membranes; Knowledge; Lipid Bilayers; Lipid Binding; Lipid Synthesis Pathway; Lipids; Liquid substance; Membrane; Methods; Molecular; Monomeric GTP-Binding Proteins; Nature; Pathology; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Phosphotransferases; Protein Kinase; Proteins; Regulatory Element; Research; Series; Signal Transduction; Signaling Molecule; Solutions; Spectrum Analysis; Study models; Substrate Specificity; System; Techniques; Technology; Testing; Time; Vesicle; base; biological research; biophysical properties; cell motility; design; in vivo; membrane polarity; network architecture; particle; polarized cell; protein activation; public health relevance; reconstitution; research study; self organization; single molecule; spatiotemporal; synthetic biology; theories

DESCRIPTION (provided by applicant): The ability of cells to regulate the localization of molecules in both time and space is a hallmark of cellular organization and signal transduction. In the case of polarized cell migration, phosphatidylinositol (PtdIns) lipids generated by kinases and phosphatases at the plasma membrane form self-organized gradients of lipids independent of the underlying actin cytoskeleton. Although many of the proteins that regulate PtdIns lipid synthesis in vivo have been identified, the molecular basis for rapid generation and propagation of lipid gradients across intracellular membranes remains unclear. Using the design principles revealed from computational modeling of two-component signaling networks, I plan to reconstitute polarized PI(4)P and PI(4,5)P2 lipid synthesis on membranes in vitro using a synthetic biology approach. To achieve this goal, I have designed a series of chimeric lipid kinases and phosphatases that have intrinsic positive feedback built into their catalytic activity. Combining these enzymatic activities results in mutual cross-negative inhibition between both enzymes and is hypothesized to support spontaneous polarization on membranes in vitro. Using this reconstitution I will then determine which biochemical features that regulate the time evolution of polarization and fine-tuning of lipid domain size. After establishing a platform to characterize the localization and activity of lipid kinases and phosphatases on fluid lipid bilayer, I will test whether polarized PI(4)P and PI(4,5)P2 lipid domains can function as spatial cues to control the recruitment and activation of proteins that regulate actin nucleation at the plasma membrane. Taken together, these experiments should reveal key principles of cell polarization and provide a new class of biochemical reconstitutions that describe the spatiotemporal organization of signaling molecules that function on intracellular membranes.

描述（由申请人提供）：细胞在时间和空间上调节分子定位的能力是细胞组织和信号转导的标志。在极化细胞迁移的情况下，磷脂酰肌醇（PtdIns）脂质产生的激酶和磷酸酶在质膜形成自组织梯度的脂质独立的底层肌动蛋白细胞骨架。虽然许多调节PtdIns脂质合成的蛋白质已被鉴定，但跨细胞内膜的脂质梯度的快速产生和传播的分子基础仍不清楚。使用从双组分信号网络的计算建模中揭示的设计原理，我计划使用合成生物学方法在体外重构膜上的极化PI（4）P和PI（4，5）P2脂质合成。为了实现这一目标，我设计了一系列嵌合脂质激酶和磷酸酶，它们的催化活性中含有内在的正反馈。 结合这些酶活性导致两种酶之间的相互交叉负抑制，并假设支持体外膜上的自发极化。使用这种重建，我将确定哪些生化功能，调节极化的时间演变和微调的脂质结构域的大小。在建立了一个平台来表征液体脂质双层上的脂质激酶和磷酸酶的定位和活性后，我将测试极化的PI（4）P和PI（4，5）P2脂质结构域是否可以作为空间线索来控制调节质膜上肌动蛋白成核的蛋白质的募集和激活。总之，这些实验应该揭示细胞极化的关键原则，并提供一类新的生化重组，描述了细胞内膜上的信号分子的时空组织。