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

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。在细胞极化迁移的情况下，由质膜上的激酶和磷酸酶产生的磷脂酰肌醇(PtdIns)脂类形成不依赖于潜在肌动蛋白细胞骨架的自组织脂质梯度。虽然已经鉴定了许多调节体内PtdIns脂质合成的蛋白质，但快速产生和传播跨细胞内膜的脂质梯度的分子基础仍然不清楚。利用双组分信号网络计算模型揭示的设计原理，我计划利用合成生物学方法在体外重建膜上极化的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的脂蛋白激酶和磷酸酶，它们的催化活性具有内在的正反馈。 将这些酶活性结合在一起会导致两种酶之间的相互交叉负抑制，并被假设支持体外膜上的自发极化。利用这种重组，我将确定哪些生化特征调节极化的时间演变和脂类结构域大小的微调。在建立了一个表征脂质双分子层上脂质激酶和磷酸酶的定位和活性的平台后，我将测试极化的PI(4)P和PI(4，5)P2类脂结构域是否可以作为空间线索来控制调控肌动蛋白在质膜上成核的蛋白质的招募和激活。综上所述，这些实验应该揭示细胞极化的关键原理，并提供一类新的生化重组，描述在细胞内膜上起作用的信号分子的时空组织。

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.