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。在极化细胞迁移的情况下，磷脂酰肌醇（PtdIns）脂质由质膜上的激酶和磷酸酶产生，形成自组织的脂质梯度，独立于潜在的肌动蛋白细胞骨架。尽管已经确定了许多调节PtdIns脂质合成的蛋白质，但细胞膜内脂质梯度快速产生和繁殖的分子基础仍不清楚。利用双组分信号网络计算建模揭示的设计原则，我计划利用合成生物学方法在体外膜上重建极化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，它们的催化活性具有内在的正反馈。

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.