Signal Integration in Neutrophil Chemotaxis

描述(申请人提供)：定向细胞迁移是单细胞生物体狩猎和交配所必需的，使先天免疫细胞能够寻找和摧毁病原体，并且对多细胞生物体的形态形成至关重要。细胞迁移调控不当与动脉粥样硬化和心脏发育缺陷密切相关。虽然我们开始了解一些与细胞迁移有关的关键成分，但我们不知道这些成分如何共同作用来组织细胞的形状和运动。为了解决这个问题，我们分析了一个关键的肌动蛋白调控因子-SCAR/WAVE复合体的空间动力学，它是后生动物和植物形态发生所必需的。我们最近发现，SCAR/WAVE复合体的Hem-1成分定位于传播的波，这些波似乎组织了一种能动的免疫细胞--人类中性粒细胞的前沿。奇怪的是，肌动蛋白既是疤痕/波浪复合体的输出也是输入：该复合体刺激肌动蛋白组装，而且肌动蛋白聚合物也需要将该复合体从膜上移除。这些相互作用似乎产生了肌动蛋白成核的传播波，这些波体现了运动细胞中形态发生的许多特性，例如细胞绕过屏障的能力和前沿突起的复杂空间组织。我们的中心假设是，Hem-1波发生器和其他信号信号之间的相互作用在空间上组织了细胞迁移过程中的肌动蛋白聚合。在这个提案中，我们将剖析组成Hem-1波动力学的信号，并研究它们与细胞形态发生和定向运动的关系。具体来说，我们会： 量化外部梯度对Hem-1波动力学的影响。我们将定量分析趋化过程中的Hem-1波动力学，以检验该领域中两个相互竞争的假设--是新突起的产生还是现有突起中的选择负责定向迁移。 2.剖析RAC与Hem-1之间的相互作用。我们同时使用微图案化和小分子二聚体来控制RAC和Hem-1在活细胞中定位的空间和时间动态，以剖析这些信号如何相互作用。 3.阐明肌动蛋白细胞骨架在Hem-1波传播中的作用。我们将结合使用肌动蛋白干扰药物和肌动蛋白成核因子的定向错位来研究肌动蛋白聚合物如何与Hem-1波动力学相结合。 4.测试HEM-1在前后串扰中的作用。我们正在使用基于微流体的药物灌流和基于小分子的二聚化来空间操纵前面(RAC/Hem-1)和后面(Rho/肌球蛋白)组织中涉及的信号，以确定这些极性调节因子如何沟通。 公共卫生评论：肌动蛋白聚合调控失调和白细胞迁移是心脏病的致病因素。白细胞在动脉粥样硬化中起着核心作用，而我们研究的运动回路对血管生成和心血管发育是必不可少的。控制细胞迁移的能力将是对抗动脉粥样硬化和其他细胞导向机制中断时发生的病理过程的有价值的工具。

DESCRIPTION (provided by applicant): Directed cell migration is required for single-celled organisms to hunt and mate, enables innate immune cells to seek and destroy pathogens, and is essential for the morphogenesis of multicellular organisms. Misregulation of cell migration is intimately involved in atherosclerosis and defective cardiac development. Though we are beginning to understand some of the key components involved in cell migration, we do not understand how these components act together to organize cell shape and movement. To address this question, we have analyzed the spatial dynamics of a key actin regulator the Scar/WAVE complex, which is required for morphogenesis in both metazoans and plants. We have recently discovered that the Hem-1 component of the Scar/WAVE complex localizes to propagating waves that appear to organize the leading edge of a motile immune cell, the human neutrophil. Curiously, actin is both an output and input to the Scar/WAVE complex: the complex stimulates actin assembly, and actin polymer is also required to remove the complex from the membrane. These reciprocal interactions appear to generate propagated waves of actin nucleation that embody many of the properties of morphogenesis in motile cells such as the ability of cells to flow around barriers and the intricate spatial organization of protrusion at the leading edge. Our central hypothesis is that the interaction between the Hem-1 wave generator and other signaling cues spatially organizes actin polymerization during cell migration. In this proposal, we will dissect the signals that organize Hem-1 wave dynamics and study their relationship to cell morphogenesis and directed motility. Specifically, we will: Quantitate the effect of external gradients on Hem-1 wave dynamics. We will quantitatively analyze Hem-1 wave dynamics during chemotaxis to test two competing hypotheses in the field-- whether generation of new protrusions or selection among existing ones is responsible for directional migration. 2. Dissect the reciprocal interactions between Rac and Hem-1. We are using both micropatterning and small molecule dimerizers to control the spatial and temporal dynamics of Rac and Hem-1 localization in living cells to dissect how these signals interact with one another. 3. Elucidate the role of the actin cytoskeleton in Hem-1 wave propagation. We will use a combination of actin perturbing drugs and targeted mislocalization of actin nucleation factors to investigate how actin polymer interfaces with Hem-1 wave dynamics. 4. Test role of Hem-1 in front/back crosstalk. We are using microfluidics-based drug perfusion and small-molecule based dimerization to spatially manipulate the signals involved in front (Rac/Hem-1) and back (Rho/myosin) organization to determine how these regulators of polarity communicate. PUBLIC HEALTH REVELANCE: Misregulation of actin polymerization and leukocyte migration are causative factors in heart disease. Leukocytes play a central role in atherosclerosis, and the motility circuit that we study is essential for angiogenesis and cardiovascular development. The ability to control cell migration would be a valuable tool for combating atherosclerosis and other pathological processes that occur upon disruption of cellular guidance mechanisms.