A Functional Genomics Approach to Uncover the Mechanisms of Neutrophil Galvanotaxis.

项目摘要/摘要 在急性炎症期间，我们的免疫细胞协调复杂但协调的缓解反应。 免疫细胞尤其擅长在复杂的细胞外环境中通过动态的 他们的肌动球蛋白细胞骨架的调节，使快速和有效的反应遍及全身。这个 细胞感知指示其迁移路径的各种化学和物理线索的能力至关重要 这一行动。对生物电流的反应迁移早已被证明，导致临床 外加电势在伤口愈合中的应用。同时也牵涉到我们的 在对感染和某些癌症转移扩散的反应中，我们对这个定向信号的理解， 所谓的趋电性或趋电性，仍然有限。本申请中提出的实验将 开发进行大规模趋化电流检测的技术，并实现全面的基因组- 确定涉及人类中性粒细胞趋化的基因和细胞机制的广泛策略。 在目标1中，我将制造一种设备，能够实现电场定向分离所需的数百万个电池 进行基因组规模的扰动分析。与托马斯·丹尼尔博士合作，我将优化设备 以及测试条件，以制定研究趋化电流的可靠方案。在这里，我将接受培训 用于分析开发的计算和工程工具。在目标2中，我将应用全基因组CRISPRi 电趋化的击倒筛选，提供了第一个全面的策略来识别涉及的关键基因 在这种迁移模式下。由于这种化验的技术挑战，将进行几轮实验 以增加我们对已确定的候选基因的信心。在目标3中，我将使用计算和 基于假说对趋化电流的细胞机制获得新见解的实验方法 从屏幕作品中产生。在这项工作中，我将与实验家肖恩·柯林斯博士合作 世卫组织是基于受体的信号和信号转导方面的专家。他将提供宝贵的指导，在 这些核心组件是大多数定向细胞迁移模式所共有的。在目标2和目标3中，我还将 加强我在分子生物学和生化技术方面的实验培训 塞里奥特实验室。 重要的是，除了这些研究机会，发展奖将为我提供更多 我目前需要的职业培训，才能开始和管理一个实验室。它还将在以下领域提供关键的职业培训 实验室领导、教学、资助撰写和科学交流。我的导师朱莉·塞里奥特博士将 提供指导，使我能够成功地过渡到独立。因此，这一奖项将 提供关键的培训，使我能够实现全面了解中性粒细胞的长期目标 运动和下游效应器功能。

Project Summary/Abstract During acute inflammation our immune cells orchestrate a complex, but coordinated mitigation response. Immune cells are especially good at navigating the complex extracellular environment through dynamic modulation of their actomyosin cytoskeletons, enabling a rapid and effective response throughout the body. The ability of cells to sense a variety of chemical and physical cues that direct their migratory paths is paramount to this action. Migration in response to bioelectric currents has long been demonstrated, leading to clinical applications in wound healing through exogenously applied electric potentials. While also implicated in our response to infections and in the metastatic spread of some cancers, our understanding of this directional cue, referred to as galvanotaxis or electrotaxis, remains limited. The experiments proposed in this application will develop the technology to perform large-scale assays of galvanotaxis and enable a comprehensive genome- wide strategy to identify the genes and cellular mechanisms involved in human neutrophil galvanotaxis. In Aim 1, I will fabricate a device that enables electric field-directed separation of the millions of cells required to perform genome-scale perturbation assays. In collaboration with Dr. Thomas Daniel, I will optimize the device and assay conditions to develop a robust protocol for studying galvanotaxis. Here I will gain training in computational and engineering tools for assay development. In Aim 2, I will apply a genome-wide CRISPRi knockdown screen of galvanotaxis, providing the first comprehensive strategy to identify the key genes involved in this mode of migration. Due to the technical challenges of such assays, several rounds of experiments will be performed to increase our confidence in identified gene candidates. In Aim 3, I will use computational and experimental approaches to gain new insights into the cellular mechanisms of galvanotaxis based on hypotheses generated from the screen work. In the course of this work, I will collaborate with experimentalist Dr. Sean Collins who is an expert in receptor-based signaling and signal transduction. He will provide invaluable guidance in these core components common to most modes of directed cell migration. Throughout Aim 2 and 3, I will also strengthen my experimental training in molecular biology and biochemical techniques through the expertise of the Theriot lab. Importantly, along with these research opportunities, the development award will provide me with additional career training that I currently need to start and manage a lab. It will also provide critical career training in laboratory leadership, teaching, grant writing and scientific communication. My mentor, Dr. Julie Theriot, will provide mentoring that will enable me to successfully transition to independence. This award will therefore provide the crucial training that will enable my longer-term goals of comprehensively understanding neutrophil motility and downstream effector functions.