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

项目总结/摘要 在急性炎症期间，我们的免疫细胞会协调一种复杂但协调的缓解反应。 免疫细胞特别擅长通过动态的免疫系统来导航复杂的细胞外环境。 调节其肌动球蛋白细胞骨架，使整个身体的快速和有效的反应。的 细胞感知各种化学和物理线索的能力，指导他们的迁移路径是至关重要的， 这一行动。响应于生物电流的迁移早已被证明，导致临床上的 通过外源施加的电势在伤口愈合中的应用。同时也牵涉到我们的 对感染的反应和某些癌症的转移扩散，我们对这种方向性线索的理解， 被称为趋电性或趋电性，仍然是有限的。本申请中提出的实验将 开发技术来进行大规模的趋电性测定，并使全面的基因组- 广泛的战略，以确定基因和细胞机制参与人类中性粒细胞趋电性。 在目标1中，我将制造一种设备，使电场定向分离所需的数百万个细胞， 进行基因组规模的扰动分析。在与托马斯丹尼尔博士的合作下，我将优化设备 和测定条件以开发用于研究趋电性的稳健方案。我将在这里接受训练， 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 敲除趋电性筛选，提供了第一个全面的策略，以确定涉及的关键基因 在这种迁移模式下。由于这种测定的技术挑战，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 实验方法，以获得新的见解细胞机制的趋电性的基础上的假设 从屏幕工作中产生。在这项工作的过程中，我将与实验学家肖恩柯林斯博士合作 他是基于受体的信号传导和信号转导方面的专家。他将提供宝贵的指导， 这些核心成分是大多数定向细胞迁移模式所共有的。在目标2和3中，我还将 通过以下专业知识加强我在分子生物学和生物化学技术方面的实验培训： Theriot实验室 重要的是，沿着这些研究机会，发展奖将为我提供额外的 职业培训，我目前需要启动和管理一个实验室。它还将提供关键的职业培训， 实验室领导，教学，资助写作和科学交流。我的导师朱莉·瑟里奥博士 提供指导，使我能够成功地过渡到独立。该奖项将 提供关键的培训，使我能够全面了解中性粒细胞的长期目标 运动性和下游效应器功能。

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.