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

揭示中性粒细胞趋电机制的功能基因组学方法。

• 批准号: 10505961
• 负责人: Nathan M Belliveau
• 金额: $ 10万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-16 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10505961
• 关键词: Actins; Actomyosin; Acute; Award; Behavior; Biochemical; Biological Assay; Biological Process; Biology; CRISPR interference; CRISPR/Cas technology; Candidate Disease Gene; Cell Death; Cells; Chemicals; Chemotaxis; Clinic; Collaborations; Collection; Communication; Complex; Computer Models; Computer Simulation; Cues; Cytoplasmic Granules; Cytoskeleton; Data; Development; Device Designs; Devices; Educational process of instructing; Engineering; Environment; Exhibits; Extracellular Matrix; Extracellular Space; Fluorescence Microscopy; Gene Targeting; Genes; Genomic DNA; Genomic approach; Goals; Grant; HL-60 Cells; HL60; Human; Human Cell Line; Immune; Infection; Inflammation; Injury; Innate Immune System; Laboratories; Leadership; Malignant Neoplasms; Mechanics; Membrane; Mentors; Molecular; Molecular Biology; Pattern; Phagocytosis; Phase; Phenotype; Process; Property; Proteins; Protocols documentation; Reporter; Research; Role; Shapes; Signal Transduction; Speed; Techniques; Technology; Testing; Training; Work; Writing; assay development; base; bioelectricity; cancer cell; career; cell motility; clinical application; cytotoxic; electric field; electrical potential; experimental study; extracellular; first responder; functional genomics; genome wide screen; genome-wide; improved; insight; knock-down; migration; neutrophil; pathogen; prevent; programs; receptor; receptor binding; response; success; suicidal; tool; voltage; wound healing

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.

项目摘要/摘要 在急性炎症期间，我们的免疫细胞会协调复杂但协调的缓解反应。 免疫细胞特别擅长通过动态导航复杂细胞外环境 调节其肌动蛋白细胞骨架，从而在整个体内产生快速有效的反应。这 细胞感知指导其迁移路径的各种化学和物理线索的能力至关重要 这个动作。长期以来一直证明了对生物电流的迁移，导致临床 通过外源施加的电势在伤口愈合中的应用。虽然也与我们有关 对感染和某些癌症的转移传播的反应，我们对这种定向提示的理解， 称为galvanotaxis或静电，仍然有限。本应用程序中提出的实验将 开发技术以进行大规模测定法，并启用全面的基因组 - 广泛的策略来识别与人类嗜中性粒细胞促成的基因和细胞机制。 在AIM 1中，我将制造一种设备，该设备能够以电场为导向的数百万个单元的分离 执行基因组尺度扰动测定。与托马斯·丹尼尔（Thomas Daniel）博士合作，我将优化设备 和测定条件以开发用于研究galvanotaxis的强大方案。在这里，我将接受培训 用于测定开发的计算和工程工具。在AIM 2中，我将应用全基因组CRISPRI Galvanotaxis的敲低屏幕，提供了第一个识别涉及关键基因的综合策略 在这种迁移方式中。由于此类测定的技术挑战，将进行几轮实验 表现为提高我们对确定的基因候选者的信心。在AIM 3中，我将使用计算和 基于假设 由屏幕工作生成。在这项工作的过程中，我将与实验家肖恩·柯林斯博士合作 谁是基于受体的信号传导和信号转导专家。他将提供宝贵的指导 这些核心成分与大多数定向细胞迁移模式共有。在整个目标2和3中，我也会 通过通过专业知识来加强我在分子生物学和生化技术方面的实验培训 Theriot实验室。 重要的是，与这些研究机会一起，开发奖将为我提供更多 我目前需要开始和管理实验室的职业培训。它还将提供关键的职业培训 实验室领导，教学，赠款写作和科学交流。我的导师朱莉·塞里奥特（Julie Theriot） 提供指导，使我能够成功过渡到独立性。因此，该奖项将 提供关键的培训，这将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。