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: 10704752
• 负责人: Nathan M Belliveau
• 金额: $ 12.5万
• 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/10704752
• 关键词: Actins; Actomyosin; Acute; Award; Behavior; Biochemical; Biological Assay; Biological Process; Biology; CRISPR interference; CRISPR/Cas technology; Candidate Disease Gene; Categories; 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; Postdoctoral Fellow; Process; Proliferating; Property; Proteins; Protocols documentation; Reporter; Research; Role; Shapes; Signal Transduction; Speed; Techniques; Technology; Testing; Training; Work; Writing; assay development; 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.

项目概要/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的机制来应对复杂的细胞外环境。 调节其肌动球蛋白细胞骨架，从而实现全身快速有效的反应。这 细胞感知引导其迁移路径的各种化学和物理线索的能力对于 这个动作。响应生物电流的迁移早已被证明，导致临床 通过外源施加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性扩散，我们对这种方向线索的理解， 称为趋电性或趋电性，仍然受到限制。本申请中提出的实验将 开发进行大规模趋电流分析的技术，并实现全面的基因组分析 识别参与人类中性粒细胞趋电性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细胞。 进行基因组规模的扰动测定。我将与 Thomas Daniel 博士合作优化该设备 和测定条件，以开发用于研究趋电性的可靠方案。在这里我将接受以下方面的培训 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 趋电性的敲低筛选，提供了第一个全面的策略来识别所涉及的关键基因 在这种迁移模式下。由于此类测定的技术挑战，将进行多轮实验 这样做是为了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 基于假设的实验方法，以获得对趋电细胞机制的新见解 从屏幕工作生成。在这项工作的过程中，我将与实验家肖恩·柯林斯博士合作 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。在整个目标 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化技术方面的实验训练 Theriot 实验室。 重要的是，除了这些研究机会外，发展奖还将为我提供额外的机会 我目前需要启动和管理实验室的职业培训。它还将提供重要的职业培训 实验室领导、教学、资助写作和科学交流。我的导师 Julie Theriot 博士将 提供指导，使我能够成功过渡到独立。因此，该奖项将 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 运动性和下游效应器功能。

项目成果

Whole-genome screens reveal regulators of differentiation state and context-dependent migration in human neutrophils.

全基因组筛选揭示了人类中性粒细胞分化状态和环境依赖性迁移的调节因子。

• 发表时间: 2023-09-18
• 影响因子: 16.6
• 作者: Belliveau, Nathan M;Footer, Matthew J;Akdoǧan, Emel;van Loon, Aaron P;Collins, Sean R;Theriot, Julie A
• 通讯作者: Theriot, Julie A