Optogenetic stimulation of TMEM16F to control phospholipid flip-flop

TMEM16F 的光遗传学刺激控制磷脂触发器

基本信息

批准号：
10601109
负责人：
Huanghe Yang
金额：
$ 23.38万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-05 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10601109
关键词：
Anemia Animal Model Apoptosis Biological Assay Biological Process Biology Biomedical Engineering Biophysics Blood coagulation Bypass C-terminal COVID-19 Calcium Caspase Cell fusion Cell membrane Cells Cellular biology Chemicals Coupled Coupling Detection Disease Engineering Environment Fertilization G-Protein-Coupled Receptors Genetic Goals HIV Health Hematology Immunology In Vitro Ion Channel Ions Light Lipids Location Malignant Neoplasms Medicine Membrane Membrane Biology Modernization Molecular Molecular Conformation Myocardial Infarction Names Oxygen Pathologic Processes Permeability Phagocytosis Phospholipids Physiologic calcification Physiological Physiological Processes Physiology Pregnancy Complications Process Property Protein Engineering Proteins Protocols documentation Research Resolution SARS-CoV-2 infection Signal Transduction Specificity Stroke Structure Time Translating Vesicle Virus Diseases cell motility cell type experience feasibility testing fluorescence imaging genetic manipulation high reward high risk in vivo Model novel optical imaging optogenetics passive transport pharmacologic phospholipid scramblase prototype repaired response spatiotemporal tool vesicular release virology voltage

Anemia Animal Model Apoptosis Biological Assay Biological Process Biology Biomedical Engineering Biophysics Blood coagulation Bypass C-terminal COVID-19 Calcium Caspase Cell fusion Cell membrane Cells Cellular biology Chemicals Coupled Coupling Detection Disease Engineering Environment Fertilization G-Protein-Coupled Receptors Genetic Goals HIV Health Hematology Immunology In Vitro Ion Channel Ions Light Lipids Location Malignant Neoplasms Medicine Membrane Membrane Biology Modernization Molecular Molecular Conformation Myocardial Infarction Names Oxygen Pathologic Processes Permeability Phagocytosis Phospholipids Physiologic calcification Physiological Physiological Processes Physiology Pregnancy Complications Process Property Protein Engineering Proteins Protocols documentation Research Resolution SARS-CoV-2 infection Signal Transduction Specificity Stroke Structure Time Translating Vesicle Virus Diseases cell motility cell type experience feasibility testing fluorescence imaging genetic manipulation high reward high risk in vivo Model novel optical imaging optogenetics passive transport pharmacologic phospholipid scramblase prototype repaired response spatiotemporal tool vesicular release virology voltage

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项目摘要

SUMMARY Phospholipid flip-flop on cell membranes can exert profound impacts on cellular signaling and functions, including apoptosis, phagocytosis, blood coagulation, membrane vesicle shedding, bone mineralization, cell-cell fusion, fertilization, viral infection including HIV and SARS-CoV2 infections. Nevertheless, how phospholipid flip-flop leads to the observed cellular responses is largely elusive. The recent identifications of phospholipid scramblases and flippases have enabled genetic manipulations of these critical phospholipid transporters, which greatly advanced our understanding on the biology of phospholipid flip-flop. However, phospholipid flip-flop is a dynamic process and the genetic manipulations only allow us to observe the end results, which hinders gaining mechanistic understanding phospholipid flip-flop in various physiological process in real time. In this application, we aim to test the feasibility of developing a genetically encoded, optogenetic toolbox to precisely control phospholipid flip-flop with light at high temporal and spatial resolution and in real time. Our proposal is based on our extensive experience on the recently discovered calcium-activated phospholipid scramblase (CaPLSase) TMEM16F at molecular and cellular levels. In response to intracellular calcium increase, TMEM16F can rapidly catalyze phospholipid flip-flop, efficiently disrupt membrane environment and trigger wide spectrum of cellular changes. Here, we will use two complementary but independent approaches to develop the optogenetic tools to control phospholipid flip-flop. First, we will coexpress various calcium- mobilizing optogenetic tools to indirectly activate TMEM16F utilizing its calcium sensing property. Second, we will engineer light sensing motifs into TMEM16F to enable direct light control of its activities. If successful, the optogenetic toolbox developed in this high-risk, high-reward application will have profound impacts and broad applications in membrane biology, cell biology, physiology, hematology, immunology, virology and medicine.

概括细胞膜上的磷脂触发器可以对细胞信号传导产生深远的影响功能，包括凋亡，吞噬作用，血液凝结，膜囊泡脱落，骨矿化，细胞融合，受精，病毒感染，包括HIV和SARS-COV2 感染。然而，磷脂触发器如何导致观察到的细胞反应是在很大程度上难以捉摸。最近对磷脂串联酶和flippases的鉴定具有启用了这些关键磷脂转运蛋白的遗传操纵我们对磷脂触发器的生物学的理解。但是，磷脂触发器是一个动态过程和遗传操作仅使我们能够观察最终结果，在各种生理过程中，阻碍理解磷脂触发器的机械理解实时。在此应用中，我们旨在测试开发遗传编码的可行性光遗传学工具箱，以精确控制磷脂触发器，并在高时空处光解决方案和实时。我们的建议基于我们在最近的丰富经验在分子和细胞水平。响应细胞内钙的增加，TMEM16F可以迅速催化磷脂触发器，有效破坏膜环境，并触发广泛的光谱细胞变化。在这里，我们将使用两种互补但独立的方法来发展控制磷脂触发器的光遗传学工具。首先，我们将共同表达各种钙动员光遗传学工具间接地激活TMEM16F，利用其钙传感特性。其次，我们将在TMEM16F中设计光图案，以使其直接控制其活动。如果成功，则在这种高风险，高级奖励中开发了光遗传学工具箱应用将对膜生物学，细胞生物学产生深远的影响和广泛的应用，生理学，血液学，免疫学，病毒学和医学。