Feedback and Crosstalk in Eukaryotic Chemotaxis

真核趋化中的反馈和串扰

基本信息

批准号：
9923130
负责人：
Takanari Inoue
金额：
$ 6万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9923130
关键词：
1-Phosphatidylinositol 3-Kinase Address Arthritis Autophagocytosis Behavior Binding Biochemical Biochemical Reaction Biological Biological Assay Biological Process Cell division Cell membrane Cell physiology Cells Chemicals Chemotactic Factors Chemotaxis Complex Computational Technique Computer Simulation Cues Development Disease Disease Progression Embryonic Development Endocytosis Esthesia Event Feedback Fibroblasts Functional disorder Genetic Goals Image Immune response Impairment In Vitro Lipids Liposomes Malignant Neoplasms Measures Mediating Membrane Migration Assay Modeling Molecular Natural regeneration Neoplasm Metastasis Pathologic Pathway interactions Physics Physiological Play Process Proteins Public Health Reporting Role Signal Transduction Signaling Molecule Site Stimulus Surface Techniques Testing Tissues Work Wound Healing amphiphysin angiogenesis base cell motility density driving force experimental study extracellular fluorescence imaging insight interdisciplinary approach knock-down loss of function member migration molecular actuator neutrophil novel physical process physical property polarized cell programs recruit response spatiotemporal tool

项目摘要

Chemotaxis occurs during a number of key physiological events, including angiogenesis, embryonic development and wound healing. It also contributes to disease progression in pathological conditions such as cancer metastasis and arthritis. The goal of the current proposal is to reveal how biochemical reactions and physical phenomena such as membrane deformation interact with one another in regulating chemotaxis. Specifically, we will focus on elucidating the role of a superfamily of membrane deforming proteins, Bin/Amphiphysin/Rvs (BAR), in distinct steps of chemotaxis. These steps include sensation of an extracellular chemical gradient, cellular amplification of the input stimulus, polarization of intracellular signaling events, and actuation of cell motility. For three BAR proteins that we already shown are involved in cell migration via gain- and loss-of-function studies, we will precisely determine how each of these BAR proteins is required for chemotaxis by performing biochemical and cell biological assays along with computational modeling. In particular, we execute the loss-of-function studies of the three BAR proteins to determine their role in any one of the aforementioned steps of chemotaxis, with an emphasis on the polarization process by performing chemotaxis and chemokinesis assays (Aim 1). We will then reveal the role of these BAR proteins specifically in one of the core polarization programs, namely a positive feedback loop that is known to consist of several signaling molecules (Aim 2). This will be achieved by conducting chemotaxis assays using both shallow and steep chemical gradient, as well as an imaging-based assay we developed to quantitatively measure the extent of feedback actuation. We also investigate sufficiency of BAR-induced membrane deformation in the positive feedback using newly established tools that can deform membrane inside living cells within seconds. Collectively, Aims 1 and 2 will characterize the crosstalk between biochemical and physical factors during the positive feedback process that drives cell polarization. We will then reveal how BAR proteins mediate the cooperative actuation of the positive feedback loop at a molecular level (Aim 3). Based both on previous reports and our own recent findings, we hypothesize that signaling molecules such as PI3K can sense membrane curvature, and therefore accumulates at local sites on the plasma membrane which have been bent by BAR proteins. To test this hypothesis, we will perform two experiments: an in vitro liposome binding assay and a cell-based localization assay. To further elucidate this non-intuitive, cooperative process on a quantitative level, parameters derived from these wet experiments will be integrated into a computational model. Combined, the work outlined here represent powerful means by which we can explore crucial, but often understudied, aspects of chemotaxis. More specifically, it will reveal the central role that membrane-deforming proteins play during cell polarization, and offer molecular insights into pathophysiological conditions where dysfunction of chemotaxis plays a significant role in disease progression, such as cancer metastasis and arthritis.

趋化性发生在许多关键的生理事件中，包括血管生成、胚胎发育发育和伤口愈合。它还有助于病理状况下的疾病进展，例如癌症转移和关节炎。当前提案的目标是揭示生化反应和膜变形等物理现象在调节趋化性方面相互作用。具体来说，我们将重点阐明膜变形蛋白超家族的作用， Bin/Amphiphyrin/Rvs (BAR)，处于不同的趋化步骤。这些步骤包括细胞外感觉化学梯度、输入刺激的细胞放大、细胞内信号事件的极化，以及细胞运动的启动。对于我们已经证明的三种 BAR 蛋白通过增益参与细胞迁移和功能丧失研究，我们将精确确定这些 BAR 蛋白中的每一种是如何被需要的通过执行生化和细胞生物学测定以及计算建模来实现趋化性。特别是，我们对三种 BAR 蛋白进行了功能丧失研究，以确定它们在上述任何一个趋化步骤，重点是通过执行极化过程趋化性和趋化作用测定（目标 1）。然后我们将揭示这些 BAR 蛋白的具体作用核心极化计划之一，即正反馈循环，已知由几个组成信号分子（目标 2）。这将通过使用浅层和浅层进行趋化性测定来实现陡峭的化学梯度，以及我们开发的基于成像的测定法来定量测量程度反馈驱动。我们还研究了 BAR 引起的膜变形的充分性使用新建立的工具进行反馈，可以在几秒钟内使活细胞内的膜变形。总的来说，目标 1 和 2 将描述积极过程中生化因素和物理因素之间的串扰。驱动细胞极化的反馈过程。然后我们将揭示 BAR 蛋白如何介导合作在分子水平上激活正反馈回路（目标 3）。基于之前的报告和我们自己的报告根据最近的发现，我们假设 PI3K 等信号分子可以感知膜曲率，并且因此聚集在质膜上被 BAR 蛋白弯曲的局部位点。测试根据这个假设，我们将进行两个实验：体外脂质体结合测定和基于细胞的定位化验。为了进一步在定量水平上阐明这种非直观的合作过程，导出了参数这些湿实验的结果将被整合到一个计算模型中。结合起来，这里概述的工作代表了我们可以探索关键但通常是的强大手段趋化性方面尚未得到充分研究。更具体地说，它将揭示膜变形的核心作用蛋白质在细胞极化过程中发挥作用，并提供对病理生理条件的分子见解趋化性功能障碍在疾病进展中起着重要作用，例如癌症转移和关节炎。