Feedback and Crosstalk in Eukaryotic Chemotaxis

真核趋化中的反馈和串扰

• 批准号: 9767252
• 负责人: Takanari Inoue
• 金额: $ 32.61万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 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/9767252
• 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

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 发育和伤口愈合。它还有助于病理状况（例如 癌症转移和关节炎。当前建议的目的是揭示生化反应和 膜变形等物理现象在调节趋化性方面相互作用。 具体而言，我们将重点阐明膜变形蛋白超家族的作用， bin/Amphiphysin/rvs（bar），以趋化性的不同步骤。这些步骤包括细胞外的感觉 化学梯度，输入刺激的细胞扩增，细胞内信号事件的极化以及 细胞运动的致动。对于我们已经显示的三种条蛋白，通过增益参与细胞迁移 - 和功能丧失研究，我们将精确确定如何需要这些条蛋白 通过进行生化和细胞生物学测定以及计算建模来进行趋化性。 特别是，我们执行了三种Bar蛋白的功能丧失研究，以确定它们在 上述趋化性步骤之一 趋化和趋化因子分析（AIM 1）。然后，我们将专门揭示这些条蛋白在 核心两极分化程序之一，即众所周知的积极反馈回路 信号分子（AIM 2）。这将通过使用浅层和 陡峭的化学梯度以及我们开发的基于成像的测定法，以定量测量程度 反馈驱动。我们还研究了阳性中条诱导的膜变形的充分性 使用新建立的工具的反馈可以在几秒钟内变形膜内部的膜。共同 目标1和2将表征阳性期间生化和物理因素之间的串扰 驱动细胞极化的反馈过程。然后，我们将揭示棒蛋白如何介导合作 在分子水平上驱动阳性反馈回路（AIM 3）。基于以前的报告和我们自己的 最近的发现，我们假设信号分子（如PI3K）可以感知膜曲率，并且 因此，在质膜上的局部位点积聚，该质膜弯曲了Bar蛋白。测试 这个假设，我们将执行两个实验：体外脂质体结合测定和基于细胞的定位 测定。为了进一步阐明这种非直觉的合作过程，该过程得出的参数 从这些湿的实验中将集成到计算模型中。 结合起来，这里概述的工作代表了我们可以探索至关重要的强大手段，但通常 研究了，趋化性方面。更具体地说，它将揭示膜形成的核心作用 蛋白质在细胞极化过程中发挥作用，并为病理生理状况提供分子见解，其中 趋化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。