Feedback and Crosstalk in Eukaryotic Chemotaxis

趋化性发生在许多关键的生理事件中，包括血管生成、胚胎发育和细胞增殖。 发育和伤口愈合。它还有助于病理条件下的疾病进展， 癌症转移和关节炎。目前的建议的目标是揭示如何生化反应和 物理现象如膜变形在调节趋化性中相互作用。 具体来说，我们将重点阐明膜变形蛋白超家族的作用， Bin/Amphiphysin/Rvs（BAR），在不同的趋化步骤中。这些步骤包括感觉细胞外 化学梯度、输入刺激的细胞放大、细胞内信号传导事件的极化，以及 细胞运动的启动。对于我们已经证明的三种BAR蛋白，它们通过增益参与细胞迁移- 和功能丧失的研究，我们将精确地确定这些BAR蛋白中的每一种是如何被需要的， 通过进行生物化学和细胞生物学测定沿着计算建模来研究趋化性。 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 任何一个上述步骤的趋化性，重点是极化过程，通过执行 趋化性和趋化作用测定（Aim 1）。然后，我们将揭示这些BAR蛋白的作用，特别是在 核心极化程序之一，即已知由几个 信号分子（Aim 2）。这将通过使用浅的和浅的两种方法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，以定量测量 反馈驱动。我们还研究了在正的条件下BAR引起的膜变形的充分性。 反馈使用新建立的工具，可以在几秒钟内变形活细胞内的膜。总的来说， 目的1和2将表征在正性免疫反应期间生物化学和物理因素之间的串扰。 反馈过程驱动细胞极化。然后，我们将揭示BAR蛋白如何介导合作 在分子水平上启动正反馈回路（目标3）。根据之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， 因此在质膜上被BAR蛋白质弯曲的局部位点上积累。测试 基于这一假设，我们将进行两个实验：体外脂质体结合试验和基于细胞的定位 比色法为了在定量水平上进一步阐明这种非直观的合作过程， 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经常 未被充分研究的趋化性更具体地说，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，并提供了对病理生理条件的分子见解， 趋化性功能障碍在疾病进展如癌症转移和关节炎中起重要作用。

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.