Feedback and Crosstalk in Eukaryotic Chemotaxis

趋化作用发生在许多关键的生理过程中，包括血管生成、胚胎 发展和伤口愈合。在病理情况下，它也会导致疾病的进展，例如 癌症转移和关节炎。目前这项提议的目标是揭示生化反应和 膜变形等物理现象在调节趋化作用中相互作用。 具体地说，我们将重点阐明膜变形蛋白超家族的作用， Bin/两体蛋白/RVS(BAR)，在不同的趋化步骤中。这些步骤包括感觉到细胞外的 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 细胞运动的驱动。对于我们已经展示的三种通过Gain参与细胞迁移的bar蛋白- 和功能丧失研究，我们将准确地确定这些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分析以及计算机建模来实现趋化性。 特别是，我们对这三种bar蛋白进行了功能丧失研究，以确定它们在 上述趋化步骤中的任何一种，重点是通过执行 趋化和趋化试验(目标1)。然后我们将揭示这些bar蛋白在 核心极化方案之一，即已知由几个 信号分子(目标2)。这将通过进行趋化分析来实现，使用浅层和 陡峭的化学梯度，以及我们开发的基于成像的分析方法来定量测量程度 反馈驱动。我们还研究了杆诱导的膜变形的充分性。 使用新建立的工具进行反馈，这些工具可以在几秒钟内使活细胞内的细胞膜变形。总而言之， 目标1和目标2将描述在积极的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蛋白是如何调节合作的 在分子水平上启动正反馈回路(目标3)。基于之前的报告和我们自己的报告 最近的发现，我们假设像PI3K这样的信号分子可以感知细胞膜的曲率，并且 因此聚集在质膜上被bar蛋白弯曲的局部位置。为了测试 在这个假设下，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 化验。为了在定量水平上进一步阐明这一非直观的合作过程，推导出参数 这些湿实验将被整合到一个计算模型中。 总而言之，这里概述的工作代表着强大的手段，我们可以通过这些手段来探索至关重要的、但往往 对趋化性的研究还不够深入。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洞察 趋化功能障碍在癌症转移和关节炎等疾病进展中起着重要作用。

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.