Nanotopographic modulation of B cell signaling activation

B细胞介导的抗体反应是免疫的重要组成部分，也是疫苗的主要靶点 发展。B细胞受体(BCR)与抗原结合触发B细胞信号激活 显示在专业抗原提呈细胞的表面。尽管可溶性抗原能够激活 B细胞，最近的研究表明，表面锚定的抗原在触发 B细胞活化。因此，抗原呈递的物理性质和机械环境 B细胞可能对bcr的激活很重要。特别是，B细胞遇到APC上的抗原，它拥有 复杂的表面，具有复杂的地形、流体膜和可变形的细胞体。从历史上看， 平面底物已被用于研究免疫细胞信号和研究其背后的机制。 信号启动。先前的研究表明，贴壁细胞和干细胞的许多行为，如 形态、运动和分化都受表面地形的影响。这些研究表明 这些细胞能够对地形信号做出反应，这反过来又会影响细胞信号。然而， 免疫细胞中信号的地形调节问题以前还没有被研究过。我们的 初步研究表明，B细胞信号和肌动蛋白动力学都受到 抗原呈递表面的纳米拓扑术。这个项目的中心假设是底物 地形影响B信号和激活。此外，界面处的细胞骨架动力学 使细胞能够感知地形，以便产生适当的信号响应。我们将测试 这一假设是通过使用允许高分辨率荧光成像的新型纳米地形表面来实现的 研究B细胞中肌动蛋白的细胞骨架动力学、bcr信号和肌动蛋白调节因子。目标1将研究 底物纳米图形对B细胞形态、肌动蛋白重组和信号激活的影响。我们 将使用非线性光学光刻技术在材料上制造具有纳米图形图案的新型表面 这使得细胞-底物界面的高分辨率活细胞成像成为可能。使用这些底物，我们将 确定底物形态是B细胞信号和细胞骨架动力学的重要调节器。 AIM 2将定义肌动蛋白重塑及其上游调控因子，它们对地形感知和 BCR信令的控制。为此，我们将使用小分子抑制剂干扰肌动蛋白细胞骨架。 以及包括WASP和N-WASP在内的调控蛋白在调节地貌传感中的基因敲除 B细胞通过肌动蛋白重塑。我们将进一步测试曲率传感蛋白在地形传感中的作用 通过控制膜的曲率。这些研究将为肌动蛋白如何 调节通路介导了地形感知，为我们提供了另一种调节B细胞信号的方法 纳米拓扑学。更广泛地说，这项工作将阐明细胞感知环境的机制 通过表面受体，这对生物医学有相当大的意义。

B cell-mediated antibody responses are an essential component of immunity and the main target of vaccine development. B cell signaling activation is triggered by the binding of B cell receptors (BCR) with antigen displayed on the surface of professional antigen presenting cells. Although soluble antigen are able to activate B cells, recent studies have shown that surface anchored antigens are significantly more efficient in triggering B cell activation. Consequently, the physical nature of antigen presentation and the mechanical environment of B cells are likely important for BCR activation. In particular, B cells encounter antigen on APC, which possess complex surfaces with convoluted topographies, a fluid membrane and deformable cell bodies. Historically, planar substrates have been used to study immune cell signaling and investigate the mechanisms behind signal initiation. Previous work has shown that many behaviors of adherent and stem cells, such as morphology, movement and differentiation, are modulated by surface topography. These studies demonstrate that cells are able to respond to topographical cues, which in turn influences cell signaling. However, the question of topographical modulation of signaling in immune cells has not been previously studied. Our preliminary studies suggest that B cell signaling and actin dynamics are both influenced by the nanotopography of the antigen-presenting surface. The central hypothesis of this project is that substrate topography influences B signaling and activation. Furthermore, cytoskeletal dynamics at the interface enable the cell to sense topography in order to generate an appropriate signaling response. We will test this hypothesis by using novel nanotopographic surfaces that allow high resolution fluorescence imaging to examine actin cytoskeletal dynamics, BCR signaling and actin regulators in B cells. Aim 1 will examine the effects of substrate nanotopography on B-cell morphology, actin reorganization and signaling activation. We will use nonlinear optical lithography to fabricate novel surfaces with nanotopographic patterns on materials that permit high-resolution live cell imaging of the cell-substrate interface. Using these substrates, we will establish that substrate topography is an important modulator of B cell signaling and cytoskeletal dynamics. Aim 2 will define actin remodeling and their upstream regulators that are critical for topography sensing and the control of BCR signaling. To do so, we will perturb the actomyosin cytoskeleton using small-molecule inhibitors and gene knockout of regulatory proteins including WASP and N-WASP in modulating topographic sensing in B cells via actin remodeling. We will further test the role of curvature sensing proteins in topographic sensing by the control of membrane curvature. These studies will provide important new insights into how actin regulatory pathways mediate topographic sensing, giving us another means of tuning B cell signaling with nanotopography. More broadly, this work will shed light on mechanisms by which cells sense their environment by surface receptors, which has considerable implications for biomedicine.