Cytoskeletal Membrane Anchors: Key Switchboards for Cellular Communication, Mecha

描述(申请人提供)：在正常和疾病状态下，细胞与其周围环境的通讯是许多生理功能的中心。它要求与细胞外配体的接触可以在细胞内“感觉”到，反之，细胞外配体-受体相互作用的强度可以由细胞内过程调节。连接细胞表面受体胞质结构域和细胞骨架的适配蛋白(“细胞骨架膜锚”)可能在这种双向通信中发挥关键的生物物理作用，即不仅作为细胞信号信使，而且作为结构元件介导并可能调节细胞对机械刺激的反应。因此，这一应用提出了一种范式，即细胞外配体-跨膜受体-细胞骨架锚-皮质肌动蛋白的串联连接代表着在细胞通信中充当关键生物物理开关的功能单元。通常，这样的串联连接暴露在物理压力下，在这种情况下，单个分子相互作用的强度等级，以及质膜和肌动蛋白皮质的物理性质，为它们的机械调节行为提供了机械基础。要理解这些复杂的机制，需要一种跨学科、从纳米到微米的方法。我们建议系统地剖析两种类型的串联跨膜连接的组成部分的个体贡献。目标1和2将建立涉及整合素LFA-1和E-和N-钙粘附素的串联连接的结构行为。目标1将通过量化各自受体：配体键的形成动力学和力相关的失效来关注细胞外结合(使用尖端的力探针工具，即我们的侧视原子力显微镜和光镊子)。目的2将研究这些受体的细胞骨架锚定的机械行为和分子决定因素。结合力量探测和荧光功能成像，Aim 3将研究这种结构组织如何与细胞功能相关。例如，我们将探索机械力-化学刺激激活白细胞的机制。这一策略将为建立对这些非凡的机械信号通路的生物学上可信和物理上现实的理解开辟新的途径，从而也朝着疾病诊断和治疗的新的自下而上的策略迈进。

DESCRIPTION (provided by applicant): A cell's communication with its surroundings is central to numerous physiological functions in both normal and diseased states. It requires that encounters with extracellular ligands can be "sensed" inside the cell, and conversely, that the strength of extracellular ligand-receptor interactions can be modulated by intracellular processes. Adapter proteins that link the cytoplasmic domains of cell-surface receptors to the cytoskeleton ("cytoskeletal membrane anchors") are likely to play a crucial biophysical role in this bidirectional communication, i.e., not only as cell-signaling messengers, but also as structural elements that mediate, and possibly regulate, a cell's response to mechanical stimuli. Accordingly, this application puts forward the paradigm that serial linkages of the form extracellular ligand - transmembrane receptor - cytoskeletal anchor - cortical actin represent functional units that act as key biophysical switchboards in cellular communication. Often such serial linkages are exposed to physical stress, in which case the strength hierarchy of individual molecular interactions, along with the physical properties of the plasma membrane and the actin cortex, provide the mechanistic foundation of their mechanoregulatory behavior. Understanding these complex mechanisms requires an interdisciplinary, nano-to-microscale approach. We propose to systematically dissect the individual contributions of the constituents of two types of serial transmembrane linkages. Aims 1 and 2 will establish the structural behavior of serial linkages involving the integrin LFA-1 and E- and N-cadherins. Aim 1 will focus on extracellular binding by quantifying the dynamics of formation and force-dependent failure of the respective receptor:ligand bonds (using cutting-edge force- probe instruments, i.e., our side-view AFM and optical tweezers). Aim 2 will examine the mechanical behavior and the molecular determinants of the cytoskeletal anchors of these receptors. Combining force probing and fluorescent functional imaging, Aim 3 will investigate how this structural organization correlates with cellula function. For example, we will explore the mechanisms of leukocyte activation by mechano-chemical stimuli. This strategy will open new avenues toward establishing a biologically plausible and physically realistic understanding of these remarkable mechanosignaling paths, and thus also toward novel bottom-up strategies for diagnosis and treatment of diseases.