Cytoskeletal Membrane Anchors: Key Switchboards for Cellular Communication, Mecha

描述（由申请人提供）：细胞与其周围环境的通信是正常和患病状态下许多生理功能的核心。它要求与细胞外配体的接触可以在细胞内被“感知”，相反，细胞外配体-受体相互作用的强度可以通过细胞内过程来调节。将细胞表面受体的胞质结构域连接到细胞骨架（“细胞骨架膜锚”）的衔接蛋白可能在这种双向通信中发挥关键的生物物理作用，即，不仅作为细胞信号的信使，而且作为介导和可能调节细胞对机械刺激的反应的结构元件。因此，本申请提出了细胞外配体-跨膜受体-细胞骨架锚-皮质肌动蛋白形式的串联连接代表在细胞通讯中充当关键生物物理开关板的功能单元的范例。通常，这种串联连接会受到物理应力，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，沿着它们的机械调节行为提供了机械基础。理解这些复杂的机制需要跨学科的纳米到微米尺度的方法。我们建议系统地剖析两种类型的串行跨膜连接的成分的个人贡献。目的1和2将建立涉及整联蛋白LFA-1和E-和N-钙粘蛋白的串联连接的结构行为。目的1将通过量化相应受体：配体键的形成和力依赖性失效的动力学来关注细胞外结合（使用尖端力探针仪器，即，我们的侧视AFM和光学镊子）。目的2将研究这些受体的细胞骨架锚的力学行为和分子决定因素。结合力探测和荧光功能成像，目标3将研究这种结构组织如何与细胞功能相关。例如，我们将探讨机械化学刺激激活白细胞的机制。这一策略将开辟新的途径，建立一个生物学上合理的和物理现实的理解这些显着的机械信号通路，从而也对新的自下而上的战略，诊断和治疗疾病。

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.