Understanding Chirality at Cell-Cell Junctions With Microscale Platforms

细胞-细胞连接的调节对于各种组织的生物学功能是必不可少的，如上皮 和内皮细胞。胚胎发育和血管生理学的最新证据表明，细胞-细胞 连接受细胞手性的调节，细胞手性是细胞的普遍但基本的属性。我们开创了 利用体外工程平台进行细胞手性研究。在这里，利用这些平台，我们将调查 与细胞-细胞连接处的手性有关的生物物理机制。虽然这一原则可以共享 在众多的细胞类型中，本研究将集中于内皮细胞和血管通透性的调节。 内皮细胞层是一个半透屏障，它严格控制蛋白质和细胞在体内的传递。 血流进入组织间隙，调节生物组织的局部环境 有机体。细胞主要通过调节细胞旁运输来实现这一重要功能，这是通过控制 细胞-细胞连接的开放和关闭。蛋白激酶C(PKC)的激活与 慢性疾病，如糖尿病和长期吸烟以及急性 败血症、急性肺损伤和病毒感染等疾病。恢复和维持血管完整性是 对身体功能和患者生存至关重要，尤其是对急性疾病。最近，我们展示了 PKC可以逆转细胞手性，从而介导内皮通透性。然而，人们对此知之甚少 PKC活化逆转内皮手性或细胞手性改变的分子机制 内皮通透性。在这个方案中，我们假设PKC通过降低水平来逆转细胞手性 肌动蛋白的交联性，细胞手性调节细胞间的连接(从而调节内皮的通透性) 通过肌动蛋白倾斜和VE-钙粘蛋白定位的生物力学机制。我们将追求以下三个目标：目标 1.确定导致多细胞手性的生物力学不对称的时间和位置 用牵引力显微镜(TFM)观察形态发生。结合2D微图案化细胞手性和TFM 对于胞体力，我们将非常详细地研究2D集体对称破缺，并询问 潜在的细胞生物力学机制。目的2.确定PKC的细胞骨架机制 诱导内皮细胞手性逆转。我们将确定福尔马林异构体和肌动蛋白交联物参与 这一过程以及PKC信号对其的调控。目的3.研究手性错配在手性失配中的作用 细胞间隙形成和内皮通透性。我们将量化肌动蛋白的结构和动力学 细胞间连接并研究细胞手性错配如何导致肌动蛋白重塑和 诱导细胞间隙的形成。 如果成功，我们将能够确定生物物理机制，允许潜在的发展 基于细胞手性的内皮功能障碍的新的、特定的治疗方法。使用从该数据库获得的数据 我们将寻求进一步的支持，并用动物模型检验我们的发现。

The regulation of cell-cell junctions is essential for the biological functions of various tissues such as epithelium and endothelium. Recent evidence in embryonic development and vascular physiology suggests that cell-cell junctions are regulated by cell chirality, a universal but fundamental property of the cell. We pioneer in research in cell chirality using engineered in vitro platforms. Here, using these platforms, we are to investigate the biophysical mechanism associated with chirality at cell-cell junctions. While the principle may be shared among many cell types, this study will focus on endothelial cells and the regulation of vascular permeability. The endothelial cell layer is a semi-permeable barrier that tightly controls the passage of proteins and cells in the bloodstream into the interstitial space and regulates the local environment of biological tissues in living organisms. Cells achieve this vital function primarily through mediating paracellular transport by controlling the opening and closure of cell-cell junctions. Protein Kinase C (PKC) activation has been associated with endothelial dysfunction in chronic conditions such as diabetes and long-term smoking as well as acute diseases such as sepsis, acute lung injury, and viral infection. Restoring and maintaining vascular integrity is critical for body function and patient survival, especially for acute diseases. Recently, we have demonstrated that PKC can reverse cell chirality, which mediates endothelial permeability. However, little is known about the molecular mechanism of how PKC activation reverses endothelial chirality or that of how cell chirality alters endothelial permeability. In this proposal, we hypothesize that PKC reverses cell chirality by reducing the level of actin crosslinking and that cell chirality regulates cell-cell junctions (and therefore endothelial permeability) biomechanically through actin tilting and VE-cadherin localization. We will pursue the following three aims: Aim 1. Identify the timing and location of biomechanical asymmetry responsible for multicellular chiral morphogenesis using traction force microscopy (TFM). Combing 2D micropatterning for cell chirality and TFM for cellular forces, we are to study in great detail of 2D collective symmetry breaking and to interrogate underlying cellular biomechanical mechanisms. Aim 2. Determine cytoskeletal mechanisms underlying PKC induced reversal of endothelial cell chirality. We will identify formin isoforms and actin crosslinkers involved in this process, and their regulation by PKC signaling. Aim 3. Investigate the role of chirality mismatch in the intercellular gap formation and endothelial permeability. We will quantify actin structure and dynamics during the intercellular junctions and examine how the mismatch of cell chirality can lead to actin remodeling and induce intercellular gap formation. If successful, we will be able to identify the biophysical mechanisms, allowing for the potential development of novel, specific therapies based on cell chirality for endothelial dysfunction. With data obtained from this proposal, we will seek further support and examine our findings with animal models.