Understanding Chirality at Cell-Cell Junctions With Microscale Platforms

细胞-细胞连接的调节对于各种组织如上皮的生物学功能是必不可少的 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 连接由细胞手性调节，细胞手性是细胞的普遍但基本的性质。我们是先锋， 使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-细胞连接处的手性相关的生物物理机制。虽然原则可以共享， 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。 内皮细胞层是一种半渗透性屏障，其严格控制蛋白质和细胞在血管中的通过。 血液流入组织间隙，调节生物组织的局部环境 有机体细胞实现这一重要功能，主要是通过调节细胞旁转运， 细胞-细胞连接的打开和关闭。蛋白激酶C（PKC）激活与 在慢性疾病如糖尿病和长期吸烟以及急性 疾病如败血症、急性肺损伤和病毒感染。恢复和维持血管完整性是 对于身体功能和患者生存至关重要，尤其是对于急性疾病。最近，我们证明了 PKC可以逆转细胞手性，介导内皮通透性。然而，人们对此知之甚少。 PKC激活如何逆转内皮手性或细胞手性如何改变的分子机制 内皮通透性在这个建议中，我们假设PKC通过降低细胞内的蛋白水平来逆转细胞的手性。 细胞手性调节细胞-细胞连接（从而调节内皮渗透性） 生物力学通过肌动蛋白倾斜和VE-钙粘蛋白定位。我们将努力实现以下三个目标： 1.确定生物力学不对称的时间和位置，负责多细胞手性 使用牵引力显微镜（TFM）观察形态发生。细胞手性和TFM的2D微图案化组合 对于细胞力，我们将非常详细地研究二维集体对称性破缺，并询问 潜在的细胞生物力学机制。目标2.确定PKC的细胞骨架机制 诱导内皮细胞手性逆转。我们将鉴定出参与免疫反应的肌动蛋白亚型和肌动蛋白交联剂。 这一过程及其通过PKC信号传导的调节。目标3.研究手性错配在 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，并研究细胞手性的错配如何导致肌动蛋白重塑， 诱导细胞间隙形成。 如果成功的话，我们将能够确定生物物理机制，允许潜在的发展， 基于细胞手性的用于内皮功能障碍的新型特异性疗法。从这个获得的数据 我们将寻求进一步的支持，并利用动物模型来检验我们的研究结果。

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.