Understanding Chirality at Cell-Cell Junctions With Microscale Platforms

利用微型平台了解细胞与细胞连接处的手性

• 批准号: 10587627
• 负责人: Leo Q. Wan
• 金额: $ 30.55万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587627
• 关键词: 3-Dimensional; Actins; Acute Disease; Acute Lung Injury; Address; Adherens Junction; Affect; Animal Model; Biological; Biological Process; Biomechanics; Biophysical Process; Blood Circulation; Blood Vessels; Cells; Cessation of life; Chronic; Crosslinker; Cytoskeleton; Data; Development; Diabetes Mellitus; Embryonic Development; Endothelial Cells; Endothelium; Engineering; Environment; Epithelium; Future; Handedness; Hydrogels; In Vitro; Intercellular Junctions; Left; Location; Maintenance; Mechanics; Mediating; Modeling; Molecular; Morphogenesis; Organism; Patients; Pattern; Permeability; Physiological; Physiology; Process; Property; Protein Isoforms; Protein Kinase C; Proteins; Regulation; Research; Role; Sepsis; Signal Transduction; Smoking; Structure; Substrate Interaction; System; Tight Junctions; Tissues; Traction Force Microscopy; Vascular Diseases; Vascular Permeabilities; Virus Diseases; adherent junction; biophysical analysis; cadherin 5; cell type; crosslink; endothelial dysfunction; fascin; interstitial; invention; mechanical force; novel; organizational structure; screening

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.

细胞 - 细胞连接的调节对于各种组织的生物学功能至关重要 和内皮。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，细胞手性是细胞的通用但基本特性。我们开拓了 使用工程化的体外平台研究细胞手性研究。在这里，使用这些平台，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。虽然可以共享原则 在许多细胞类型中，这项研究将集中于内皮细胞和血管渗透性的调节。 内皮细胞层是一个半渗透的屏障，它紧紧控制蛋白质和细胞中的通过 血液进入天质空间，并调节生物中生物组织的局部环境 有机体。细胞主要通过控制副细胞运输来实现这一重要功能 细胞电池连接的开放和闭合。蛋白激酶C（PKC）激活与 在糖尿病和长期吸烟等慢性疾病中的内皮功能障碍以及急性 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 对于身体功能和患者生存至关重要，特别是对于急性疾病。最近，我们已经证明了 PKC可以逆转细胞手性，从而介导内皮渗透性。但是，关于 PKC激活如何逆转内皮手性或细胞手性改变的分子机制 内皮渗透性。在此提案中，我们假设PKC通过降低水平逆转了细胞手性 肌动蛋白的交联和细胞手性调节细胞 - 细胞连接（因此内皮通透性） 通过肌动蛋白倾斜和VE-钙粘蛋白定位在生物力学上。我们将追求以下三个目标：目标 1。确定负责多细胞手性的生物力学不对称的时间和位置 使用牵引力显微镜（TFM）的形态发生。梳理2D微图案，用于细胞手性和TFM 对于细胞力，我们将详细研究2D集体对称性破坏并询问 潜在的细胞生物力学机制。 AIM 2。确定PKC的细胞骨架机制 诱发内皮细胞手性的逆转。我们将确定参与参与 这个过程及其对PKC信号的调节。目标3。调查手性不匹配在 细胞间间隙形成和内皮渗透性。我们将量化肌动蛋白的结构和动态 细胞间连接处并检查细胞手性不匹配如何导致肌动蛋白的重塑和 诱导细胞间间隙形成。 如果成功，我们将能够识别生物物理机制，从而允许潜在的发展 基于细胞手性的新型特定疗法，用于内皮功能障碍。从中获得的数据 提案，我们将寻求进一步的支持，并通过动物模型来检查我们的发现。