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.

细胞与细胞连接的调节对于上皮等各种组织的生物学功能至关重要 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 连接受到细胞手性的调节，这是细胞的普遍但基本的特性。我们率先在 使用工程体外平台研究细胞手性。在这里，利用这些平台，我们要调查 与细胞-细胞连接处的手性相关的生物物理机制。虽然原理可能是共享的 在许多细胞类型中，本研究将重点关注内皮细胞和血管通透性的调节。 内皮细胞层是一个半透性屏障，严格控制蛋白质和细胞的通过 血液进入细胞间隙，调节生物组织的局部环境 有机体。细胞主要通过控制旁细胞运输来实现这一重要功能 细胞与细胞连接的打开和关闭。蛋白激酶 C (PKC) 激活与 糖尿病和长期吸烟等慢性疾病以及急性疾病中的内皮功能障碍 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 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。在这个提议中，我们假设 PKC 通过降低细胞手性水平来逆转细胞手性。 肌动蛋白交联以及细胞手性调节细胞间连接（从而调节内皮通透性） 生物力学上通过肌动蛋白倾斜和 VE-钙粘蛋白定位。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 使用牵引力显微镜（TFM）进行形态发生。结合细胞手性和 TFM 的 2D 微图案 对于细胞力，我们将详细研究二维集体对称性破缺并探究 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。我们将鉴定参与的福尔米亚型和肌动蛋白交联剂 这个过程及其受 PKC 信号传导的调节。目标 3. 研究手性失配在 细胞间间隙的形成和内皮细胞的通透性。我们将量化肌动蛋白结构和动力学 细胞间连接并检查细胞手性的不匹配如何导致肌动蛋白重塑和 诱导细胞间隙形成。 如果成功，我们将能够确定生物物理机制，从而实现潜在的发展 基于细胞手性治疗内皮功能障碍的新颖、特异性疗法。通过由此获得的数据 根据建议，我们将寻求进一步的支持并用动物模型检验我们的发现。