Understanding Chirality at Cell-Cell Junctions With Microscale Platforms - 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.
