Decoding Force Transduction Mechanisms of Leukocyte Specific Integrin LFA-1 in Immune Responses

PROJECT SUMMARY The primary goal of this research is to unravel the force transduction mechanisms of CD18 integrins and their role in immune cell adhesion and signaling. LFA-1 (CD11a/CD18, αLβ2) is essential for immune cell function, mediating critical processes such as cell migration, cell-cell adhesion, endothelial extravasation and the formation of immunological synapses. However, despite its importance, the molecular mechanisms by which LFA-1 transduces mechanical forces into biochemical signals remain poorly understood. This study aims to fill this knowledge gap by exploring how specific cytoplasmic proteins interact with LFA-1 to regulate its mechanical properties and signaling capabilities during immune cell adhesion. The long-term objective of this research is to deepen our understanding of immune cell adhesion and mechanotransduction pathways, which are crucial for normal immune function and play a key role in the development and progression of cardiovascular and pulmonary disease. The specific aims of this project are: (1) to elucidate the temporal dynamics of cytoplasmic protein associations with LFA-1 during adhesion formation and maturation. By focusing on the regulatory role of cytoplasmic proteins, this research will identify key molecular players that control integrin affinity and mechanical signaling. (2) to dissect the force transduction mechanisms of LFA-1 integrin by quantifying molecular orientation and force during immune cell function. To achieve these aims, the research will utilize cutting-edge techniques in advanced microscopy, including polarization anisotropy microscopy to measure LFA-1's molecular orientation and force. Additionally, the project will employ APEX2 proximity labeling coupled with mass spectrometry to uncover the temporal sequence of cytoplasmic protein interactions with LFA-1 during adhesion. CRISPR-based knockout strategies will then be used to test the functional significance of these proteins in regulating integrin activity and immune cell adhesion. The combination of these approaches will provide a comprehensive map of the mechanotransduction pathways and protein dynamics that govern LFA-1 integrin function. The expected outcomes of this research will enhance our fundamental understanding of how immune cells use mechanical information to control adhesion and immune function. By identifying new regulatory proteins involved in LFA-1- mediated force transduction, this work may also uncover novel therapeutic targets for treating immune-related cardiovascular and pulmonary diseases. In the long term, these findings have the potential to lead to new strategies for controlling immune cell behavior, improving treatments for a variety of conditions where immune dysfunction contributes to disease progression.