Non-conventional signaling by α5 integrin in blood and endothelial cells

SUMMARY – Integrins interact with their ligands to induce intracellular signals that mediate cellular activities such as cell adhesion, spreading, and migration, which are the essential cellular activities for the function of blood and endothelial cells. These signaling events are typically mediated by the cytoplasmic tail of integrin β subunit, while the role of α integrin cytoplasmic tail in integrin function remains relatively underexplored. Our research found that the fibronectin receptor integrin α5β1, via its α cytoplasmic tail, is involved in the formation of tunneling nanotubes (TNTs), a novel type of cellular structure for cell-to-cell communication. Remarkably, our research found that the α5β1-mediated TNT formation can be induced by the spike protein of coronavirus SARS- CoV-2. We observed the spike-induced and α5-dependent TNT formation in model cell lines and primary human blood and endothelial cells. Furthermore, we found that α5β1 integrin can mediate the spike-induced proinflammatory response in human blood and endothelial cells, which may contribute to the thrombotic events in COVID-19. TNTs are long actin-rich cell membrane protrusions that have been increasingly recognized as functional subcellular structures for long-distance dynamic intercellular connection. Functioning as conduits between connected cells, TNTs can transport cytoplasmic components like small molecules, proteins, vesicles, and mitochondria intercellularly. Accumulating evidence suggests that TNTs are involved in the progress of many pathological conditions such as cancer, inflammation, and neurodegenerative diseases. Bacteria and virus pathogens also exploit TNTs for cell-to-cell transmission. TNTs can form under cell stress conditions such as inflammation and virus infection. However, the cellular mechanisms regulating TNT formation remain largely unknown. This is mainly because little to nothing is known about the cell surface receptors directly responsible for TNT biogenesis. Our discovery of α5β1 integrin as a functional signaling receptor for TNT formation provides a powerful platform for investigating TNT biology. Mechanistically, we found that both α5β1-mediated TNT formation and proinflammatory response require the participation of α5 cytoplasmic tail, suggesting that these two processes are interconnected events. Moreover, our protein interaction data suggest a direct binding between α5β1 and the spike protein, which is independent of the classical integrin recognition Arg-Gly-Asp (RGD) motif. Based on these promising data, this application aims to elucidate the cellular mechanisms governing the non-conventional signaling function of α5β1 integrin in TNT formation and inflammation in blood and endothelial cells. Multifaced biochemical, biophysical, structural and cell biology approaches will be used to identify intracellular molecules and signaling pathways involved in the α5β1-mediated TNT formation (Aim 1) and inflammation (Aim 2) and to characterize the non-RGD dependent α5β1 and ligand interaction (Aim 3). The outcome of this study will advance both integrin and TNT biology and uncover potential therapeutic targets for modulating TNTs and inflammation in various diseases.