The Role of End Binding Protein 3 in Regulating the Permeability of the Endothelial Barrier

The endothelium forms a semi-permeable barrier that restricts the passage of protein-rich fluids into the underlying tissues and is critical for maintaining tissue-fluid homeostasis, transmigration of blood cells, and gas exchange. Disruption of the endothelial barrier, through the disassembly of adherens junctions (AJs), results in the accumulation of protein rich fluids in the interstitium, a condition that is frequently associated with pulmonary edema. The work seeks to further the understanding of the role of end binding protein 3 (EB3), a core element of plus end tracking protein (+TIP) complex of microtubules (MT) in cell signaling and the regulation of the actin cytoskeletal activity and dynamics of AJs. EB3 at the growing MT tips has the ability to transduce signals by providing a scaffold at the growing end of MTs as well as through its ability to regulate dynamic reorganization of MTs. We demonstrate that growing MT tips serve as the binding platform for inositol 1,4,5-trisphosphate receptors (IP3Rs), the Ca2+ gating channel on the endoplasmic reticulum (ER) membrane. EB3 interacts with IP3R3 and triggers receptor clustering, modulating ER Ca2+ release. Targeting EB3-IP3R interactions in ECs prevents adhesions acto-myosin contractile response and suppresses vascular leak suggesting that MT-dependent amplification of Ca2+ signaling is a significant component of pathological response that can be targeted therapeutically. We then identified a pivotal function of VE-cadherin homophilic interaction in modulating endothelial barrier through the tuning of MT dynamics. We demonstrate that VE-cadherin outside-in signaling regulates cytosolic Ca2+ homeostasis and EB3 phosphorylation, which are required for assembly of AJs. We observed that loss of VE-cadherin-mediated adhesion induced the activation of Src and phospholipase Cγ2, which mediated Ca2+ release from ER stores resulting in the phosphorylation of serine 162 in EB3. This phospho-switch was required to destabilize the EB3 dimer, suppress microtubule MT growth, and assemble AJs. It is our intent that by understanding how EB3 regulates barrier permeability, we will provide novel insights into the mechanisms of dysregulation of lung fluid homeostasis.