We study a signaling pathway involved in endothelial cell migration [6] as well as maintenance of tight junctions between cells. One of the pivotal proteins in this pathway is a RhoA GTPase activator, the guanine exchange factor Syx (Synectin-binding exchange factor; synectin is a versatile adaptor protein). We found that syx deletion in the mouse and its knockdown in the zebrafish drastically and specifically inhibited arterial angiogenesis – namely the sprouting of arterioles from pre-existing arteries [10]. We are currently identifying the molecular mechanisms underlying the defective angiogenesis in the zebrafish and mouse. Syx associates with angiomotin, a protein that regulates intercellular junctions and EC migration in response to VEGF. Strikingly, knockdown of angiomotin in the zebrafish produced a morphologically identical block in intersegmental vessel (ISV) sprouting to syx knockdown, suggesting the two proteins share a common signaling pathway. Moreover, Syx and angiomotin are functionally similar: they regulate both EC migration and tight junctions. Our results indicate that both proteins associate with early endosomes, thus implying that they undergo trafficking between tight junctions and early endosomes. Consequently, we hypothesize that the trafficking of Syx and angiomotin-carrying vesicles between the tight junctions and the plasma membrane regulates the VEGF-driven directional migration of endothelial cells.
We are currently focusing on neuropilin-1, a receptor that relays both attractive and repellent signals from VEGF-A or semaphoring 3C, respectively. We found that neuropilin-1 undergoes endocytosis via a different pathway in response to each ligand [9]. VEGF-A induced clathrin-dependent endocytosis of neuropilin-1 and VEGF receptor 2, while semaphorin 3C induced lipid raft-dependent endocytosis. Neuropilin-1 and VEGFR2 were clustered together in quiescent cells and remained so during post-endocytic trafficking after either VEGF-A or semaphorin 3C treatment. Neuropilin-1 trafficking depended on its interaction with the PDZ adaptor protein synectin, since, as we have previously established [8], the latter is required for coupling of internalized receptors to myosin VI, a molecular motor involved in trafficking. We found that neuropilin-1 trafficking was required for both the stimulation and suppression of EC migration by VEGF and by semaphorin, respectively [9]. We are pursuing the hypothesis that neuropilin-1 uptake is required for the regulation of EC migration by VEGF-A and semaphoring 3C, and for neuropilin-1 function as a guidance receptor in the vascular system.
Lerner Research Institute
Cleveland Clinic, Mail Code NB21
9500 Euclid Avenue
Cleveland, Ohio 44195
