Abstract 722: Inhibition of Endothelial Cell Migration by Semaphorin 3C Requires Neuropilin-1 Uptake via Lipid Rafts
Introduction: Semaphorins play important roles in the morphogenesis of the vascular system. In particular, semaphorin 3C (sema3C) was shown to be required for the development of the aortic arch and the cardiac outflow tract (Dev Cell, 7: 107–16, 2004). Sema3C binding to the cell surface is mediated by two receptors, neuropilin-1 (Npn1), and plexin D1, but its signaling mechanism downstream of these receptors is not well understood. Recent findings suggest that Npn1 uptake is involved in the response to semaphorin and VEGF (Blood, 107: 3892–901, 2004). Npn1 binds the PDZ-domain protein synectin, an adaptor for the unconventional myosin VI, a retrograde actin-based molecular motor (PNAS, 103: 12735– 40, 2006). Similar to other cell surface receptors, it is conceivable that Npn1 uptake mediates the cell response to sema3C.
Objective: We set out to characterize the endocytic pathway of Npn1, to determine the dependence of Npn1 uptake on synectin, and to test the hypothesis that sema3C signaling depends on Npn1 uptake.
Methods and results: We found that sema3C inhibited the migration of mouse primary endothelial cells (ECs) in Transwell assays by 40%, but it did not inhibit the migration of ECs from synectin−/− mice. In order to correlate the reduced effect of sema3C on synectin−/− EC migration to Npn1 uptake rate, we measured Npn1 endocytosis in wild type (WT) and synectin−/− ECs in response to sema3C. In pulse-chase assays, Npn1 uptake in synectin−/−EC was slower by 70% compared to WT EC. The internalized Npn1 did not collocate with clathrin and AP1α, ruling out clathrin-dependent endocytosis. Similarly, Npn1 did not collocate with caveolin-1. Npn1 did collocate extensively, however, with the lipid-raft marker cholera toxin-β subunit (CTβ). Before the initiation of uptake, Npn1 and CTβ collocated along the cell edges in both WT and synectin−/− EC, but 10 min later Npn1 collocated with perinuclear early endosomes in 73% of WT EC, but only in 19% of synectin−/− ECs. Similarly, 30 min after uptake started, Npn1 recycled back to the cell edges in 57% of WT ECs, but only in 10% of synectin−/−ECs.
Conclusions: These results establish that Npn1 undergoes endocytosis via lipid rafts, rather than by clathrin, and that sema3C signaling is dependent on Npn1 uptake.