title: Endothelial nitric oxide synthase is segregated from caveolin-1 and localizes to the leading edge of migrating cells.
abstract: The enzyme endothelial Nitric Oxide Synthase (eNOS) is involved in key physiological and pathological processes, including cell motility and apoptosis. It is widely believed that at the cell surface eNOS is localized in caveolae, where caveolin-1 negatively regulates its activity, however, there are still uncertainties on its intracellular distribution. Here, we applied high resolution confocal microscopy to investigate the surface distribution of eNOS in transfected HeLa cells and in human umbilical vein endothelial cells (HUVEC) endogenously expressing the enzyme. In confluent and non-confluent HUVEC and HeLa cells, we failed to detect substantial colocalization between eNOS and caveolin-1 at the cell surface. Instead, in non-confluent cells, eNOS was concentrated in ruffles and at the leading edge of migrating cells, colocalizing with actin filaments and with the raft marker ganglioside G(M1), and well segregated from caveolin-1, which was restricted to the posterior region of the cells. Treatments that disrupted microfilaments caused loss of eNOS from the cell surface and decreased Ca(2+)-stimulated activity, suggesting a role of the cytoskeleton in the localization and function of the enzyme. Our results provide a morphological correlate for the role of eNOS in cell migration and raise questions on the site of interaction between eNOS and caveolin-1.
authors.affiliation: Department of Pharmaco-Biological Science, University of Catanzaro Magna Graecia, 88021 Catanzaro, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Bulotta
authors.forename: Andrea
authors.initials: A
authors.lastname: Cerullo
authors.forename: Rico
authors.initials: R
authors.lastname: Barsacchi
authors.forename: Clara
authors.initials: C
authors.lastname: De Palma
authors.forename: Domenicantonio
authors.initials: D
authors.lastname: Rotiroti
authors.forename: Emilio
authors.initials: E
authors.lastname: Clementi
authors.forename: Nica
authors.initials: N
authors.lastname: Borgese
mesh_terms: D000199:Actins / Q000502:physiology; D000818:Animals; D002417:Cattle; D051242:Caveolin 1 / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000502:physiology*; D012680:Sensitivity and Specificity
chemical_list: D000199:Actins; D051242:Caveolin 1; D052250:Nitric Oxide Synthase Type III