RESUMEN
Although different nanosized materials, including quantum dots (QDs), are intended to be used for biomedical applications, their interactions with microvessels and their inflammatory potential are largely unknown. In this in vivo study we report that leukocyte recruitment is modulated in the presence of quantum dots. We found that the surface chemistry of QDs strongly affects their localization in postcapillary venules, their uptake by perivascular macrophages, and their potential to modify steps of leukocyte recruitment.
Asunto(s)
Adhesión Celular , Movimiento Celular , Leucocitos/citología , Puntos Cuánticos , Proteínas Sanguíneas , Macrófagos/citología , Mastocitos/citología , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Propiedades de SuperficieRESUMEN
Annexin 2 is a Ca2+- and phospholipid-binding protein previously identified on endosomal membranes and the plasma membrane. Inferred from this location and its stimulatory effect on membrane transport annexin 2 has been proposed to play a role in the structural organization and dynamics of endosomal membranes. Validation of this view requires a detailed analysis of the distribution of annexin 2 over the endosomal compartment and a characterization of the parameters governing this distribution. Towards this end we have devised an immunoisolation protocol to purify annexin 2-positive membrane vesicles from subcellular fractions of BHK cells containing early endosomes. We show that this approach leads to the isolation of intact endosomal vesicles containing internalized fluid-phase marker and that the immunoisolated membranes are positive for the transferrin receptor and Rab4 but not for the early endosomal antigen EEA1. A distinct and non-uniform distribution of annexin 2 over the early endosomal compartment is also observed in immunoelectron microscopy analyses of whole-mount specimens of BHK cells. Annexin 2 antibodies labeled transferrin receptor-containing tubular early endosomal structures, but not EEAl-positive endosomal vacuoles. We also observed that the Ca2+-independent association of annexin 2 with endosomal membranes was disrupted by the cholesterol-binding glycerid saponin, while Ca2+ could trigger annexin 2 binding to saponin-treated endosomal membranes. Thus, either Ca2+- or cholesterol-stabilized membrane domains are required for the binding of annexin 2 to endosomes suggesting that both factors may regulate this interaction.
Asunto(s)
Anexina A2/metabolismo , Calcio/metabolismo , Colesterol/metabolismo , Endosomas/metabolismo , Animales , Sitios de Unión , Línea Celular , Cricetinae , Membranas Intracelulares/metabolismo , Microscopía Fluorescente , Microscopía InmunoelectrónicaRESUMEN
Studying the mode of interaction between actin and actin-binding proteins, we constructed a chimaeric protein consisting of the sequence for bovine profilin I (P), to which the sequence for the actin-binding domain of Dictyostelium discoideum alpha-actinin (alphaA1-2) was fused N-terminally. The resulting hybrid clone was expressed in Escherichia coli, and the chimaeric protein, alphaA1-2P, purified by affinity chromatography on poly-(L-proline) (PLP) columns and identified using specific antibodies. High resolution electron microscopy demonstrated that this protein consists of two discrete subdomains. In biochemical, viscometric and electron microscopic analyses, we showed that both modules in this molecule are biologically active. The chimaera binds to poly-(L-proline) and inhibits the polymerization of G-actin in KCl, which is consistent with the assumption that the profilin part is intact. Inhibition of actin polymerization in KCl was stronger than that of the parental profilin, and the Kd value of its interaction with rabbit skeletal muscle actin, as determined by falling ball viscometry, was smaller (mean value 0.5 x 10(-6) M, as compared to 1.9 x 10(-6) M for bovine profilin). In 2mM MgCl2, the actin polymerized rapidly, consistent with the interpretation that under these conditions the chimaera, like profilin, is less efficient as an actin-sequestering agent. In the presence of alphaA1-2P, the resulting filaments were decorated with particles projecting from the filament axis. We conclude that under these conditions the alphaA1-2 domain of alphaA1-2P is preferentially active, attaching the chimaeric particles laterally to the filaments. Hence, the parental modules combined in alphaA1-2P permit this molecule to switch from a G-actin- to an F-actin-binding form.
Asunto(s)
Actinina/metabolismo , Actinas/metabolismo , Proteínas Contráctiles , Proteínas de Microfilamentos/metabolismo , Actinina/genética , Actinina/ultraestructura , Animales , Sitios de Unión , Bovinos , Magnesio , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/ultraestructura , Péptidos/metabolismo , Profilinas , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructuraRESUMEN
Annexin II, a member of a family of Ca2+ and membrane binding proteins, has been implicated in regulating membrane organization and membrane transport during endocytosis and Ca2+ regulated secretion. To characterize the mechanistic aspects of the annexin. II action we studied parameters which determine the endosomal association of annexin II. Immunoblot analysis of subcellular membrane fractions prepared from BHK cells in the presence of a Ca2+ chelating agent reveals that annexin II remains associated with endosomal membranes under such conditions. This annexin II behaviour is atypical for the Ca2+ regulated annexins and is corroborated by the finding that ectopically expressed annexin II mutants with inactivated Ca2+ binding sites continue to co-fractionate with endosomal membranes. The Ca(2+)-independent membrane association of annexin II is also not affected by introducing mutations interfering with the complex formation of annexin II with its intracellular protein ligand p11. However, a deletion of the unique N-terminal domain of annexin II, in particular the sequence spanning residues 15 to 24, abolishes the Ca(2+)-independent association of the protein with endosomes. These results describe a novel, Ca(2+)-independent type of annexin-membrane interaction and provide a first explanation for the observed preference of different annexins for different cellular membranes. In the case of annexin II this specificity could be mediated through specific membrane receptors interacting with a unique sequence in the annexin II molecule.