Assuntos
Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Cinesinas/genética , Mitose/genética , Proteínas de Xenopus/genética , Animais , Humanos , Carioferinas/fisiologia , Lamina Tipo B/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Proteína ran de Ligação ao GTP/fisiologiaRESUMO
Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.
Assuntos
Pareamento Cromossômico/genética , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinesinas/metabolismo , Mitose/genética , beta Carioferinas/fisiologia , Proteína ran de Ligação ao GTP/genética , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromossomos/genética , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Cinesinas/genética , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Sinais de Localização Nuclear/genética , Fosforilação , Transporte Proteico/genética , Fuso Acromático/genética , alfa Carioferinas/genética , beta Carioferinas/genéticaRESUMO
Immune dysfunction in patients with iron overload has been reported. Iron disturbed CD2 expression on T-cells, cell-mediated immunity by Th1 cells and monocyte functions including phagocytosis and natural killer activity. In the present study, we examined the effects of iron and desferrioxamine (DFX, an iron chelator) on generation of multinucleated giant cells (MGC) by human monocytes in vitro. Human monocytes were isolated from venous blood and cultured with concanavalin A (Con A) stimulation with additives, ferric citrate (Fe-citrate) or sodium citrate (Na-citrate) or DFX for 4 days. The cells were fixed and subjected to Wright staining. MGC formation was observed under light microscopy. Con A induced MGC formation in a dose-dependent manner, and reached a plateau after 3 days of incubation. MGC formation was suppressed when Con A-stimulated monocytes were cultured with the co-addition of Fe-citrate but not Na-citrate only in the early phase of culture (less than 24 hours). DFX also suppressed MGC formation in a dose-dependent manner. Using flow cytometry analysis, the co-addition of Fe-citrate significantly suppressed CD18 (beta2 integrin) and CD54 (ICAM-I) but not CD11a (alpha integrin) expression on Con A-stimulated monocytes. Iron supressed the generation of MGC by human monocytes in vitro. These observations suggested that iron might affect MGC generation by down-regulation of adhesion molecule expression on monocytes.
