RESUMO
ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. In the present study, we comprehensively examined the cellular and subcellular localization of Arf6 in adult mouse peripheral tissues by immunofluorescence and immunoelectron microscopy using the heat-induced antigen retrieval method with Tris-EDTA buffer (pH 9.0). Marked immunolabeling of Arf6 was observed particularly in epithelial cells of several tissues including the esophagus, stomach, small and large intestines, trachea, kidney, epididymis, oviduct, and uterus. In most epithelial cells of simple or pseudostratified epithelia, Arf6 exhibited predominant localization to the basolateral membrane and a subpopulation of endosomes. At an electron microscopic level, Arf6 was localized along the basolateral membrane, with dense accumulation at interdigitating processes and infoldings. Arf6 was present in a ring-like appearance at intercellular bridges in spermatogonia and spermatocytes in the testis and at the Flemming body of cytokinetic somatic cells in the ovarian follicle, thymus, and spleen. The present study provides anatomical clues to help understand the physiological roles of Arf6 at the whole animal level.
Assuntos
Fatores de Ribosilação do ADP/análise , Epididimo/química , Intestino Delgado/química , Rim/química , Oviductos/química , Testículo/química , Fator 6 de Ribosilação do ADP , Animais , Reações Antígeno-Anticorpo , Feminino , Imunofluorescência , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Microscopia ImunoeletrônicaRESUMO
IQ-ArfGEF/BRAG1 is a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), which are implicated in membrane trafficking and actin cytoskeleton dynamics. In this study, we examined the immunohistochemical localization of IQ-ArfGEF/BRAG1 in the adult mouse retina using light and electron microscopy. IQ-ArfGEF/BRAG1 was distributed in a punctate manner and colocalized well with RIBEYE in both the outer and inner plexiform layers. Immunoelectron microscopic analysis showed that IQ-ArfGEF/BRAG1 was localized at the synaptic ribbons of photoreceptors. When heterologously expressed in HeLa cells, IQ-ArfGEF/BRAG1 was recruited to RIBEYE-containing clusters and formed an immunoprecipitable complex with RIBEYE. Furthermore, immunoprecipitation analysis showed that anti-IQ-ArfGEF/BRAG1 antibody efficiently pulled down RIBEYE from retinal lysates. These findings indicate that IQ-ArfGEF/BRAG1 is a novel component of retinal synaptic ribbons and forms a protein complex with RIBEYE.