RESUMEN
The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic ß-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.
Asunto(s)
Vesículas Citoplasmáticas/metabolismo , Retículo Endoplásmico/metabolismo , Ribosomas/metabolismo , Animales , Transporte Biológico , Microscopía por Crioelectrón , Vesículas Citoplasmáticas/ultraestructura , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Ratones , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Imagen Molecular , Especificidad de Órganos , Ratas , Ribosomas/ultraestructura , Estrés FisiológicoRESUMEN
The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved Förster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved Förster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.
