RESUMEN
Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+ Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.
Asunto(s)
Citocinesis , Estratos Germinativos/ultraestructura , Morfogénesis/genética , Células Madre Pluripotentes/ultraestructura , Actinas/genética , Actinas/metabolismo , Biomarcadores/metabolismo , Calcio/metabolismo , Calnexina/genética , Calnexina/metabolismo , Línea Celular , Polaridad Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Expresión Génica , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Humanos , Interfase , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Mitosis , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Análisis de la Célula Individual , Imagen de Lapso de TiempoRESUMEN
Cyclooxygenases (COX-1 and COX-2) are N-glycosylated, endoplasmic reticulum-resident, integral membrane proteins that catalyze the committed step in prostanoid synthesis. COX-1 is constitutively expressed in many types of cells, whereas COX-2 is usually expressed inducibly and transiently. The control of COX-2 protein expression occurs at several levels, and overexpression of COX-2 is associated with pathologies such as colon cancer. Here we have investigated COX-2 protein degradation and demonstrate that it can occur through two independent pathways. One pathway is initiated by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. We provide evidence from site-directed mutagenesis that a 27-amino acid instability motif (27-IM) regulates posttranslational N-glycosylation of Asn-594. This motif begins with Glu-586 8 residues upstream of the N-glycosylation site and ends with Lys-612 near the C terminus at Leu-618. Key elements of the 27-IM include a helix involving residues Glu-586 to Ser-596 with Asn-594 near the end of this helix and residues Leu-610 and Leu-611, which are located in an apparently unstructured downstream region of the 27-IM. The last 16 residues of the 27-IM, including Leu-610 and Leu-611, appear to promote N-glycosylation of Asn-594 perhaps by causing this residue to become exposed to appropriate glycosyl transferases. A second pathway for COX-2 protein degradation is initiated by substrate-dependent suicide inactivation. Suicide-inactivated protein is then degraded. The biochemical steps have not been resolved, but substrate-dependent degradation is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases. The pathway involving the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled to the rate of substrate turnover.