RESUMO
The actin cortex that lines the plasma membrane of most eukaryotic cells resists external mechanical forces and plays critical roles in a variety of cellular processes including morphogenesis, cytokinesis, and cell migration. Despite its ubiquity and significance, we understand relatively little about the composition, dynamics, and structure of the actin cortex. Adenomatous polyposis coli (APC) proteins regulate the actin and microtubule cytoskeletons through a variety of mechanisms, and in some contexts, APC proteins are cortically enriched. Here we show that APC2 regulates cortical actin dynamics in the follicular epithelium and the nurse cells of the Drosophila ovary and in addition affects the distribution of cortical actin at the apical side of the follicular epithelium. To understand how APC2 influences these properties of the actin cortex, we investigated the mechanisms controlling the cortical localization of APC2 in S2 cultured cells. We previously showed that the N-terminal half of APC2 containing the Armadillo repeats and the C-terminal 30 amino acids (C30) are together necessary and sufficient for APC2's cortical localization. Our work presented here supports a model that cortical localization of APC2 is governed in part by self-association through the N-terminal APC Self-Association Domain (ASAD) and a highly conserved coiled-coil within the C30 domain.
Assuntos
Actinas/metabolismo , Proteínas do Domínio Armadillo/metabolismo , Proteínas de Drosophila/metabolismo , Ovário/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Drosophila , Feminino , Ligação Proteica , Multimerização ProteicaRESUMO
Teratoma formation in xenografts is a sufficiently stringent pluripotency assay for stem cells. However, little is known about the composition and spatial relationships of tissues within teratomas that may provide clues about development and platforms for studying organ development. Additionally, teratoma formation and analysis lack standards for reporting as assays of pluripotency. Three of 27 total teratomas derived from pedigreed primate embryonic stem cells underwent quantitative three-dimensional high-resolution magnetic resonance microscopy (MRM). Teratomas were subsequently serially sectioned and tissue types identified, semiquantitated, and correlated with MRM images. All teratomas demonstrated tissue derivatives from the three germ layers and approximately 23 different tissue types were identified. Certain tissue groups attempted to form organs more frequently (e.g., trachea/bronchi, small intestine). MRM discriminated some tissues readily (e.g., bone, adipose, cartilage) while other tissue types with like MR intensities could not be distinguished. Semiquantitative histopathological analysis of teratomas demonstrates the ability to delineate multiple tissues as derived from ectoderm, mesoderm, or endoderm and to use this information for comparison to other teratomas. MRM provides rapid quantitative imaging of intact teratomas that complements histology and identifies sites of interest for additional biological studies.
Assuntos
Células-Tronco Embrionárias/patologia , Teratoma/patologia , Animais , Microscopia/instrumentação , Microscopia/métodos , PrimatasRESUMO
Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells, including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate, as well as reversing those decisions during induced pluripotency (iPS), have focused largely on transcriptomic controls. Here, we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore, p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However, expression of constitutively active p70 S6K, but not wild-type p70 S6K, induces differentiation. Additionally, hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/fisiologia , Células-Tronco Embrionárias/efeitos dos fármacos , Ativação Enzimática , Humanos , Microscopia Eletrônica de Transmissão , Células-Tronco Pluripotentes/efeitos dos fármacos , Interferência de RNA , Sirolimo/farmacologiaRESUMO
Here we have developed protocols using the baboon as a complementary alternative Old World Primate to rhesus and other macaques which have severe limitations in their availability. Baboons are not limited as research resources, they are evolutionarily closer to humans, and the multiple generations of pedigreed colonies which display complex human disease phenotypes all support their further optimization as an invaluable primate model. Since neither baboon-assisted reproductive technologies nor baboon embryonic stem cells (ESCs) have been reported, here we describe the first derivations and characterization of baboon ESC lines from IVF-generated blastocysts. Two ESCs lines (BabESC-4 and BabESC-15) display ESC morphology, express pluripotency markers (Oct-4, hTert, Nanog, Sox-2, Rex-1, TRA1-60, TRA1-81), and maintain stable euploid female karyotypes with parentage confirmed independently. They have been grown continuously for >430 and 290 days, respectively. Teratomas from both lines have all three germ layers. Availabilities of these BabESCs represent another important resource for stem cell biologists.