RESUMO
Neural stem and progenitor cells in the developing cerebral cortex, but also when grown in culture, display a range of distinct phenomena during cytokinesis. Cleavage furrow ingression in neural progenitor cells can bisect their basal processes and, later on, result in midbody formation at the apical surface. After abscission, these midbodies are released as membrane-bound particles into the extracellular space, in contrast to uptake and degradation of postabscission midbodies in other cell types. Whether these cellular dynamics are unique to neural stem cells, or more ubiquitously found, and what biological significance these processes have for cell differentiation or cell-cell communication, are open questions that require a combination of approaches. Here, we discuss techniques to study the specific membrane dynamics underlying the basal process splitting and postabscission midbody release in neural stem cells. We provide some basic concepts and protocols to isolate, enrich and stain released midbodies, and follow midbody dynamics over time. Moreover, we discuss techniques to prepare cortical sections for high-voltage electron microscopy to visualize the fine basal processes of progenitor cells.
Assuntos
Separação Celular/métodos , Rastreamento de Células/métodos , Citocinese/genética , Células-Tronco Neurais/ultraestrutura , Animais , Diferenciação Celular/genética , Divisão Celular/genética , Membrana Celular , Córtex Cerebral/ultraestrutura , Células HeLa , Humanos , Microscopia de FluorescênciaRESUMO
Saccharomyces cerevisiae Sly1 protein is a member of the Sec1/Munc18-family proteins, which are essential for vesicular trafficking, but their exact biological roles are yet to be determined. A temperature-sensitive sly1 mutant arrests the vesicular transport from the ER to Golgi compartments at 37 degrees C. We screened for multicopy suppressor genes that restore the colony formation of the sly1(ts) mutant to discover functionally interacting components. The suppressor genes obtained were classified as: (1) those that encode a multifunctional suppressor, SSD1; (2) heat shock proteins, SSB1 and SSB2; (3) cell surface proteins, WSC1, WSC2 and MID2; (4) ER-Golgi transport proteins, USO1 and BET1; and (5) an as-yet-uncharacterized protein, HSD1 (high-copy suppressor of SLY1 defect 1). By epitope tagging of the gene product, we found that Hsd1 protein is an ER-resident membrane protein. Its overproduction induced enlargement of ER-like membrane structures.
Assuntos
Proteínas de Transporte/genética , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Genes Supressores , Complexo de Golgi/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Mapeamento de Epitopos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Genes Fúngicos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas Munc18 , Mutação , Transporte Proteico , Proteínas Qc-SNARE , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMO
Targeting of vesicles to the acceptor membrane in protein transport depends on membrane proteins called SNAREs. Saccharomyces cerevisiae Golgi t-SNARE Sed5 protein and its neural cognate syntaxin 1 have similar three alpha-helices which are predicted to form coiled coils. We dissected the helices of Sed5 and found several characteristics unexpectedly distinct from those of syntaxin 1. Most importantly, only the N-terminal helix is responsible for the binding of Sly1 protein while almost the entire molecule of syntaxin is necessary for the binding of the cognate, Munc-18. The N-terminal region of Sed5 protein also binds to the C-terminal helix and Sly1 protein interfered this binding.
