RESUMO
Intracellular RNA localization is a widespread and dynamic phenomenon that compartmentalizes gene expression and contributes to the functional polarization of cells. Thus far, mechanisms of RNA localization identified in Drosophila have been based on a few RNAs in different tissues, and a comprehensive mechanistic analysis of RNA localization in a single tissue is lacking. Here, by subcellular spatial transcriptomics we identify RNAs localized in the apical and basal domains of the columnar follicular epithelium (FE) and we analyze the mechanisms mediating their localization. Whereas the dynein/BicD/Egl machinery controls apical RNA localization, basally-targeted RNAs require kinesin-1 to overcome a default dynein-mediated transport. Moreover, a non-canonical, translation- and dynein-dependent mechanism mediates apical localization of a subgroup of dynein-activating adaptor-encoding RNAs (BicD, Bsg25D, hook). Altogether, our study identifies at least three mechanisms underlying RNA localization in the FE, and suggests a possible link between RNA localization and dynein/dynactin/adaptor complex formation in vivo.
Assuntos
Proteínas de Drosophila , Dineínas , Animais , Dineínas/genética , Dineínas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Complexo Dinactina/metabolismo , Cinesinas , Transcriptoma , RNA Mensageiro/metabolismo , Drosophila/genética , Drosophila/metabolismo , RNA/genética , RNA/metabolismo , Microtúbulos/metabolismoRESUMO
Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.
Assuntos
Drosophila melanogaster/ultraestrutura , Células da Granulosa/ultraestrutura , Glândulas Mamárias Animais/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Coloração e Rotulagem/métodos , Células Tecais/ultraestrutura , Traqueia/ultraestrutura , Animais , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Expressão Gênica , Genes Reporter , Células da Granulosa/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Larva/metabolismo , Larva/ultraestrutura , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Glândulas Mamárias Animais/metabolismo , Camundongos , Microscopia Eletrônica de Varredura/instrumentação , Organoides/metabolismo , Organoides/ultraestrutura , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Células Tecais/metabolismo , Traqueia/metabolismo , Fluxo de Trabalho , Proteína Vermelha FluorescenteRESUMO
Despite the significant advances in the comprehension of stem cell control network, the nature of extrinsic signals regulating their dynamic remains to be understood. In this paper, we take advantage of the stem cell repopulation process that follows low-dose X-ray treatment in planarians to identify genes, preferentially enriched in differentiated cells, whose expression is activated during the process. Genetic silencing of some of them impaired the stem cell repopulation, suggesting a tight extrinsic control of stem cell activity.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Células-Tronco/efeitos da radiação , Animais , Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Modelos Biológicos , Planárias/genética , Planárias/efeitos da radiação , RNA Mensageiro/genética , Regeneração/genética , Raios XRESUMO
Despite increasing evidence indicates polyamines as a convergence point for signaling pathways, including cell growth and differentiation, a unifying concept to interpret their role is still missing. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is tightly regulated by a complex molecular machinery, and the demonstration of the existence of multiple ODC paralogs, lacking decarboxylation activity, suggests additional layers of complexity to the intricate ODC regulatory pathway. Because of their extraordinary regenerative abilities and abundance of stem cells, planarians have potential to contribute to our understanding of polyamine function in an in vivo context. We undertook a study on ODC function in planarians and we found six planarian ODCs (ODC1-6). Five out of six ODC homologs carry substitutions of key aminoacids for enzymatic activity, which makes them theoretically unable to decarboxylate ornithine. Silencing of ODC5 and 6 produced a complex phenotype, by prompting animals to an aberrant response, following chronic injury without tissue removal. Phenotype is neither rescued by putrescine, nor mimicked by difluoromethylornithine treatment. Moreover, the co-silencing of other genes of the ODC regulatory pathway did not modulate phenotype outcome or severity, thus suggesting that the function/s of these ODC-like proteins might be unrelated to decarboxylase activity and putrescine production.
