RESUMO
Decades of research have not yet fully explained the mechanisms of epithelial self-organization and 3D packing. Single-cell analysis of large 3D epithelial libraries is crucial for understanding the assembly and function of whole tissues. Combining 3D epithelial imaging with advanced deep-learning segmentation methods is essential for enabling this high-content analysis. We introduce CartoCell, a deep-learning-based pipeline that uses small datasets to generate accurate labels for hundreds of whole 3D epithelial cysts. Our method detects the realistic morphology of epithelial cells and their contacts in the 3D structure of the tissue. CartoCell enables the quantification of geometric and packing features at the cellular level. Our single-cell cartography approach then maps the distribution of these features on 2D plots and 3D surface maps, revealing cell morphology patterns in epithelial cysts. Additionally, we show that CartoCell can be adapted to other types of epithelial tissues.
Assuntos
Cistos , Imageamento Tridimensional , Humanos , Imageamento Tridimensional/métodos , Processamento de Imagem Assistida por Computador/métodos , Epitélio , Células EpiteliaisRESUMO
Although developmental signalling pathways control tumourigenic growth, the cellular mechanisms that abnormally proliferating cells rely on are still largely unknown. Drosophila melanogaster is a genetically tractable model that is used to study how specific genetic changes confer advantageous tumourigenic traits. Despite recent efforts, the role of deubiquitylating enzymes in cancer is particularly understudied. We performed a Drosophila in vivo RNAi screen to identify deubiquitylating enzymes that modulate RasV12-induced hyperplastic growth. We identified the spliceosome core component Prp8 as a crucial regulator of Ras-, EGFR-, Notch- or RET-driven hyperplasia. Loss of prp8 function alone decreased cell proliferation, increased cell death, and affected cell differentiation and polarity. In hyperplasia, Prp8 supported tissue overgrowth independently of caspase-dependent cell death. The depletion of prp8 efficiently blocked Ras-, EGFR- and Notch-driven tumours but, in contrast, enhanced tumours that were driven by oncogenic RET, suggesting a context-specific role in hyperplasia. These data show, for the first time, that Prp8 regulates hyperplasia, and extend recent observations on the potential role of the spliceosome in cancer. Our findings suggest that targeting Prp8 could be beneficial in specific tumour types.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Oncogenes , Fatores de Processamento de RNA/metabolismo , Actinas/metabolismo , Animais , Carcinogênese , Morte Celular , Diferenciação Celular , Polaridade Celular , Proliferação de Células , Olho/crescimento & desenvolvimento , Olho/metabolismo , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Técnicas de Silenciamento de Genes , Hiperplasia , Invasividade Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Especificidade de Órgãos , Fenótipo , Interferência de RNA , Proteínas ras/metabolismoRESUMO
Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Here, using a multidisciplinary approach, we identify the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). We show that elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. Our results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements.
Assuntos
Drosophila melanogaster/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Animais , Animais Geneticamente Modificados , Movimento Celular/genética , Extensões da Superfície Celular/genética , Células Cultivadas , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Repressão Enzimática/genética , Feminino , Morfogênese/genética , Proteínas Proto-Oncogênicas c-vav/genética , RNA Interferente Pequeno/genética , Deleção de Sequência/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Stationary-to-migratory transitions of epithelial cells have a key role in development and tumour progression. Border cell migration is a powerful system in which to investigate this transition in living organisms. Here, we identify the Ste20-like kinase misshapen (msn) as a novel regulator of border-cell migration in Drosophila. Expression of msn in border cells is independent of the transcription factor slow border cells and of inputs from all pathways that are known to control border-cell migration. The msn gene functions to modulate the levels and/or distribution of Drosophila E-cadherin to promote the invasive migratory behaviour of border cells.