RESUMO
Thorax fusion occurs in the midline of the Drosophila pupal notum and involves epithelial cell delamination requiring apoptotic signaling. By genetic screening, we found that NADPH oxidases (Nox and Duox) associated with superoxide anion (OË-2) are responsible for caspase-3 activation and delamination. We observed that Nox is upregulated in cells that undergo delamination and that delamination depends on caspase activation. However, the cell morphology and the almost complete lack of propidium iodide incorporation suggested little membrane disruption and signified apoptotic modulation. These results demonstrate that most delaminating cells undergo caspase activation, but this activation is not sufficient for apoptosis. We showed that the expression of Catalase, encoding an H2O2 scavenger in the cytosol, increases delamination and induces apoptotic nuclear fragmentation in caspase-3-activated cells. These findings suggest that the roles of OË-2 and intracellular H2O2 for delamination differs before and after caspase-3 activation, which involves live cell delamination.
RESUMO
To achieve highly selective ablation of lacZ-positive cells in a biological milieu in vivo, we developed an activatable photosensitizer, SPiDER-killer-ßGal, targeted to ß-galactosidase encoded by the lacZ reporter gene. Hydrolysis of SPiDER-killer-ßGal by ß-galactosidase simultaneously activates both its photosensitizing ability and its reactivity to nucleophiles, so that the phototoxic products generated by light irradiation are trapped inside the lacZ-positive cells. The combination of SPiDER-killer-ßGal and light irradiation specifically killed lacZ-positive cells in coculture with cells without lacZ expression. Furthermore, ß-galactosidase-expressing cells in the posterior region of cultured Drosophila wing discs and in pupal notum of live Drosophila pupae were selectively killed with single-cell resolution. This photosensitizer should be useful for specific ablation of targeted cells in living organisms, for example, to investigate cellular functions in complex networks.
RESUMO
Non-apoptotic caspase activation involves multiple cellular events. However, the link between visible non-apoptotic caspase activation and its function in living organisms has not yet been revealed. Here, we visualized sub-lethal activation of apoptotic signaling with the combination of a sensitive indicator for caspase 3 activation and in vivo live-imaging analysis of Drosophila During thorax closure in pupal development, caspase 3 activation was specifically observed at the leading edge cells, with no signs of apoptosis. Inhibition of caspase activation led to an increase in thorax closing speed, which suggests a role of non-apoptotic caspase activity in cell motility. Importantly, sub-lethal activation of caspase 3 was also observed during wound closure at the fusion sites at which thorax closure had previously taken place. Further genetic analysis revealed that the activation of the initiator caspase Dronc is coupled with the generation of reactive oxygen species. The activation of Dronc also regulates myosin levels and delays wound healing. Our findings suggest a possible function for non-apoptotic caspase activation in the fine-tuning of cell migratory behavior during epithelial closure.
Assuntos
Apoptose , Caspases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Epitélio/embriologia , Tórax/embriologia , Cicatrização , Animais , Movimento Celular , Cruzamentos Genéticos , Ativação Enzimática , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , MAP Quinase Quinase 4/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tórax/metabolismoRESUMO
Tissue closure involves the coordinated unidirectional movement of a group of cells without loss of cell-cell contact. However, the molecular mechanisms controlling the tissue closure are not fully understood. Here, we demonstrate that Lamin C, the sole A-type lamin in Drosophila, contributes to the process of thorax closure in pupa. High expression of Lamin C was observed at the leading front of the migrating wing imaginal discs. Live imaging analysis revealed that knockdown of Lamin C in the thorax region affected the coordinated movement of the leading front, resulting in incomplete tissue fusion required for formation of the adult thorax. The closure defect due to knockdown of Lamin C correlated with insufficient accumulation of F-actin at the front. Our study indicates a link between A-type lamin and the cell migration behavior during tissue closure.