To achieve highly
selective ablation of lacZ-positive
cells in a biological milieu in vivo, we developed
an activatable photosensitizer, SPiDER-killer-βGal, targeted
to β-galactosidase encoded by the lacZ reporter
gene. Hydrolysis of SPiDER-killer-βGal by β-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-βGal and light irradiation
specifically killed lacZ-positive cells in coculture
with cells without lacZ expression. Furthermore,
β-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.
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.
Summary
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 (O˙
-
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 H
2
O
2
scavenger in the cytosol, increases delamination and induces apoptotic nuclear fragmentation in caspase-3-activated cells. These findings suggest that the roles of O˙
-
2
and intracellular H
2
O
2
for delamination differs before and after caspase-3 activation, which involves live cell delamination.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.