Apoptosis is a mechanism that removes abnormal or no longer needed cells. Caspases, a class of cysteine protease, are key element of apoptosis, and they cleave various polypeptides resulting in cellular structural changes. Despite the importance of caspases, the mechanisms regulating caspases and proteins cleaved by caspases remains to be elucidated. Moreover, apoptotic subprogrammes, such as cell shrinking and the emission of pro-engulfment signals are not clearly understood yet.
To find caspase substrates and elucidate genetic pathways related to caspases, we generated a transgenic fly overexpressing Drosophila caspase, dcp-1, in its eye using GAL4/UAS system. Flies carrying one copy of UAS-dcp-1 and gmr-GAL4 exhibited a distinct phenotype in eye, in which the level of pigment was reduced. Transgenic flies carrying another Drosophila caspases, dronc and dredd, were also generated. Flies carrying one copy of UAS-dronc and gmr-GAL4 exhibited ablated eye. And flies carrying one copy of UAS-dredd and gmr-GAL4 exhibit rough eye.
Since RPR and HID have been known as proapoptotic proteins in fly, we crossed UAS-dcp-1 flies to gmr-rpr, gmr-hid flies. Flies coexpressing dcp-1 and rpr in their eye was died as pupa, while flies coexpressing dcp-1 and hid in their eye exhibited almost same phenotype compared to the eye of gmr-hid flies. We also crossed gmr-dcp-1 flies to gmr-p35 flies since p35 has been known as a specific inhibitor of caspases. The eye phenotype induced by dcp-1 was suppressed by crossing these flies to gmr-p35 flies. These results imply that DCP-1 genetically interacts with RPR and p35 but not with HID.
To perform genetic screening, we generated gmr-dcp-1 flies, which gmr-GAL4 and UAS-dcp-1 is in the same second chromosome. Because two copies of gmr-dcp-1 caused a reduced fertility, male flies carrying one copy of gmr-dcp-1 were crossed with females from EP library or female flies containing various signal transduction genes. We scree...