Independent roles of autophagy and apoptosis in post-apocrine-secretion cell death of salivary glands during Drosophila metamorphosis

The Drosophila larval salivary glands (SGs) serve as an outstanding model to understand developmental transitions in secretory epithelial cells. They have been instrumental in understanding the regulatory mechanisms that lead to the exocytosis of SG glue (Sgs) just prior to pupariation during the late larval period, and gaining insights into apocrine secretion (AS), a non-canonical, non-vesicular transport and secretion, which occurs later in the late prepupal period to provide the immunoprotective exuvial fluid that surrounds the pupae during its metamorphosis into an adult fly. AS is the SGs major, evolutionarily conserved secretory function, as unlike Sgs exocytosis, AS is found across the Diptera. Since the SGs undergo hormonally controlled programmed cell death (PCD) shortly after they complete AS, a key question is how the regulation of AS and PCD are related. To address this, it is essential to clarify the mechanism of PCD. Earlier proposals that it occurs via autophagic cell death (ACD) or autophagy dependent cell death (ADCD) did not incorporate or explain any of the dynamic post-Sgs cellular processes that are associated with AS. Here we present three lines of evidence that the SGs die using an apoptosis-prone mechanism during which the endoplasmic reticulum (ER) becomes fragmented into vesicles that wholly fill the cytoplasm. First, although a low level of autophagy can be seen in the SGs, inducing autophagy with rapamycin does not accelerate PCD. Second, while the genetic removal of Atg1 or RNAi silencing of the Atg3, Atg5, Atg6, Atg7, and Atg12 genes inhibits the development of autophagy, this does not prevent vesiculation and subsequent SG-cell death. Third, the Drosophila SGs exhibit externalization of phosphatidylserine and attract phagocytic macrophages, both of which are pivotal hallmarks of apoptosis. Therefore, we propose that the entire Drosophila SG organ dies using a non-autophagic mechanism which utilizes key features of the apoptotic pathway.