Apoptosis in Drosophila is governed by caspases, inhibitor of apoptosis proteins (IAPs), and IAP antagonists. Using AlphaFold3, we modeled full-length 3D structures of the IAP antagonists Reaper, Hid, Grim, Sickle, and Jafrac2, as well as DIAP1 and dBruce, and their binary and higher-order complexes. We predict a paradoxical role for the N-terminal methionine of Reaper in stabilizing Reaper/Hid complexes and inhibiting DIAP1 binding. Our models reveal that Reaper uniquely engages both BIR1 and BIR2 domains of DIAP1, guided by α-helical residues in its backbone, while all other IAP antagonists preferentially target BIR2. Higher-order assemblies show how Reaper and Hid as well as Reaper and Grim cooperatively engage DIAP1 and allosterically modulate its E3 ligase activity. We present the first full-length model of dBruce and its inhibitory interaction with Rpr. These findings provide a comprehensive structural framework for apoptosis regulation in Drosophila, and offer new insights into conserved mechanisms of caspase control and IAP antagonism across species.

Caspases, traditionally viewed as mediators of apoptosis and tumor suppressors, have also been shown to promote cell proliferation and to contribute to tumor growth. For example, the initiator caspase Dronc (the Drosophila orthologue of Caspase-9) can trigger apoptosis-induced proliferation (AiP), a process where apoptotic cells generate mitogenic signals for compensatory proliferation independently of their apoptotic function. AiP is crucial for homeostatic cell turnover, wound healing, and tissue regeneration. Previously, we established that Dronc activates the NADPH oxidase DUOX at the plasma membrane, resulting in the production of extracellular reactive oxygen species (ROS) which are required for AiP. However, the mechanism by which Dronc activates DUOX has remained elusive. Here, we identified Dronc-dependent Ca2+ entry into the cytosol as a significant factor for DUOX activation and AiP. Three cell surface Ca2+ channels of the TRP family mediate Ca2+ influx in a non-redundant fashion. Additionally, calcium-induced calcium release (CICR) from the ER was identified as another source of cytosolic Ca2+ during AiP. Notably, DUOX itself acts as a Ca2+ effector in AiP, requiring Ca2+ binding for its activation. These findings highlight the importance of Ca2+ signaling in AiP and provide insights into how similar signaling mechanisms might operate in vertebrates.

Immune cells play essential roles in maintaining tissue homeostasis and responding to abnormal growth, but how innate immune cells adapt to chronic apoptotic signaling remains poorly understood. In Drosophila melanogaster, hemocytes, particularly plasmatocytes, are recruited to tumor-like overgrowths, yet their transcriptional diversity and lineage dynamics under these conditions remain undefined. Here, we apply single-cell RNA sequencing to nearly 50,000 circulating and sessile hemocytes from larvae bearing undead overgrown eye discs, a model of regenerative overgrowth driven by sustained caspase activity. We resolve 17 transcriptionally distinct hemocyte clusters, including known lineages and 13 previously unrecognized plasmatocyte subtypes. Interestingly, specific plasmatocyte populations are differentially expanded or depleted under overgrowth conditions. Notably, we identify a matrix-remodeling plasmatocyte population marked by high expression of Jonah-family serine proteases. Pseudotime analysis reveals unexpected plasmatocyte plasticity and two novel terminally differentiated effector states. These findings define the immune landscape of tumor-like overgrowth and establish Drosophila as a platform for dissecting innate immune responses to tissue stress and dysregulated growth in vivo.

The Hippo pathway is classically viewed as a tumor suppressor that limits tissue growth by inhibiting the transcriptional co-activator Yorkie/YAP. Accordingly, Hippo activation is expected to suppress proliferation and can promote apoptosis. In this issue, Honda and colleagues challenge the prevailing unidirectional view of Hippo signaling as a purely tumor-suppressive pathway by showing that the outcome of Hippo activation is highly context-dependent in the Drosophila wing disc (Honda et al, 2026). In the wing pouch, Hippo activation leads primarily to growth suppression, as expected. In contrast, in the hinge and ventral notum, Hippo-activated cells survive and act as “oncogenic niche” cells that stimulate tumorigenic proliferation in neighboring cells. This tumor-promoting effect is mediated by non-apoptotic activity of the initiator caspase Dronc, induction of Wnt and EGF signaling, and metabolic support provided by the amino-acid transporters Sat1 and Sat2, which together drive mTOR activation in nearby cells. These findings reveal how growth suppression in one cell population can paradoxically drive tumor growth in another, reframing Hippo activity as a regulator of tumor-promoting microenvironments. A study in this issue challenges the prevailing unidirectional view of Hippo signaling as a purely tumor-suppressive pathway by showing that the outcome of Hippo activation is highly context-dependent in the Drosophila wing disc.

Cell death, compensatory proliferation, and cell competition are fundamental interconnected processes that shape how tissues develop, maintain homeostasis, and regenerate. In this review, I highlight how cell death (apoptosis) not only eliminates excess and damaged cells but can also initiate compensatory proliferation, an adaptive response that occurs following cell loss. I examine cell competition, a quality-control mechanism that removes less fit loser cells in favor of healthier winner neighbors. Cell competition is intricately linked to cell death and compensatory proliferation. I present the history of these processes, discuss the most important examples, and reveal the key molecular mechanisms that underlie them. I incorporate findings from Caenorhabditis elegans, Drosophila melanogaster, vertebrates, and other models to underscore the conservation of the key molecular signaling events. I also discuss how misregulation of these processes can contribute to pathological conditions, including cancer.

Caspases are best known for promoting apoptosis, yet their role in tissue regeneration by compensatory proliferation remains unclear. Using Drosophila wing discs and a delayed reporter for the initiator caspase-9 ortholog Dronc activity, we identify two apoptosis-resistant epithelial cell populations that mediate regeneration after ionizing radiation: Dronc-activating (DARE) and non-activating (NARE) cells. Dronc activity in DARE cells, independent of Dark and effector caspases, drives regeneration both cell-autonomously and non-cell-autonomously. The TNFR in DARE cells, Wengen, likely activated by ROS, strongly promotes DARE proliferation, while TNF/Eiger and TNFR Grindelwald moderately suppress it. Downstream, p38 MAPK is the main signaling essential for DARE and NARE cell proliferation. Myo1D ensures DARE survival by preventing lethal effector caspase activation, whereas Myo7A/Crinkled supports moderate caspase activity. Dying cells trigger DARE induction, and both DARE and NARE transmit apoptosis resistance to progeny, with DARE progeny showing enhanced resistance. Maintaining balanced DARE-NARE proliferation is crucial for proper regeneration, growth, and differentiation, insights that may be relevant to radiation-resistant cells in cancer therapy.

An enzyme known as caspase, which initiates apoptosis, has a central role in the regeneration of cells and repair of tissue that can occur after necrosis.

We conducted an EMS mutagenesis screen on chromosome arm 2L to identify recessive suppressors of GMR-hid-induced apoptosis in the Drosophila eye. Through this screen, we recovered three alleles of the lysine demethylase gene Kdm5. Kdm5, a member of the JmjC-domain-containing protein family, possesses histone demethylase activity towards H3K4me3. Our data suggest that Kdm5 specifically regulates Hid-induced cell death during development, as we did not observe control of Reaper- or Grim-induced cell death by Kdm5. Interestingly, GMR-hid-induced apoptosis is suppressed independently of Kdm5’s demethylase activity. Our findings indicate that Rbf and dMyc are necessary for Kdm5 mosaics to suppress GMR-hid-induced cell death. Moreover, Kdm5 mosaics failed to suppress apoptosis induced by a mutant form of Hid that is resistant to inhibition by Erk-type MAPK activity. Additionally, Kdm5 dominantly enhances the wing phenotype of an activated MAPK mutant. These results collectively suggest that Kdm5 controls Hid-induced apoptosis by regulating the Rbf, dMyc, and MAPK pathways.

Protein aggregation is a common pathological occurrence in neurodegenerative diseases. This often leads to neuroinflammation, which exacerbates the aggregation and progression of diseases like Parkinson’s and Alzheimer’s. Here, we focus on immune responses and neurotoxicity in a Parkinson’s disease model in Drosophila. Mutations in the SNCA gene that encodes the alpha (α)-Synuclein protein have been linked to familial Parkinson’s disease, disrupting autophagy regulation in neuronal cells and promoting the formation of Lewy bodies, a hallmark of Parkinson’s pathology. This results in the loss of dopaminergic neurons, manifesting as movement disorders. α-Synuclein aggregation triggers innate immune responses by activating microglial cells, leading to phagocytic activity and the expression of neuroprotective antimicrobial peptides (AMPs). However, sustained AMP expression or chronic inflammation resulting from inadequate microglial phagocytosis can induce neuronal toxicity and apoptosis, leading to severe dopaminergic neuron loss. This review underscores the mechanistic connection between immune response pathways and α-Synuclein-mediated neurodegeneration using Drosophila models. Furthermore, we extensively explore factors influencing neuroinflammation and key immune signaling pathways implicated in neurodegenerative diseases, particularly Parkinson’s disease. Given the limited success of traditional treatments, recent research has focused on therapies targeting inflammatory signaling pathways. Some of these approaches have shown promising results in animal models and clinical trials. We provide an overview of current therapeutic strategies showing potential in treating neurodegenerative diseases, offering new avenues for future research and treatment development.

Caspases, well-known for their role in executing apoptosis, also participate in various non-apoptotic processes. Despite this, their involvement in promoting compensatory proliferation - a key aspect of tissue regeneration following extensive cell death - has been a subject of ongoing ambiguity. In our study, we investigate compensatory proliferation in the Drosophila wing imaginal disc following ionizing radiation, a model epithelial tissue that has been a pioneering system for studying this regenerative response. Using a delayed genetic reporter to monitor the activity of the initiator caspase-2/9 ortholog, Dronc, we identified two populations of apoptosis-resistant epithelial cells involved in compensatory proliferation: those that activate Dronc (termed DARE cells) and those that do not (NARE cells). We show that DARE cells pass their apoptosis-resistance trait to their daughter cells, suggesting a molecular memory. We demonstrate that Dronc in DARE cells, but not the apoptosome adapter Dark and the effector caspases, promotes compensatory proliferation both within these cells and in NARE cells through a non-cell-autonomous mechanism. We found that Myo1D, an unconventional myosin interacting with Dronc, is essential for the survival of DARE cells by preventing the lethal activation of effector caspases and subsequent apoptosis. In contrast, Myo7A/Crinkled, another unconventional myosin that interacts with Dronc, promotes effector caspase activation in DARE cells. We demonstrate that the TNFR>JNK signaling pathway in DARE cells directly regulates their proliferation, which in turn influences NARE cell proliferation. Consequently, we show that maintaining proliferative homeostasis between DARE and NARE cells is vital for balanced tissue regeneration. Given the widespread use of ionizing irradiation in cancer treatment and prevention, our findings have potential implications for understanding treatment-resistant cells and cancer recurrence.

Since the discovery of caspases in 1993 (Yuan et al, 1993), they have largely been regarded as essential Cys proteases in apoptosis. However, more recently, it has become evident that caspases also have non-apoptotic functions in the so-called caspase-dependent non-lethal processes (CDPs) including cell proliferation and cell differentiation (Aram et al, 2017). While most of the non-apoptotic functions were characterized under normal developmental conditions, a new study by Galasso et al took this examination a step further-to the analysis of moderate stress conditions. The authors now report that caspases have a non-apoptotic prosurvival function in Drosophila ovarian somatic cells exposed to moderate heat stress by controlling Hedgehog signaling and autophagy (Galasso et al, 2023).

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. Here, we show that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, we define a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP.

Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although our knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. Here, we identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP.

Although there are over a dozen types of cell death known, there is clearly more to discover in this field. In this issue of PLOS Biology, erebosis is identified as a new type of cell death involved in tissue homeostasis of the adult Drosophila intestine.

The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc+ in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.