Appropriate cell-cell signaling is crucial for proper tissue homeostasis. Protein sorting of cell surface receptors at the early endosome is important for both the delivery of the signal and the inactivation of the receptor, and its alteration can cause malignancies including cancer. In a genetic screen for suppressors of the pro-apoptotic gene hid in Drosophila, we identified two alleles of vps25, a component of the ESCRT machinery required for protein sorting at the early endosome. Paradoxically, although vps25 mosaics were identified as suppressors of hid-induced apoptosis, vps25 mutant cells die. However, we provide evidence that a non-autonomous increase of Diap1 protein levels, an inhibitor of apoptosis, accounts for the suppression of hid. Furthermore, before they die, vps25 mutant clones trigger non-autonomous proliferation through a failure to downregulate Notch signaling, which activates the mitogenic JAK/STAT pathway. Hid and JNK contribute to apoptosis of vps25 mutant cells. Inhibition of cell death in vps25 clones causes dramatic overgrowth phenotypes. In addition, Hippo signaling is increased in vps25 clones, and hippo mutants block apoptosis in vps25 clones. In summary, the phenotypic analysis of vps25 mutants highlights the importance of receptor downregulation by endosomal protein sorting for appropriate tissue homeostasis, and may serve as a model for human cancer.

Cell shape changes require the coordination of actin and microtubule cytoskeletons. The molecular mechanisms by which such coordination is achieved remain obscure, particularly in the context of epithelial cells within developing vertebrate embryos. We have identified a novel role for the actin-binding protein Shroom3 as a regulator of the microtubule cytoskeleton during epithelial morphogenesis. We show that Shroom3 is sufficient and also necessary to induce a redistribution of the microtubule regulator ␥-tubulin. Moreover, this change in ␥-tubulin distribution underlies the assembly of aligned arrays of microtubules that drive apicobasal cell elongation. Finally, experiments with the related protein, Shroom1, demonstrate that ␥-tubulin regulation is a conserved feature of this protein family. Together, the data demonstrate that Shroom family proteins govern epithelial cell behaviors by coordinating the assembly of both microtubule and actin cytoskeletons.

The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell‐culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c‐p and cyt c‐d. Loss of cyt c‐p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c‐d mutants are viable but male sterile. Here, we show that cyt c‐d regulates developmental apoptosis in the pupal eye. cyt c‐d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease‐activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c‐d—as with ark and dronc—regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.

Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitinconjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.

In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway. Subsequently, the CED-4-like genes Apaf-1 (apoptotic protease activating factor-1) in mammals and ARK (Apaf-1-related killer, also known as Dark, D-Apaf-1 and Hac-1) in Drosophila were discovered.3-6 CED-4-like proteins are characterized by the presence of a N-terminal caspase activation and recruitment domain (CARD), and a nucleotide oligomerization domain (NOD, also referred to as CED-4 homology domain and nucleotide-binding domain). Based on structural analysis of inactive, ADP-bound WD40-depleted Apaf-1, the NOD was divided into several distinct domains: a/b domain, helical domain I (HD1), winged-helix domain (WHD), and helical domain II (HD2).7 Apaf-1 and ARK also contain in their C-terminal half a series of WD40 repeats which are not found in CED-4 (reviewed in Cain et al. 8 ). WD40 repeats are short B40 amino-acid motifs, often terminating in a Trp-Asp (WD) dipeptide, and are thought to form a circularized b-propeller structure. WD40-repeat containing proteins coordinate multi-protein complex assemblies, where the repeating units serve as a rigid scaffold for protein interactions. 9Among the CED-4-like proteins, Apaf-1 is best characterized both biochemically and structurally. Apaf-1 functions to multimerize and activate Caspase-9, an initiator caspase that triggers apoptosis when activated. 8 The CARD of Apaf-1 is required for homotypic interaction with the CARD of Caspase-9.10 However, inactive Apaf-1 is a globular monomer that is kept in an auto-inhibited state by complex formation between the CARD and the WD40 repeats, 11 thus preventing CARD-CARD interactions. Hence, the WD40 repeats have an inhibitory function for Apaf-1 activity in the absence of apoptotic signals. Consistently, deletion of the WD40 repeats results in the activation of Apaf-1. 12,13 In response to apoptotic stimuli, cytochrome c is released from mitochondria and binds to the WD40 repeats, thus displacing the CARD from WD40 inhibition. This displacement allows binding of dATP/ATP to the NOD, which then promotes assem...