An in vivo model of apoptosis: linking cell behaviours and caspase substrates in embryos lacking DIAP1

Dhianjali Chandraratna 1 Nicola Lawrence 1 2 David P. Welchman 0 Bndicte Sanson 0 1 0 Department of Physiology, Development and Neuroscience, University of Cambridge , Anatomy Building, Downing Street, Cambridge CB2 3DY , UK 1 Department of Genetics, University of Cambridge , Downing Street, Cambridge CB2 3EH , UK 2 Present address: Gurdon Institute , Tennis Court Road, Cambridge CB2 1QN , UK Summary The apoptotic phenotype is characterised by dynamic changes in cell behaviours such as cell rounding and blebbing, followed by chromatin condensation and cell fragmentation. Whereas the biochemical pathways leading to caspase activation have been actively studied, much less is known about how caspase activity changes cell behaviours during apoptosis. Here, we address this question using early Drosophila melanogaster embryos ce lacking DIAP1. Reflecting its central role in the inhibition ne of apoptosis, loss of DIAP1 causes massive caspase ic activation. We generated DIAP1-depleted embryos by S either using homozygous null mutants for thread, the gene lle coding DIAP1, or by ectopically expressing in early C embryos the RGH protein Reaper, which inhibits DIAP1. fo We show that (1) all cells in embryos lacking DIAP1 follow l synchronously the stereotypic temporal sequence of a n behaviours described for apoptotic mammalian cells and ru (2) these cell behaviours specifically require caspase o J - Introduction Drosophila melanogaster provides an excellent model in which to investigate programmed cell death, for its ease of study and because of the high level of conservation of apoptotic mechanisms with mammals (Adrain et al., 2006; Cashio et al., 2005). Here we focus on analysing the apoptotic phenotype of embryos lacking DIAP1, one of the Drosophila inhibitor of apoptosis proteins (IAPs). IAPs were initially characterised in baculovirus as proteins, such as p35, that suppressed apoptosis in infected host cells. Since then IAPs have been found in Caenorhabditis elegans, Drosophila and vertebrates. IAPs contain baculovirus IAP repeat (BIR) domains, which can suppress apoptosis by directly binding caspases. Most IAPs also contain a RING domain, which provides E3 ubiquitin ligase activity (Stennicke et al., 2002; Vaux and Silke, 2005). In Drosophila, DIAP1 constitutes a central point of control during apoptosis. DIAP1 binds to and inhibits the apical caspase DRONC as well as the effector caspases Drice and Dcp-1 (Hawkins et al., 1999; Meier et al., 2000; Wang et al., 1999). There is evidence that binding of DIAP1 to caspases induces their ubiquitylation-dependent degradation (Chai et al., 2003; Wilson et al., 2002). Inhibition of the caspase cascade by DIAP1 is relieved by pro-apoptotic factors such as Reaper, Grim and Hid, which are clustered in region H99 of the genome. In embryos deficient for H99, developmental activity and are not merely a consequence of cellular stress. Next, we analyse the dynamic changes in the localisation of actomyosin, Discs large, Bazooka and DE-cadherin in the course of apoptosis. We show that early changes in Bazooka and Discs large correlate with early processing of these proteins by caspases. DE-cadherin and Myosin light chain do not appear to be cleaved, but their altered localisation can be explained by cleavage of known regulators. This illustrates how embryos lacking DIAP1 can be used to characterise apoptotic changes in the context of an embryo, thus providing an unprecedented in vivo model in which thousands of cells initiate apoptosis simultaneously. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/15/2594/DC1 apoptosis is completely abolished (White et al., 1994). The RGH (Reaper, Grim and Hid) proteins have been reported to induce cell death by a variety of mechanisms. First, they are able to bind to the second BIR domain of DIAP1 and thereby release the caspases bound to it (Goyal et al., 2000; Wang et al., 1999; Wu et al., 2001). Second, the RGH proteins stimulate DIAP1 protein degradation by regulating its ubiquitylation (Holley et al., 2002; Ryoo et al., 2002; Wilson et al., 2002; Yoo, 2005; Yoo et al., 2002). Third, the RGH proteins act as general translational repressors, and because DIAP1 has a shorter half-life than most caspases, this promotes cell death (Colon-Ramos et al., 2006; Holley et al., 2002; Yoo et al., 2002). In the absence of DIAP1, the caspase cascade is fully activated. For example, loss of thread, the gene coding for DIAP1, causes massive apoptosis in early Drosophila embryos (Wang et al., 1999). Caspase activation in the absence of DIAP1 requires Dark, the homologue of mammalian Apaf-1 (Quinn et al., 2000; Rodriguez et al., 1999). Dark and DRONC form an apoptosome (Yu et al., 2006), which in turn is thought to activate effector caspases such as Drice or Dcp-1. Although there have been spectacular advances in understanding the pathways that lead to caspase activation, how caspases accomplish the cellular changes of apoptosis is less well understood. Caspase activation promotes the proteolytic cleavage of many proteins (Fischer et al., 2003), and this is thought to cause the characteristic phenotypes displayed by the apoptotic cells, such as cell rounding and blebbing, chromatin condensation and cell fragmentation (Mills et al., 1999). Although the consequences of cleaving some individual proteins have been well characterised (e.g. Adrain et al., 2004; Brancolini et al., 1997; Coleman et al., 2001; Freeman, 1996; Lane et al., 2002; Lane et al., 2001; Lowe et al., 2004; Sebbagh et al., 2001; Slee et al., 2001), the majority of reported caspase cleavages have not been linked to a specific apoptotic phenotype. It seems likely that only some of the cleaved substrates are key to producing the apoptotic phenotype whereas others will be innocent bystanders (Martin, 2002). In addition, some cleavage events might be required for the early phenotypes of apoptosis (cell rounding and blebbing), whereas others might be responsible for the later phenotypes (such as chromatin condensation and cell fragmentation). Because it is difficult to obtain synchronous populations of mammalian cells entering the apoptotic program, it has been hard to investigate the timing in these systems (Mills et al., 1999). We have established an alternative method to address these questions using early Drosophila embryos lacking DIAP1 (i.e. embryos homozygous null for thread, or ectopically expressing Reaper). We confirm that the sequence of extranuclear changes described for mammalian cells (Mills et al., 1999) occurs in ce embryos lacking DIAP1. Crucially, all cells enter apoptosis n simultaneously, which enabled us to identify specific caspase e ic cleavages and relate them in vivo to the behaviour of several S components of the cytoskeleton and epithelial polarity. This lle establishes embryos lacking DIAP1 as a powerful in vivo C model to investigate the consequence of caspase cleavage in fo apoptoti (...truncated)