Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. An overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity is necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation is triggered by mitochondria from apoptotic cells but identical preparations from healthy cells are inactive. These observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases (Varkey, 1999; full text of article).
The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. This study demonstrates that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals (Dorstyn, 2002; full text of article).
In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Silencing expression of either or both DC3 and DC4 has no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. Loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 fail to activate caspases in Drosophila cell lysates, but, remarkably, induce caspase activation in extracts from human cells. Overall, these results argue that DARK-mediated DRONC activation occurs independently of cytochrome c (Dorstyn, 2004; full text of article).
The data clearly show that neither of the two cytochrome c species in Drosophila are required for caspase activation or apoptosis. Previous studies reported that a P-element insertion in the dc3 gene (bln1) results in loss of DRICE activity in testis (Arama, 2003). However, a recent report indicates that the bln1 P-element insertion also disrupts a number of other genes (Huh, 2004), thus questioning whether DC3 is responsible for DRICE activity. Additionally, DRICE activation during spermatogenesis appears to be independent of DARK and DRONC (Huh, 2004). If DC3 is required for caspase activation in Drosophila, a loss of function mutation in dc3 should lead to severe developmental defects and lethality. Furthermore, although a tissue-specific function has been suggested for DC3, it is unlikely that DC3 functions only during spermatogenesis, given its ubiquitous expression. Although disruption of the dc4 gene is embryonic lethal, DC4 cannot induce caspase activation and apoptosis in Drosophila cells (Dorstyn, 2004).
The question remains: how does DARK mediates DRONC activation? One possibility is that other factors can substitute for cytochrome c function during apoptosis. Alternatively, removal of DIAP1 from DRONC may be sufficient to allow an interaction with DARK and activation. Given that transcription plays a major role in developmental PCD in Drosophila, changes in the concentration of DIAP1, DRONC, and DARK proteins could facilitate caspase activation in the fly. These studies, combined with published work, demonstrate that Drosophila and mammalian cytochrome c proteins are functionally similar since they can both mediate respiration and Apaf-1 activation in mammalian cell lysates. Therefore, the requirement for cytochrome c in caspase activation in mammals is likely to have evolved late in evolution (Dorstyn, 2004).
Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, it has been demonstrated that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases are inhibited, induce caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract did not activate caspases, nor did it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduced caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid is sufficient to activate caspases in cytosolic extract, and this activity is not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also inducew apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors (Means, 2005; full text of article).
In vertebrates, mitochondria play an important role in the control of apoptosis by activating the apoptosome, a multiprotein complex that includes caspase-9, Apaf-1, and cytochrome C. Drosophila possesses one Apaf-1 orthologue known either as Hac-1, Dark, or Dapaf-1 which, like its mammalian counterpart, is important in multiple apoptotic pathways. In addition, Drosophila also has a caspase-9 orthologue, Dronc, which, similar to the vertebrate caspase-9, contains a caspase recruitment domain (CARD), and functions in a variety of cell death pathways. Whether Ark and Dronc are required for spermatid individualization was examined. For this purpose, several EMS-derived loss-of-function alleles of both ark and dronc were examined. ark and dronc mutant flies display highly similar phenotypes and most mutant animals die during pupariation. However, some adult 'escapers' emerge that are both male and female sterile. Both ark and dronc mutants displayed severe defects during the spermatid individualization process. In particular, ark and dronc mutant spermatids failed to extrude much of their cytoplasm into a CB, leaving trails of the cytoplasm in what should have been the postindividualized region of the spermatids. Consequently, ark-/- and dronc-/- CBs and WBs are highly reduced in size or appear flat, and frequently a large portion of the spermatids' cytoplasm is retained behind in a 'mini' CB structure (white arrowhead in, which often contains part of the IC. The size of the CBs and WBs in ark and dronc mutants is on average only half the size of their wild-type counterparts. These phenotypes are reminiscent of testes that ectopically express the caspase inhibitor gene p35 (Arama, 2003). These results suggest that ark and dronc are required for normal caspase activation and the initiation of an apoptosis-like process essential for spermatid individualization. However, whereas no caspase-3-like activity was detected in cyt-c-d-/- mutants, some activation of caspase-3 was detected in ark and dronc mutant testes. This suggests that some of the cytochrome-C-mediated caspase-3 activation is independent of apoptosome components. Alternatively, it is possible that the ark and dronc alleles used in this study are not complete nulls and therefore retain some residual function that allows a small amount of cytochrome C-induced caspase activation (Arama, 2006).
The 14-3-3 proteins are highly conserved molecules that function as intracellular adaptors in a variety of biological processes, such as signal transduction, cell cycle control, and apoptosis. This study shows that a 14-3-3 protein is a heat-shock protein (Hsp) that protects cells against physiological stress as its new cellular function. In Drosophila cells, the 14-3-3zeta is up-regulated under heat stress conditions, a process mediated by a heat shock transcription factor. As the biological action linked to heat stress, 14-3-3zeta interacted with apocytochrome c, a mitochondrial precursor protein of cytochrome c, in heat-treated cells, and the suppression of 14-3-3zeta expression by RNA interference resulted in the formation of significant amounts of aggregated apocytochrome c in the cytosol. The aggregated apocytochrome c was converted to a soluble form by the addition of 14-3-3zeta protein and ATP in vitro. 14-3-3zeta also resolubilized heat-aggregated citrate synthase and facilitated its reactivation in cooperation with Hsp70/Hsp40 in vitro. These observations provide the first direct evidence that a 14-3-3 protein functions as a stress-induced molecular chaperone that dissolves and renaturalizes thermal-aggregated proteins (Yano, 2006; full text of article).
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