Rescue results showing that the two cytochrome genes can functionally rescue each other, raise the question of why cyt-c-d-/- males are sterile if both cytochrome c genes are functionally equivalent. One possible explanation is distinct expression of the two genes, namely that cyt-c-d is testis-specific, whereas cyt-c-p may be restricted to the soma. To examine this possibility, the distribution of transcripts from both cytochrome c genes were examined in the testis and the soma. For this purpose, comparative RT–PCR experiments were performed using specific primers in the unique 5' and 3' UTR sequences of cyt-c-d and cyt-c-p. While cyt-c-p was highly expressed in the soma, cyt-c-d is only weakly expressed there (represented by adult females that lack testes). In contrast, cyt-c-d expression was much higher in testes than cyt-c-p. The low levels of cyt-c-p in testes is attributed to the somatic cells present in this tissue. Furthermore, although the expression of cyt-c-d in the soma of both males and females is much lower than the levels of cyt-c-p, cyt-c-d levels are much higher in adult males than in females, suggesting that the male germ cells provide the main contribution of cyt-c-d in the adult. These results suggest that the distinct phenotypes of cyt-c-d and cyt-c-p are mainly due to their restricted differential expression in the testis and the soma, respectively (Arama, 2006).
In addition to germ cells, the testis also contains somatic cells, such as the testicular wall, muscles cells, and cyst cells. To determine which testicular cell types express cyt-c-d, comparative RT–PCR analyses were first performed with RNA from reproductive tracts of oskar male mutants that are defective in germline development and lack germ cells in the adults. While both cytochrome c genes were expressed in wild type, only cyt-c-p was detected in the germ-cell-less reproductive tracts of sons of oskar-/-. This indicates that cyt-c-d expression is restricted to the germ cells of the adult male. Next, the developmental stage at which cyt-c-d is expressed in the male germ line was examined. For this purpose, advantage was taken of the fact that testes of adult flies and third instar larvae differ in their repertoire of germ cells. While adult testes contain germ cells in a variety of developmental stages, the most developmentally advanced germ cells present in third instar larval testes are premeiotic spermatocytes. Interestingly, the patterns of cyt-c-d expression in both adult and larval testes are identical, demonstrating that cyt-c-d mRNA accumulates before the entry of spermatocytes into meiosis (Arama, 2006).
The activation of apoptotic effector caspases, as visualized by CM1-staining, is not restricted to the male germ cells but can also be detected in nurse cells during oogenesis. The possibility is considered that caspase activation in this system is also influenced by cyt-c-d. However, no abnormalities during oogenesis were detected in cyt-c-d-/- flies and the females are fertile. Consistent with this idea, comparative RT–PCR analysis of adult ovaries revealed expression of cyt-c-p but not cyt-c-d (Arama, 2006).
To study the pattern of cytochrome C-d expression in the testis, polyclonal antibodies were raised against four peptides covering the entire length of the protein. Consistent with the findings that no cyt-c-d RNA is expressed in cyt-c-dbln1 homozygote flies, almost no signal was detected after staining testes of this mutant with the anti-cytochrome C-d antibody. Staining wild-type testes with this antibody revealed a grainy pattern of cytochrome C-d signal along the entire length of elongating spermatids and elongated spermatids. Once an individualization complex (IC) was assembled in the vicinity of the nuclei, an increase in cytochrome C-d staining was detected with the highest intensity found next to the IC. During the caudal translocation of the IC, a significant portion of cytochrome C-d is depleted from the newly individualized part of the spermatids into the CB. Eventually, the newly formed WBs accumulate high levels of cytochrome C-d (Arama, 2006).
To test whether this antibody could also crossreact with cytochrome C-p, testes of cyt-c-d mutant lines were tested that were rescued by transgenic cyt-c-p expression in germ cells. Similar to the cytochrome C-d expression in wild type or after ectopic expression in mutant testes, ectopic cytochrome-C-p expression was also detectable as grainy staining along the entire length of elongated spermatids as well as in CBs and WBs. These results demonstrate that the antibody can detect both forms of the Drosophila cytochrome C molecules. The lack of staining found in cyt-c-dbln1 elongating spermatids is consistent with the idea that only cyt-c-d and not cyt-c-p is expressed in mature spermatids (Arama, 2006).
Effector caspases, such as drICE, can display Caspase-3-like (DEVDase) cleaving activity. Therefore, it was asked whether wild-type adult testes also contain DEVDase activity, and whether this activity is affected in cyt-c-d mutant testes. Lysates of wild-type testes indeed display detectable levels of DEVDase activity, which were significantly reduced upon treatment with the potent DEVDase inhibitor Z-VAD.fmk. Importantly, this activity was highly reduced in cyt-c-dZ2-1091 mutant testes. These results provide independent evidence for effector caspase activity in wild-type sperm, and they support a role of cytochrome C-d in caspase activation in this system (Arama, 2006).
Because of the cytological proximity between cyt-c-d and cyt-c-p (241 bp maximum between the end of the 3' UTR of cyt-c-d and the beginning of the 5' UTR of cyt-c-p) there is a possibility that the bln1 P-element insertion in cyt-c-d might also interfere with the expression of cyt-c-p (Huh, 2004). In order to determine whether cyt-c-p expression was altered in cyt-c-dbln1, RT–PCR analyses was carried out with RNA from wild-type (yw) and cyt-c-dbln1 adult flies using two sets of primers for each gene specific for either the 5' UTRs of cyt-c-d and cyt-c-p. In agreement with Northern results, no cyt-c-d RNA was detected in cyt-c-dbln1 flies, confirming that bln1 is a null allele of cyt-c-d. In contrast, cyt-c-p is expressed in both wild-type and cyt-c-dbln1 flies (Arama, 2006).
Although the sequence of cyt-c-d and cyt-c-p proteins is highly conserved, they are not identical. In addition, mutations in each gene display distinct phenotypes (Arama, 2003). This raises the possibility that both proteins may have distinct functions in respiration (cytochrome C-p) and caspase activation/apoptosis (cytochrome C-d). To test this hypothesis, it was first asked whether expression of cyt-c-p in developing spermatids is able to substitute for the loss of cyt-c-d. In order to drive expression of transgenes in the male germ line, an expression vector was constructed composed of the hsp83 promoter followed by the 5' and 3' UTRs of cyt-c-d, which are important for the proper temporal regulation of cyt-c-d translation in spermatids, and next the coding regions of either cyt-c-d or cyt-c-p were inserted between both UTRs and transgenic flies were generated with these constructs. At least three independent transgenic lines for each of these constructs were crossed to cyt-c-dbln1 or cyt-c-dZ2-1091 flies, and the presence of the appropriate transgene was confirmed by genomic PCR. To validate expression of the transgenes, RT–PCR analysis was inserted with testes RNA in a cyt-c-dbln1-/- background. Finally, the ability of these transgenes to rescue caspase activation, spermatid individualization, and male sterility was examined in cyt-c-dbln1 and cyt-c-dZ2-1091 flies. As a control, transgenic flies containing 'empty vector' (including the hsp83-promotor with the 5' and 3' UTRs of cyt-c-d but without a coding region) were also generated. As expected, no caspase activation was detected in testes of these control flies. In contrast, a transgene with the cyt-c-d open reading frame (ORF) fully rescued CM1-staining, spermatid individualization, and male fertility. This firmly establishes that both the caspase and sterility phenotypes seen in cyt-c-dbln1 and cyt-c-dZ2-1091 mutant flies are strictly due to the loss of cytochrome c function, with no detectable contribution from adjacent genes (Arama, 2006).
The ability of cyt-c-p to functionally substitute for the loss of cyt-c-d was tested. Surprisingly, transgenic expression of cyt-c-p was equally effective in rescuing all defects in cyt-c-dbln1 or and cyt-c-dZ2-1091 males. It is concluded that both proteins have similar biochemical properties to promote caspase activation and spermatid individualization (Arama, 2006).
l(2)k13905 flies contain a P-element insertion in the 5' UTR of cyt-c-p and die as late embryos or early first instar larva (Arama, 2003). Using RT–PCR, it was found that only cyt-c-p expression was detected in early first instar wild-type larvae, while a dramatic reduction was observed in the cyt-c-pk13905 mutants. These results are consistent with the phenotypes of cyt-c-d (viable but male sterile) and cyt-c-p (early lethal) mutants (Arama, 2006).
Lethality of cyt-c-pk13905 homozygotes as well as trans-heterozygotes to Df(2L)Exel6039, a deletion in the region that includes both cyt-c-p and cyt-c-d, is consistent with the idea that cyt-c-p encodes the major cytochrome C responsible for respiration. Whether cyt-c-d could also function in respiration and rescue the early lethality of cyt-c-p-/- flies was examined. Both cytochrome C proteins were ectopically expressed in cyt-c-pk13905 mutants using the GAL4-UAS system. The Tub-Gal4 driver line was used to drive cyt-c-p and cyt-c-d expression throughout the lifespan of the fly. Notably, one copy of either the UAS-cyt-c-p or the UAS-cyt-c-d transgenes together with one copy of the driver completely rescued the lethality of cyt-c-pk13905/Df(2L)Exel6039 flies. It is concluded that both cytochrome C proteins of Drosophila can function in electron transfer/respiration. The complete absence of cyt-c-p from the rescued adult flies is consistent with the idea that cyt-c-pk13905 is a null allele of cyt-c-p. The faint expression of cyt-c-p in cyt-c-pk13905 homozygote and cyt-c-pk13905/Df(2L)Exel6039 trans-heterozygote mutants detected in early first instar larvae only after 30 PCR cycles is attributed to remnants of maternal contribution. This also explains how cyt-c-p-/- mutant embryos can reach the early first instar larval stage without any zygotic contribution. Finally, it was not possible to rescue the lethality of flies homozygous for the cyt-c-pk13905 allele, suggesting that the k13905 chromosome carries an additional unrelated lethal mutation (Arama, 2006).
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. This study shows 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, it was found that cyt c-d—as with ark and dronc—regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, these results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells (Mendes, 2006).
In response to apoptotic stimuli, mammalian cells release cytochrome c (Cyt c) from the mitochondria into the cytoplasm where it binds to apoptotic protease-activating factor 1 (Apaf 1). This leads to the recruitment of the zymogen form of caspase 9 to a catalytically active multi-protein complex called the apoptosome (Jiang, 2004). Once activated, the apoptosome, consisting of Cyt c, dATP, Apaf 1 and caspase 9, can cleave and activate downstream caspases, including caspase 3. Genetically modified mice have demonstrated the in vivo importance of several components of this pathway, including Apaf 1, caspase 9, caspase 3 and Cyt c. However, these studies have also shown a surprising degree of complexity and raised questions about how apoptosis is activated in the absence of canonical apoptosome components (Hao, 2005; Mendes, 2006 and references therein).
In Drosophila, the mechanisms leading to the activation of the Apaf 1 homologue are controversial (Kornbluth, 2005). Similar to its mammalian homologue, Drosophila Ark (also called Hac 1/Dapaf 1/Dark) contains a series of WD40 repeats, which, in vitro, can bind Drosophila Cyt c and form an apoptosome-like complex and induce caspase activation. RNA interference knock-down experiments (Zimmermann, 2002; Dorstyn, 2004), however, failed to support a role for cyt c in the apoptosis of S2 culture cells (Mendes, 2006).
The Drosophila genome contains two closely related but distinct cyt c genes: cyt c-d and cyt c-p (Limbach, 1985). cyt c-p is involved in mitochondrial respiration and viability, whereas cyt c-d is required for caspase activation and sperm differentiation (Arama, 2003; Arama, 2006). In the sperm, caspase activation does not lead to cell death, but to sperm maturation. This study reports on the role of cyt c-d in apoptosis during normal development of the Drosophila retina (Mendes, 2006).
In the developing eye, superfluous interommatidial cells (IOCs) and perimeter ommatidial cells (POCs) are eliminated by apoptosis, allowing the precise rearrangement of ommatidia into a honeycomb-like formation. Antibodies raised against the membrane-bound protein Armadillo (Arm) allow the visualization of each cell in the eye lattice. By 42 h after puparium formation (APF), a fixed number of IOCs form an hexagonal array around each photoreceptor cell cluster, comprising four cone cells, three bristle cells, two primary, six secondary and three tertiary pigment cells (Mendes, 2006 and references therein).
To examine the role of the cyt c locus in cell death during pupal eye development, the number of IOCs were compared between wild-type and several cyt c-d male-sterile and viable, loss-of-function alleles at a stage in which cyt c-d is expressed. This analysis focused on the cyt c-dZ2-1091 allele, since it bears a point mutation that creates a stop codon in the cyt c-d coding region, which does not affect neighbouring open reading frames. At 42 h APF, a time when IOC death is normally complete, cyt c-dZ2-1091-/- mutant retinas showed extra cells in the secondary or tertiary position. A more pronounced phenotype was observed in cyt c-dZ2-1091/Df(2L)H20 retinas, indicating that another neighbouring gene included in Df(2L)H20 contributes to the regulation of IOC death, or that cyt c-dZ2-1091 might be a hypomorphic allele. In addition, the extra IOC phenotype observed in cyt c-dZ2-1091-/- was rescued by the ectopic expression of cyt c-d in the developing retina. Mutant retinas for the other cyt c-d alleles also showed extra IOCs (Mendes, 2006).
To further characterize the role of cyt c-d, the number of extra IOCs at different stages of pupal development were counted in the mutant retina: at 22 h APF, cyt c-dZ2-1091-/- retinas already showed extra IOCs; at 48 h APF, they still occasionally showed extra IOCs compared with wild type. The decrease in the number of extra IOCs between 22 and 48 h APF indicates that IOC death is delayed and not completely suppressed in the cyt c-d mutant. Considering that the IOC death process terminates at 36 h APF, it was estimated that IOC apoptosis in cyt c-dZ2-1091-/- retinas can be delayed up to 12 h. It is proposed that the cyt c-d gene is required for the 'on-time' apoptosis of IOCs during pupal development (Mendes, 2006).
The results show that cyt c-d regulates IOC apoptosis in pupal retinas. It was then asked whether cyt c-d also regulates POC apoptosis. Ommatidia at the edge of the eye (perimeter ommatidia) contain photoreceptor, cone and pigment cells that die by apoptosis. Between 36 and 44 h APF, 80–100 ommatidia are eliminated, allowing the formation of a normal eye edge. POCs were visualized using an anti-Arm antibody in staged cyt c-d-/- and wild-type retinas. By 38 h APF, POC elimination has just begun, with numerous small ommatidial clusters along the edge of the retinas. By 40 h APF, many wild-type POCs have been eliminated. By contrast, cyt c-dZ2-1091-/- retinal edges showed more clusters of malformed ommatidia in a thick layer of IOCs. The same phenotype was also visible in all the other cyt c-d alleles. By 54 h APF, POC elimination is complete both in wild-type and mutant retinas. These results indicate that cyt c-d promotes not only IOC elimination but also POC death (Mendes, 2006).
The detection of cyt c-d expression is challenging, since none of the available antibodies allows distinguishing between the two cyt c species or visualize the release of Cyt c during apoptosis of ommatidial cells. The analysis of cyt c RNA transcripts showed that cyt c-p is the prevailing form expressed throughout development and adulthood. The cyt c-d transcript, however, seems to be mainly restricted to the testis (Arama, 2006). Both cyt c transcripts are present at the time of IOC elimination in the retina. This supports the possibility that the two Cyt c proteins can function in the elimination of superfluous retinal cells during pupation. The fact that physiological amounts of cyt c-p cannot substitute for the loss of cyt c-d suggests that the full apoptogenic function of cyt c requires the expression of both cyt c genes (Mendes, 2006).
Elimination of both cyt c genes in the retina might lead to a more pronounced phenotype than cyt c-d mutation alone. Unfortunately, this hypothesis is extremely difficult to test, given the general requirement of cyt c-p for cell survival. In addition, the possibility that the loss of both cyt c genes would lead to a phenotype as pronounced as the complete inhibition of death observed in retina expressing p35is not favored because in cyt c-dZ2-1091/Df(2L)H20 flies, in which cyt c-d is lost and only one copy of cyt c-p is functional, IOC death is delayed to a level comparable with that in cyt c-d mutants (Mendes, 2006).
Apoptosis is delayed in the cyt c-d-/- retina. This could be due to a direct role of cyt c-d in the apoptotic process or an indirect consequence of an impaired respiratory function in the mutant retina. To address the latter possibility, ATP levels were measured in several wild-type strains and cyt c-d mutants. No significant difference was found between wild-type and cyt c-d mutants, ruling out an effect in the bioenergetics levels as the cause of extra cells in cyt c-d-/- retina. To eliminate any consequence in retinal development, cyt c-dZ2-1091-/- larval and pupal eyes were stained with antibodies against several specific differentiation markers. cyt c-dZ2-1091-/- larval eye discs stained against Elav (neuronal marker), Boss (R8-specific marker) and Spalt-major (R3, R4, R7, R8 and cone cell marker) appeared as in the wild-type control. Moreover, tangential plastic sections of cyt c-dZ2-1091-/- adult eyes presented the normal number and arrangement of photoreceptor cells (Mendes, 2006).
Other retinal cell types, including primary pigment, cone and bristle cells, visualized at pupal stages in cyt c-d mutant, appeared normal in shape and number. cyt c-dZ2-1091-/- retinas were stained at different stages of pupal development (24, 27, 30 and 42 h APF) with an anti-Homothorax (Hth) antibody, which stains secondary and tertiary pigment cell nuclei. All secondary and tertiary cells expressed hth, suggesting that the extra IOCs differentiate normally. Thus, the only phenotype associated with cyt c-d mutations is the appearance of extra secondary and tertiary cells in the eye lattice, with no disruption of early retinal development. For this reason, the cyt c-d mutation can be classified as lattice-specific. To determine whether cyt c-d is required for development progression, the dynamic IOC rearrangement and maturation was measured in staged cyt c-dZ2-1091-/- and wild-type retinas. Despite the presence of extra IOCs in cyt c-dZ2-1091-/-, the process of cell sorting and IOC maturation occurs similarly to wild-type retinas (20–27 h APF). Thus, the dynamic rearrangement and maturation of IOCs are not delayed in cyt c-dZ2-1091-/- retinas, eliminating any significant effect of cyt c-d mutations on the progression of retinal cell differentiation (Mendes, 2006).
Together, these results demonstrate that cyt c-d is not required for respiration, differentiation or developmental progression in the pupal eye, providing the first genetic evidence for a physiological role of Drosophila cyt c in the regulation of developmental apoptosis (Mendes, 2006).
cyt c-d is required for apoptosis progression during pupal eye development in Drosophila. It was asked whether the other homologues of the apoptosome components, Ark and Dronc, are also required for apoptosis in this model. ark and dronc loss-of-function mutant alleles were used; both ark and dronc alleles are strong loss-of-function or null alleles leading to apoptosis defects at early stages in development and lethality (Mendes, 2006).
Using the flipase (FLP)/FLP recombinase target (FRT) technique, ark and dronc mutant clones were generated in the eye. In these clones, visualized by the absence of green fluorescent protein (GFP), an excess was counted of 5.20 and 4.36 IOCs/ommatidium, respectively. These values are comparable with those observed in pupal retinas in which the caspase inhibitor, p35, is ectopically expressed under control of the ubiquitous eye promoter, GMR, showing 5.06 extra IOCs/ommatidium. These values are higher than the total estimated number of IOCs that are dying between 18 and 36 h APF (about 3.5 IOCs/ommatidium). This is probably due to the fact that, in those mutant situations, unwanted IOCs are also rescued during larval development and early pupal development (<18 h APF) (Mendes, 2006).
The number of extra IOCs obtained in dronc mutant clones is similar to the value observed in the retinas of dronc mutant escapers. In addition, clonal analysis showed that only the mutant tissue for ark or dronc, exhibit extra IOCs, not the surrounding non-mutant tissue. This indicates that ark and dronc are required cell-autonomously for IOC apoptosis. In addition, it was found that the combination of cyt c-d mutations and the expression of Dronc dominant negative in the retina induces synergistic reduction of IOC death, suggesting the proximity of these genes in the same pathway (Mendes, 2006).
The role of ark and dronc in POC apoptosis was examined. In ark and dronc mutant clones, POCs are rescued and TUNEL is blocked. Moreover, the mutant retinas present extra POCs that are never eliminated, as seen in GMR-p35. Thus, Dronc has a pivotal role as an initiator caspase in the pupal retina—which differs from embryonic tissues—in which Dronc is required for most, but not all, cell death (Mendes, 2006).
To rule out the possibility that developmental defects in ark or dronc mutant retinas indirectly affect cell death, retinal cell differentiation was examined in ark and dronc mutant clones using several larval and pupal eye differentiation markers. It was found that retinal cell differentiation is normal in ark and dronc mutant clones (Mendes, 2006).
Together, these results demonstrate that ark and dronc are required for the initiation and/or execution of IOC and POC apoptosis, placing these genes hierarchically at the top of the apoptotic cascade during pupal eye development (Mendes, 2006).
To further explore the role of cyt c-d in the regulation of caspase activation, the elimination of sensory organs (macrochaetes) was used as a model. A recent study proposed that caspase activation does not lead to apoptosis but inhibits the Wingless pathway to ensure the correct number of sensory organ precursors (SOPs). Consistently, loss-of-function mutations in ark or dronc lead to the appearance of extra bristles on the Drosophila notum. To determine the role of cyt c-d during SOP development, the number of posterior scutellar bristles on the thorax of cyt c-d mutant flies was counted. In all the cyt c-d mutant alleles examined, a significant number of flies was found that had one extra bristle. Using a recently characterized allele of ark (arkN5), an extra bristle cell phenotype was observed. As for the extra IOCs, ark has a more pronounced phenotype than cyt c-d mutants, suggesting that similar mechanisms lead to caspase activation in the two models. Together, these results provide further support that cyt c-d promotes caspase activation required for accurate developmental progression (Mendes, 2006).
Extra bristles were identified in flies mutant for the executioner caspase dcp 1 (dcp-1prev1), for which no role in the regulation of bristle cell number has yet been reported. Interestingly, no extra bristle phenotype was found in drICE mutant (drICE17), suggesting that dcp 1 could be the main executioner caspase in this model (Mendes, 2006).
In most tissues, with the exception of developing testis, SOP and retina, cyt c-d has no apparent role in caspase activation or apoptosis, suggesting that apoptosis can occur in the absence of this protein. The existence of a Cyt-c-independent pathway for apoptosis in Drosophila was previously proposed on the basis of RNA interference studies in Drosophila cell lines or a cell-free system, showing that apoptosis can occur independent of Cyt c function but requires Ark (Zimmermann, 2002; Dorstyn, 2004; Means, 2005). Therefore, at least in some models, Ark-dependent caspase activation might be either constitutive or regulated by other pathways. In support of the latter, ectopic expression of Ark is not sufficient to trigger apoptosis in vivo, suggesting that Ark must be activated to function (Akdemir, 2006). Likewise, analysis of mice devoid of Cyt c apoptogenic function (K72A) indicates that caspase activation in thymocytes can occur independently of Cyt c (Hao, 2005). Mammalian Apaf 1 might either have some constitutive activity or might be regulated by factors other than Cyt c (Mendes, 2006).
Conversely, the results indicate that a Cyt-c-dependent mechanism for apoptosis in the retina might be necessary for the rapid removal of a precise number of cells during development. An even stricter requirement is observed during sperm or SOP development, in which imbalanced caspase activation or loss of cyt c-d function leads to male sterility and extra bristle cells, respectively (Arama, 2006). The results indicate that Cyt c is able to promote the activation of Ark to form an apoptosome that leads to Dronc activation and cell death. In support of this hypothesis, Dronc is recruited into a >700 kDa complex in Drosophila cell extracts supplemented with Cyt c and dATP (Dorstyn, 2002), similar to the mammalian apoptosome. In addition, Ark interacts with Cyt c, an interaction dependent on the WD40 domain of Ark. However, recent structural data suggest that Ark does not require Cyt c to form an apoptosome-like structure (Yu, 2006). Although that study used horse and not Drosophila Cyt c for the apoptosome assembly, Drosophila apoptosome formation might not require Cyt c. If so, it raises a question on the inhibitory function of the WD40 domain of Ark. The WD40 is conserved between vertebrates and Drosophila but not Caenorhabditis elegans, in which it is thought to maintain Apaf 1 in an inactive conformation that is relieved on Cyt c binding. How Ark activation in vivo is dependent on Cyt c awaits further analysis (Mendes, 2006).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Cytochrome
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Hao, Z., et al. (2005). Specific ablation of the apoptotic functions of cytochrome C reveals a differential requirement for cytochrome C and Apaf-1 in apoptosis. Cell 121: 579-591. PubMed citation: 15907471
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Huh, J. R., et al. (2004). Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization. PLoS Biol 2: E15
Inoue, S., Inoue, H., Hiroyoshi, T., Matsubara, H. and Yamanaka T (1986). Developmental variation and amino acid sequences of cytochromes c of the fruit fly Drosophila melanogaster and the flesh fly Boettcherisca peregrina. J. Biochem. (Tokyo) 100: 955-965. Medline abstract: 3029051
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Kanuka, H., et al. (1999). Control of the cell death pathway by Dapaf-1, a Drosophila Apaf-1/CED-4-related caspase activator. Mol Cell 4: 757-769. Medline abstract: 10619023
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date revised: 15 March 2007
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