The two Drosophila cytochrome C proteins can function in both respiration and caspase activation

Abstract

Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, cyt-c-d and cyt-c-p. We show that only cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis.

Figure 1

(A–F) Mutations in cyt-c-d block caspase activation and spermatid individualization. Visualization of active drICE with anti-cleaved caspase-3 antibody (CM1; green) in wild-type (A), cyt-c-d^bln1 (B), cyt-c-d^Z2−1091 (C), cyt-c-d^Z2−1091/DF(2L)Exel6039 (D), cyt-c-d^Z2−1091/cyt-c-d^bln1 (E), and Z2-2468 (F). Whereas CM1-positive elongated spermatid cysts at different individualization stages can be readily seen in wild-type testes (A; white arrow pointing at a CB), no CM1-staining was detected in spermatids of flies homozygous for the P-element allele, bln¹ (B) and the point mutation allele, Z2-1091 (C). Similarly, spermatids of Z2-1091 flies either trans-heterozygous to the small deficiency Df(2L)Exel6039 (D) or to the bln¹ allele (E) also displayed no CM1 staining. In contrast, the vast majority of male-sterile mutants with spermatid individualization defects display strong CM1 positive cysts (e.g. Z2-2468; F). To visualize all the spermatids, the testes were counter-stained with phalloidin that binds F-actin (red). (G) Caspase-3-like (DEVDase) activity is detected in wild-type testes, and is blocked either after treatment with the caspase-3 inhibitor Z-VAD.fmk or in cyt-c-d^Z2−1091−/− mutant testes. DEVDase activity, presented as relative luminescence units (RLU), was determined on Ac-DEVD-pNA substrate in 62 wild-type (yw) or cyt-c-d^Z2−1091−/− mutant testes treated with Z-VAD or left untreated (DMSO). Readings were obtained every 2 min, and each time interval represents an average (mean±s.e.m.) of five readings (for more details also see the Supplementary data). Note that the levels of DEVDase activity in cyt-c-d^Z2−1091−/− mutant testes are highly similar to the corresponding levels in wild-type testes that were treated with Z-VAD. (H) Alignment of the predicted protein sequences of cytochrome C-d (c-d) and cytochrome C-p (c-p). Identical residues are indicated in the consensus (cons.) line. Cytochrome C-d and cytochrome C-p share 72% identity and 82% similarity. The tryptophan (W; red) at position 62 of cytochrome C-d is mutated to a stop codon in the Z2-1091 allele. (I) RT–PCR analyses of cyt-c-d and cyt-c-p expression. After reverse transcription with adult flies RNA, PCR was performed using two sets of specific primers spanning the unique 5′ (upper panel) and 3′ (lower panel) UTRs of both cyt-c-d and cyt-c-p. Whereas cyt-c-d and cyt-c-p are expressed in wild-type flies (YW), only cyt-c-p is expressed in the bln¹ flies, confirming that bln¹ is a null allele of cyt-c-d.

Figure 2

Both cyt-c-d and cyt-c-p can rescue the male sterile phenotypes of cyt-c-d^−/− flies. (A) Schematic structure of the rescue constructs for cyt-c-d^−/− male sterile flies. The promoter region (dark blue) and a portion of the 5′ UTR (light blue) of the hsp83 gene were fused to the 5′ UTR followed by the 3′ UTR sequences of cyt-c-d and served as a control (I). The precise coding region sequences of either cyt-c-d (II) or cyt-c-p (III) were subcloned in between the 5′ and 3′ UTRs. (B–E) Ectopic expression of either cyt-c-d or cyt-c-p rescues caspase activation, spermatid individualization, and sterility of cyt-c-d^bln1−/− male flies. Similar to WT (B), CM1-positive spermatids (green), CBs (white arrows), and WBs (yellow arrows) are readily detected in transgenic lines of the cyt-c-d^bln1−/− background expressing either cyt-c-d (D) or cyt-c-p (E) coding regions. In contrast, no CM1-positive cysts are found in cyt-c-d^bln1−/− flies that ectopically express the control construct of cyt-c-d 5′–3′ UTRs alone (C). To visualize all the spermatids and the ICs, the testes were counter-stained with phalloidin that binds F-actin (spermatids are in weak red; ICs are in strong red or yellow and associated with CM1-positive spermatids; note that remnants of the testis sheath layer autofluoresce in strong red in B). Scale bars 200 μm (B–E, upper panels), and 100 μm (D, E, lower panels). (F) Integrations of the appropriate constructs into the genome were confirmed by genomic PCR analyses. The relative locations of the primers, which are indicated with forward black and reverse red (cyt-c-d; A, II) or reverse orange (cyt-c-p, A, III) arrows were used to amplify the fragments seen in the upper and middle panels in (F), respectively. For loading control, the dcp-1 gene was amplified (lower panel in F). (G, H) Transcriptional expression from the transgenes was confirmed by RT–PCR analyses on RNA from testes of the indicated genotypes. The relative locations of the primers are indicated with black arrows in (A). Representative figures demonstrating exogenous expression of cyt-c-d 5′-3′ UTR sequences alone (G), and the exogenous expression of cyt-c-d coding region flanked by its UTR sequences (H). ‘RT+Taq' and ‘Taq' indicate reactions with reverse transcriptase or without it, respectively, to control for possible genomic DNA contamination. (H) Primers corresponding to the unique 5′ and 3′ UTRs of cyt-c-p and primers corresponding to the exogenous cyt-c-d sequences (see Materials and methods and black arrows in A) were used in the same reaction.

Figure 3

cyt-c-d is mainly expressed in the testis, while in contrast cyt-c-p is mainly expressed in the soma. (A) Schematic structure of the Drosophila cytochrome c genes. cyt-c-d and cyt-c-p display similar genomic organization of two exons (thick black and gray bars, respectively) separated by a relatively large intron (thin bar). In both of them, the coding region is restricted to the second exon (between the ATG and the stop codons). The locations of the primers used in the comparative RT–PCR experiments in (B–E) are indicated by arrows. (B) Analysis of cyt-c-d versus cyt-c-p expression in the testis and the soma. The above primers (arrows in A) to amplify either a 543-bp cyt-c-d fragment or a 483-bp cyt-c-p fragment were added to one reaction master-mix. The reaction was stopped at different cycle points to identify the linear amplification phase (20, 25, and 30 cycles are indicated). Note that the relative expression levels of cyt-c-d (strong) and cyt-c-p (weak) in the testis are switched in the soma, which is represented by adult female flies. (C) In the soma of both males and females, the expression levels of cyt-c-d are much lower than the levels of cyt-c-p. However, cyt-c-d levels are higher in adult males than in females. Note the PCR cycle number at which a band first becomes visible. (D) The expression of cyt-c-d is restricted to the male germ cells. While the expression of cyt-c-p is not affected in sons of oskar agametic testes, no cyt-c-d expression was detected. (E) cyt-c-d is expressed in premeiotic cells comprising the larval testis but not in ovaries. (F) cyt-c-p is exclusively expressed in first instar WT larva and is almost completely absent from the cyt-c-p^k13905−/−.

Figure 4

Both cyt-c-p and cyt-c-d can completely rescue the lethality/respiration defect of cyt-c-p^−/− embryos. A premix of RT–PCR reaction was designed to amplify endogenous cyt-c-p and/or transgenic cyt-c-d from testes of wild-type (WT) or rescued cyt-c-p^−/− adult flies that express transgenic cyt-c-d under the control of the tubulin promoter (cyt-c-p^k13905/Df(2L)Exel6039; tub-Gal4/UAS-cyt-c-d). To confirm that the rescued flies are of the right genotypes, we performed RT–PCR analyses with wild-type and the rescued adult flies using specific primers for the endogenous cyt-c-p as well as the transgenic cyt-c-d. Note that the strong cytochrome c transcript expression in cyt-c-p^−/− adult flies originated from the cyt-c-d transgene.

Figure 5

Expression of the testis-specific cytochrome C protein, cytochrome C-d, during spermatogenesis. Nuclei are stained blue (DAPI), and the ICs (which mark the sites of spermatid individualization) are either red or orange (phalloidin). (A) In wild-type testis, cytochrome C-d (green) accumulates along the length of elongating and elongated spermatids in a typical mitochondrial grainy pattern, (B) while this signal is almost abolished in cyt-c-d^bln1−/− mutant spermatids. (C) The expression of cytochrome C-d becomes more intense after the assembly of the IC with the highest expression detected in the preindividualized region of the spermatids just adjacent to the IC (arrowheads). After the caudal translocation of the IC, the remaining cytochrome C-d is highly reduced in the postindividualized part of the spermatids (white arrow). (D) As the IC progresses, the CB (arrowhead) collects the spermatids' bulk cytoplasm and much of the cytochrome C-d from the postindividualized parts of the spermatids. (E) Eventually, strong cytochrome C-d signal was detected in the WBs (arrowheads), which contain the discarded cytoplasm. (F, G) Staining cyt-c-d depleted testes (cyt-c-d^bln −/−), which ectopically express the cyt-c-p rescue construct, revealed that the antibodies raised against cytochrome C-d (polyclonal anti-cyt-C-d) can also react with cytochrome C-p. Similar to cytochrome C-d staining in wild type (A), cytochrome C-p expression (green) was detected along the entire length of elongated spermatids (F) and in the WBs (arrowheads in G). (H) The ectopically expressed cytochrome C-d is also detected in the rescued cyt-c-d^bln1−/− mutant testes (arrowhead pointing to a CB). Scale bars 20 μm.

Figure 6

Mutations in Drosophila homologues of the apoptosome complex, ark and dronc, cause spermatid individualization defects. CM1 is in green and the IC appears in red in all the panels. The direction of the caudal individualization movement is from top to bottom (A, B, C, E, F, J, K), or from left to right (D, G, H, I, L). (G, H, and I) A whole testis view. (A, G) WT CB. The extruded cytoplasm is contained within the oval-shaped CB, which is marked by CM1 staining in green (yellow arrowheads pointing at the ICs within the CBs). Importantly, CM1 staining is absent from the post-individualized portion of the spermatids (above the CB), while it is still apparent in the preindividualized portion (white arrowhead). (B, C) In ark and (H, I) dronc mutants, the CBs are frequently reduced in size or appear flat (yellow arrowhead in B and H, respectively) due to a failure in the appropriate collection of the cytoplasm of the spermatids. The retained cytoplasm is clearly visualized as a ‘trail' of residual cytoplasm (marked by the green CM1 staining) along the entire length of what was supposed to have been the postindividualized portion of the spermatids (B, C and H, I; white or yellow arrows following the ‘cytoplasmic trails'). Frequently, a large portion of the spermatids' cytoplasm is retained behind in ‘mini' CB structures, which often contain part of the IC (white arrowheads in B, and H). Note that in wild-type testes, ‘cytoplasmic trails' do not exist in the post-individualized portion of the spermatids (left to the CBs, which are indicated by yellow arrowheads in G). The surface area of the flattened advanced CBs and WBs in ark (E, and F) and dronc (K, and L) mutants were measured and compared to the wild-type counterparts (D, and J, respectively). The CBs and WBs in (F) corresponds to the ones in (C), and the WB in (E) corresponds to the WB in (B), which is marked by a yellow arrowhead, asterisks in (K and L) correspond to the asterisks in (H and I), respectively. The actual surface area appears in square micrometer next to the CBs and WBs. Note that the surface area of ark and dronc mutant WBs vary from cyst to cyst but are always highly reduced compared to wild type. Scale bars 50 μm. (A, B, and C) and (G, H, and I) are displayed in the same magnification.