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Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization.

Huh JR, Vernooy SY, Yu H, Yan N, Shi Y, Guo M, Hay BA - PLoS Biol. (2003)

Bottom Line: This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC.A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization.Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, USA.

ABSTRACT
Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. Here we demonstrate that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.

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driceless Males Lack Active Drice Staining and Show Defects in Individualization(A) Testis from driceless male stained with active DRICE. Active DRICE staining is eliminated.(B) Elongated cysts from driceless male. AXO49 staining (blue) outlines the location of three cystic bulges. Individualization complexes (arrows) are marked with phalloidin (red).(C) Example of a cyst from a driceless male in which individualization has proceeded normally.(D) Example of a cyst from a driceless male in which individualization has failed to occur.(E) Boxed area in (D) shown at higher magnification. A region in which individualization has failed is outlined with a dashed line.
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pbio-0020015-g006: driceless Males Lack Active Drice Staining and Show Defects in Individualization(A) Testis from driceless male stained with active DRICE. Active DRICE staining is eliminated.(B) Elongated cysts from driceless male. AXO49 staining (blue) outlines the location of three cystic bulges. Individualization complexes (arrows) are marked with phalloidin (red).(C) Example of a cyst from a driceless male in which individualization has proceeded normally.(D) Example of a cyst from a driceless male in which individualization has failed to occur.(E) Boxed area in (D) shown at higher magnification. A region in which individualization has failed is outlined with a dashed line.

Mentions: Finally, we noted that DRICE activation was insensitive to inhibition (but perhaps not to complete elimination) of ARK and DRONC; to complete loss of HID, DREDD, or FADD; and to expression of the potent general caspase inhibitors DIAP1, p35, or p49 (see Figures 1–4; data not shown). This, together with the observation that DRONC and DRICE were activated in distinct spatial and temporal patterns (see Figure 3A–3D), suggests that DRICE activation occurs through an unknown HID-, ARK-, DRONC-, dFADD-, and DREDD-independent mechanism. It has been proposed that DRICE activation in spermatids is essential for fertility and that DRICE activation is mediated by an isoform of cytochrome c, cytochrome c-d (cyt-c-d), based on the observation that males homozygous for a P-element insertion (bln1) in the cyt-c-d gene were sterile and lacked active DRICE staining in testis (Arama et al. 2003). However, as illustrated in Figure 5, the region surrounding the bln1 insertion contains multiple transcription units. In addition, cysts from bln1 males showed multiple defects in spermatogenesis prior to individualization, including failure to carry out polyglycylation of axonemal microtubules (Figure 5C and 5E), and aberrant development of the major and mitochondrial derivatives (Figure 5F–5H). These observations leave it unclear whether cyt-c-d is in any direct sense required for DRICE activation or whether DRICE is required for fertility. We serendipitously identified a line of flies carrying an X chromosome mutation (driceless) in which DRICE activation during spermatid individualization was completely eliminated (Figure 6A) (see Materials and Methods for details). Testis from these flies contained large cystic bulges in which individualization complexes were present as a coordinated front, as in wild-type (Figure 6B). In contrast to bln1 males, driceless males were fertile and investment cones moved apically. As expected from this phenotype, some cysts from driceless males underwent individualization normally (approximately 50%) (Figure 6C). However, in others, individualization failed completely (Figure 6D and 6E).


Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization.

Huh JR, Vernooy SY, Yu H, Yan N, Shi Y, Guo M, Hay BA - PLoS Biol. (2003)

driceless Males Lack Active Drice Staining and Show Defects in Individualization(A) Testis from driceless male stained with active DRICE. Active DRICE staining is eliminated.(B) Elongated cysts from driceless male. AXO49 staining (blue) outlines the location of three cystic bulges. Individualization complexes (arrows) are marked with phalloidin (red).(C) Example of a cyst from a driceless male in which individualization has proceeded normally.(D) Example of a cyst from a driceless male in which individualization has failed to occur.(E) Boxed area in (D) shown at higher magnification. A region in which individualization has failed is outlined with a dashed line.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC300883&req=5

pbio-0020015-g006: driceless Males Lack Active Drice Staining and Show Defects in Individualization(A) Testis from driceless male stained with active DRICE. Active DRICE staining is eliminated.(B) Elongated cysts from driceless male. AXO49 staining (blue) outlines the location of three cystic bulges. Individualization complexes (arrows) are marked with phalloidin (red).(C) Example of a cyst from a driceless male in which individualization has proceeded normally.(D) Example of a cyst from a driceless male in which individualization has failed to occur.(E) Boxed area in (D) shown at higher magnification. A region in which individualization has failed is outlined with a dashed line.
Mentions: Finally, we noted that DRICE activation was insensitive to inhibition (but perhaps not to complete elimination) of ARK and DRONC; to complete loss of HID, DREDD, or FADD; and to expression of the potent general caspase inhibitors DIAP1, p35, or p49 (see Figures 1–4; data not shown). This, together with the observation that DRONC and DRICE were activated in distinct spatial and temporal patterns (see Figure 3A–3D), suggests that DRICE activation occurs through an unknown HID-, ARK-, DRONC-, dFADD-, and DREDD-independent mechanism. It has been proposed that DRICE activation in spermatids is essential for fertility and that DRICE activation is mediated by an isoform of cytochrome c, cytochrome c-d (cyt-c-d), based on the observation that males homozygous for a P-element insertion (bln1) in the cyt-c-d gene were sterile and lacked active DRICE staining in testis (Arama et al. 2003). However, as illustrated in Figure 5, the region surrounding the bln1 insertion contains multiple transcription units. In addition, cysts from bln1 males showed multiple defects in spermatogenesis prior to individualization, including failure to carry out polyglycylation of axonemal microtubules (Figure 5C and 5E), and aberrant development of the major and mitochondrial derivatives (Figure 5F–5H). These observations leave it unclear whether cyt-c-d is in any direct sense required for DRICE activation or whether DRICE is required for fertility. We serendipitously identified a line of flies carrying an X chromosome mutation (driceless) in which DRICE activation during spermatid individualization was completely eliminated (Figure 6A) (see Materials and Methods for details). Testis from these flies contained large cystic bulges in which individualization complexes were present as a coordinated front, as in wild-type (Figure 6B). In contrast to bln1 males, driceless males were fertile and investment cones moved apically. As expected from this phenotype, some cysts from driceless males underwent individualization normally (approximately 50%) (Figure 6C). However, in others, individualization failed completely (Figure 6D and 6E).

Bottom Line: This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC.A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization.Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, USA.

ABSTRACT
Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. Here we demonstrate that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.

Show MeSH
Related in: MedlinePlus