scotti: Biological Overview | References
Gene name - scotti
Cytological map position- 88B7-88B7
Function - signaling
Symbol - soti
FlyBase ID: FBgn0038225
Genetic map position - 3R:10,206,105..10,207,421 [-]
Classification - novel protein
Cellular location - cytoplasmic
Caspases are executioners of apoptosis but also participate in a variety of vital cellular processes. This study has identified Soti, an inhibitor of the Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during Drosophila spermatid terminal differentiation (individualization). Evidence is provided that the giant inhibitor of apoptosis-like protein dBruce is a target for the Cullin-3-based complex, and that Soti competes with dBruce for binding to Klhl10, the E3 substrate recruitment subunit. Soti is expressed in a subcellular gradient within spermatids and in turn promotes proper formation of a similar dBruce gradient. Consequently, caspase activation occurs in an inverse graded fashion, such that the regions of the developing spermatid that are the last to individualize experience the lowest levels of activated caspases. These findings elucidate how the spatial regulation of caspase activation can permit caspase-dependent differentiation while preventing full-blown apoptosis (Kaplan, 2010).
Programmed cell death is one of the most fundamental processes in biology. A morphologically distinct form of this active cellular suicide process, dubbed apoptosis, serves to eliminate unwanted and potentially dangerous cells during development and tissue homeostasis in virtually all multicellular organisms. Members of the caspase family of proteases are the central executioners of apoptosis. Caspases start off as inactive proenzymes and are activated upon proteolytic cleavage by other caspases. Apoptotic caspases can also participate in a variety of vital cellular processes, including differentiation, signaling, and cellular remodeling. However, the mechanisms that protect these cells against excessive caspase activation and undesirable death have remained obscure (Kaplan, 2010).
In both insects and mammals, spermatids eliminate their bulk cytoplasmic content as they undergo terminal differentiation. In Drosophila, an actin-based individualization complex (IC) slides caudally along a group of 64 interconnected spermatids, promoting their separation from each other and the removal of most of their cytoplasm and organelles into a membrane-bound sack called the cystic bulge (CB), which is eventually discarded as a waste bag (WB). This vital process, known as spermatid individualization, is reminiscent of apoptosis and requires apoptotic proteins including active caspases. However, the mechanisms that restrict caspase activation in spermatids, as opposed to their full-blown activation during apoptosis, are poorly understood (Kaplan, 2010).
The isolation of a Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during spermatid individualization has been described (Arama, 2007). Ubiquitin E3 ligases tag cellular proteins with ubiquitin, thereby affecting protein localization, interaction, or turnover by the proteasome. The Cullin-RING ubiquitin ligases (CRLs) comprise the largest class of E3 enzymes, conserved from yeast to human. Cullin family proteins serve as scaffolds for two functional subunits: a catalytic module, composed of a small RING domain protein that recruits the ubiquitin-conjugating E2 enzyme, and an adaptor subunit which binds to the substrate and brings it within proximity to the catalytic module. In Cullin-3-based E3 ligase complexes, BTB-domain proteins interact with Cullin-3 via the eponymous domain, while they bind to substrates through additional protein-protein interaction domains, such as MATH or Kelch domains. A large body of evidence indicates that substrate specificity and the time of ubiquitination are determined by posttranslational modifications of the substrates and the large repertoire of the adaptor proteins. In addition, the Cullins themselves are subject to different types of posttranslational regulation. Most notably, they are activated by a covalent attachment of a ubiquitin-like protein Nedd8 (Kaplan, 2010).
This study has identified a small protein called Soti that specifically binds to Klhl10, the adaptor protein of a Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during the nonapoptotic process of spermatid individualization. Soti acts as a pseudosubstrate inhibitor of this E3 complex and inactivation of Soti leads to elevated levels of active effector caspases and progressive severity of individualization defects in spermatids. Furthermore, the giant inhibitor of apoptosis (IAP)-like protein dBruce is targeted by this E3 complex, and this effect is antagonized by Soti. Finally, immunofluorescence studies reveal that Soti is expressed in a distal-to-proximal gradient, which promotes a similar distribution of dBruce in spermatids. Consequently, activation of caspases is restricted in both space and time, displaying a proximal-to-distal complementary gradient at the onset of individualization (Kaplan, 2010).
The current study provides insight into how some cells can utilize active caspases to promote vital cellular processes but still avoid unwanted death. According to this model, during early and advanced spermatid developmental stages, a gradient of Soti is generated, allowing graded activation of the Cullin-3-based E3 ubiquitin ligase complex in the opposite direction. This E3 complex then targets dBruce, promoting its distribution in a similar gradient as that of Soti. Subsequently, caspase activation occurs in a complementary gradient descending from proximal to distal. Since the removal of the cytoplasm and caspases also occurs in the direction of proximal to distal, the regions of the developing spermatid that are the last to individualize are also those that are the most protected against activated caspases. This setting ensures that each spermatidal domain encounters similar transient levels of activated caspases throughout the process of individualization (Kaplan, 2010).
The gradual regulation of caspase activity in spermatids is attributed to the outstanding length of Drosophila spermatids (a phenomenon called sperm gigantism). Spermatozoa of Drosophila melanogaster are about 1.9 mm long and other Drosophilids can produce sperm up to 58 mm long. Spermatids in Drosophila individualize over the course of 12 hr through a constant rate of proximal-to-distal individualization complex movement and clearance of the cytoplasmic content (including the active caspases) into a cystic bulge. Since it takes a few hours for the active effector caspasesto kill a cell, spermatids had to develop an efficient mechanism to prevent prolonged exposure of the more distal cellular regions to caspase activity. A gradient of a caspase inhibitor, descending from distal to proximal, is therefore an elegant mechanism to ensure a level of caspase activity that is sufficient to drive spermatid differentiation, yet not high enough to engage an apoptotic program (Kaplan, 2010).
Ubiquitination may target dBruce for either degradation or active redistribution. Because of technical limitations of the in vivo system, the biochemical analyses were performed in a heterologous system using truncated dBruce versions, and thus, we cannot completely rule out the possibility that at least some of the ubiquitinated dBruce is degraded by the proteasome. However, the genetic data support a model where dBruce may be redistributed by an active translocation mechanism, as hyperactivation of the Cullin-3-based complex, following Soti inactivation, leads to accumulation of dBruce at tail ends of spermatids and not to its elimination. This idea is also indirectly supported from the experiment in the eye system, showing that transgenic expression of the dBruce mini-gene enhanced the small eye phenotype caused by expression of Klhl10, suggesting that the Cullin-3-based complex does not target the dBruce mini-gene for degradation in this system. Consistent with this notion, accumulating evidence indicates that the ubiquitin 'code' on target proteins can be read by a large number of ubiquitinbinding proteins, which translate the ubiquitin code to specific cellular outputs, such as protein redistribution. Interestingly, a recent report suggests that Cullin-3-based polyubiquitination of caspase- 8 promotes its aggregation, which subsequently leads to processing and full activation of this protease. Furthermore, another Cullin-3-based ubiquitin ligase complex was shown to regulate the dynamic localization of the Aurora B kinase on mitotic chromosomes. Therefore, Cullin-3-based ubiquitin ligase complexes appear to promote also nondegradative ubiquitination and redistribution of proteins (Kaplan, 2010).
Cullin-RING ubiquitin ligases (CRLs) bind to substrates via adaptor proteins. However, adaptor proteins can also bind to pseudosubstrate inhibitors in a manner which is reminiscent of an E3-substrate-type interaction. Several lines of evidence strongly suggest that Soti is a pseudosubstrate inhibitor of the Cullin-3-based E3 ubiquitin ligase complex in spermatids. (1) The interaction between Klhl10 and Soti is an E3-substrate-type interaction. (2) Soti is not a substrate for this E3 complex. (3) dBruce polypeptides can outcompete with Soti for binding to Klhl10. Finally, Soti is a potent inhibitor of this E3 complex. Therefore, the mechanism of regulation by pseudosubstrates may represent a more common mechanism for modulation of CRL activity than has been previously appreciated (Kaplan, 2010).
Two alternative protein degradation pathways were recently described: N-terminal ubiquitination (NTU) and degradation 'by default'. Whereas the former promotes degradation of proteins by ubiquitination at N-terminal residues, the latter targets proteins for degradation by a ubiquitin-independent, 20S proteasome-dependent mechanism. Although these results cannot conclusively distinguish between these two pathways, two notable mechanistic traits of degradation 'by default' can be also attributed to Soti, including the targeting of intrinsically disordered proteins and their protection by binding to other proteins ('nannies'). Using the FoldIndex tool, Soti was predicted to be intrinsically disordered, while it is stabilized by attachment of a structured Myc-tag to its N terminus. Furthermore, Soti is highly unstable in the absence of its binding partner Klhl10, suggesting that Klhl10 functions as a 'nanny' for its own inhibitor. In conclusion, this study has uncovered a mechanism that restricts caspase activation during the vital process of spermatid individualization. This process appears to be conserved both anatomically and molecularly from Drosophila to mammals (reviewed in detail in Feinstein-Rotkopf and Arama, 2009). Moreover, several recent studies suggest that a similar Klhl10-Cul3 complex is essential for late spermatogenesis in mammals. Therefore, although the mammalian sperm is about 30 times shorter than in Drosophila, similar mechanisms (albeit scaled-down) for regulation of caspase activation may also exist during mammalian spermatogenesis. Further studies of the link between the ubiquitin pathway and apoptotic proteins during sperm differentiation in Drosophila may, therefore, provide new insights into the etiology of some forms of human infertility (Kaplan, 2010).
Post-meiotic transcription was accepted to be essentially absent from Drosophila spermatogenesis. This study identified 24 Drosophila genes whose mRNAs are most abundant in elongating spermatids. By single-cyst quantitative RT-PCR, post-meiotic transcription of these genes was demonstrated. It is concluded that transcription stops in Drosophila late primary spermatocytes, then is reactivated by two pathways for a few loci just before histone --> transition protein --> protamine chromatin remodelling in spermiogenesis. These mRNAs localise to a small region at the distal elongating end of the spermatid bundles, thus they represent a new class of sub-cellularly localised mRNAs. Mutants for a post-meiotically transcribed gene (scotti), are male sterile, and show spermatid individualisation defects, indicating a function in late spermiogenesis (Barreau, 2008).
Approximately 4 kb of genomic DNA, including the entire scotti (soti, a comet) ORF, were deleted by FLP-mediated recombination between flanking FRT-containing transposons. soti homozygous mutants were viable and female fertile, but male sterile. Phase contrast microscopy indicated no gross defects in soti testes organisation or spermatid elongation; however, empty seminal vesicles indicated spermiogenesis defects. Within each individualising spermatid cyst, 64 actin-rich investment cones move together as an individualisation complex, pushing ahead a cystic bulge of excess cytoplasm and organelles. This cytoplasm is discarded from spermatid distal ends as a waste bag. Waste bags were completely absent from mutant testes, and cystic bulges were rarely seen. FITC-phalloidin labelling revealed that investment cones formed normally in soti mutant males; however, nuclei failed to remain tightly clustered and were displaced distally along the cyst. Although investment cones progressed away from the nuclei in mutants, investment cone coupling within individualisation complexes was lost, and cones never progressed the full length of mutant spermatids. Thus, soti function is required for spermatid individualisation (Barreau, 2008).
Search PubMed for articles about Drosophila Scotti
Arama, E., Bader, M., Rieckhof, G.E., and Steller, H. (2007). A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila. PLoS Biol. 5: e251. PubMed ID: 17880263
Barreau, C., Benson, E., Gudmannsdottir, E., Newton, F. and White-Cooper, H. (2008). Post-meiotic transcription in Drosophila testes. Development 135(11): 1897-902. PubMed ID: 18434411
Feinstein-Rotkopf, Y. and Arama, E. (2007). Can't live without them, can live with them: roles of caspases during vital cellular processes. Apoptosis 14(8): 980-95. PubMed ID: 19373560
Kaplan, Y., et al. (2010). Gradients of a ubiquitin E3 ligase inhibitor and a caspase inhibitor determine differentiation or death in spermatids. Dev. Cell 19(1): 160-73. PubMed ID: 20643358
date revised: 5 January 2011
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