The Mitochondria Limit the Rate of Caspase Activation During Spermatid Terminal Differentiation in Drosophila

• Main Author: Aram, Lior
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2016
• Subjects: Bioinformatics; Cellular biology; Developmental biology; Immunology; Microbiology
• ISBN: 9798597015934
• DOI: 10.34933/wis.000023

Partial cellular destruction during cell remodeling often involves active apoptotic caspases, but how cells avoid excessive caspase activity and unwanted cell death has been poorly understood. Here we use the caspase-mediated sperm remodeling process in Drosophila called individualization to investigate this striking phenomenon. In this dissertation, we have identified a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase (SCS) β subunit (A-Sβ), which binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. A-Sβ is localized to mitochondria during all stages of spermatogenesis, but its levels significantly increase at the onset of spermatid individualization. We demonstrate that inactivation of A-Sβ abrogates caspase activation and spermatid individualization, a phenotype reminiscent of mutations in the CRL3 complex. Spermatid mitochondrial fractionation analyses revealed that a relatively large portion of the CRL3 complex is also localized to the mitochondria, and that this depends, at least in part, on A-Sβ. Moreover, the mitochondrial portion of the complex was preferentially neddylated/activated, and A-Sβ could enhance the activity of the CRL3 complex in a Drosophila transgenesis system and in cultured cells. Immuno-electron microscopy (EM) also supports the model by which A-Sβ is localized to the mitochondrial surface in spermatids. We then identified two major isoforms of A-Sβ, one of which is testis-specific, and we showed that the latter, but not the somatic isoform, is the one localized to the surface of the mitochondria. We demonstrate that this activating arm (ASβ) of the CRL3 complex competes with the inhibitory arm mediated by Soti, the specific pseudosubstrate inhibitor of the complex, for binding to the CRL3 complex, suggesting an interplay between these two regulatory arms of the complex in spermatids. Finally, ubiquitination assays and in situ domain swapping between A-Sβ and the GTP-specific SCSβ (G-Sβ), which function redundantly in the Krebs cycle, uncouple between the metabolic and structural roles of A-Sβ in spermatids. Altogether, these results support a model in which the source of the CRL3 complex activation is limited to the vicinity of the mitochondria, consequently reducing the rate of caspase activation by at least 60%.