Effete-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila

Abstract

Increasing evidence supports the idea that the regulation of stem cells requires both extrinsic and intrinsic mechanisms. However, much less is known about how intrinsic signals regulate the fate of stem cells. Studies on germline stem cells (GSCs) in the Drosophila ovary have provided novel insights into the regulatory mechanisms of stem cell maintenance. In this study, we demonstrate that a ubiquitin-dependent pathway mediated by the Drosophila eff gene, which encodes the E2 ubiquitinconjugating enzyme Effete (Eff), plays an essential role in GSC maintenance. We show that Eff both physically and genetically interacts with dAPC2, a key component of the anaphase-promoting complex (APC), which acts as a multisubunit E3 ligase and plays an essential role in targeting mitotic regulators for degradation during exit from mitosis. This interaction indicates that Eff regulates the APC/C-mediated proteolysis pathway in GSCs. Moreover, we show that expression of a stable form of Cyclin A, but not full-length Cyclin A, results in GSC loss. Finally we show that, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Collectively, our data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila. Given that the regulation of mitotic Cyclins is evolutionarily conserved between flies and mammals, our study also implies that a similar mechanism may be conserved in mammals.

Ci antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation

“…To further test this, we took advantage of the temperature-dependent activity of the Gal4 to spatio-temporally overexpress Sd in ECs, since overexpression of Sd usually causes animal lethality under normal temperature (25 °C). Following the method described previously [33], crosses were performed and the resulting progenies were initially raised at 18 °C until adult eclosion, at which point they were shifted to 29 °C. As shown in Supplementary information, Figure S2D, overexpression of Sd in ECs using c587-gal4 did not produce apparent germarium phenotype.…”

“…The following primary antibody dilutions were used: rabbit anti-GFP (1:2 000; Invitrogen), mouse anti-Hts (1:1 500; DSHB), rabbit and mouse anti-BamC (1:2 000; [33]), rabbit anti-Vasa (1:2 000; Santa Cruz), rabbit anti-HA(1:2 000; CST), rabbit anti-Hpo (1:500; [36]), rabbit anti-Yki (1:1 000; [19]), rat anti-Ci (1:2 000; DHSB), and mouse anti-β-Gal (1:1 000; Promega). The following secondary antibodies were used at a 1:1 500 dilution: goat anti-mouse Alexa568, goat anti-rabbit Alexa488 and goat anti-rat Alexa647 (all from Molecular Probes).…”

Dual roles of Hh signaling in the regulation of somatic stem cell self-renewal and germline stem cell maintenance in Drosophila testis

“…Although the pMad staining varied among individual testes, which is consistent with a previous report [9], the overall trend indicates that the BMP signaling activity in GSCs could be positively regulated by the Hh signaling activity in CySCs. Furthermore, we examined the endogenous expression of bag of marbles (bam) gene, which is repressed by Mad in GSCs and is expressed only in differentiated spermatogonia [10]. Compared with normal testis (dashed lines in Figure 1T lines in Figure 1U), indicating that the diminished Hh activity in CySCs caused a premature differentiation of GSCs.…”

Bam and Otu can regulate stem cell fate by stabilizing cyclin A

“…Bam strongly interacted with free ubiquitin, suggesting that Bam could bind proteins bearing a ubiquitin chain, which can be marked for degradation. Previous work by the authors had shown that ubiquitinmediated degradation of CycA was necessary for GSC maintenance (8). Expression of a nondegradable CycA in GSCs leads to their loss by differentiation, a phenotype identical to the ectopic expression of Bam (8).…”

“…Previous work by the authors had shown that ubiquitinmediated degradation of CycA was necessary for GSC maintenance (8). Expression of a nondegradable CycA in GSCs leads to their loss by differentiation, a phenotype identical to the ectopic expression of Bam (8). In PNAS, Ji et al (7) discover that CycA can be coimmunoprecipitated with Bam from S2 cells and ovarian extracts.…”