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ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

Abstract

The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used1,2. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation.

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Figure 1: The ATP synthase has an essential role during stem cell differentiation.
Figure 2: Phenotypic characterization of ATP synthase knockdown ovaries.
Figure 3: Knockdown (KD) of ATP synthase, but not other members of the oxidative phosphorylation system, causes germline defects.
Figure 4: ATP synthase is upregulated in differentiating cysts and its function is required for cyst differentiation.
Figure 5: ATP synthase dimers are required for crista maturation during stem cell differentiation.

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Acknowledgements

We thank C. Malone, M. Murphy, J. Carroll, A. Sfeir, E. Skolnik, R. Cinalli, A. Zamparini and A. Blum for comments on the manuscript and advice. We thank F. Liang, C. Petzold and K. Dancel of the NYULMC OCS Microscopy Core for their assistance with transmission electron microscopy, and the NYULMC Immune Monitoring Core supported in part by NCATS NIH grant UL1 TR00038 and NCI NIH grant P30CA016087. We acknowledge the DGRC supported by NIH 2P40OD010949-10A1 and Bloomington Stock Center for reagents. F.K.T. was supported by EMBO and HFSP long-term fellowships, C.G.S. by NIH F31/HD080380, T.R.H. by CIHR, J.R.K.S. by NIH F32/GM082169, B.C. by a PhD fellowship from the Boehringer Ingelheim Fonds and J.B.P. by ACS award 121614-PF-11-277-01-RMC. This work was supported by the NIH (5R01GM062534) and a kind gift from K. W. Davis to G.J.H. G.J.H. is an investigator of the HHMI. R.L. is an HHMI investigator and is supported by NIH R01/R37HD41900.

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Authors and Affiliations

Authors

Contributions

F.K.T., T.R.H., R.L., C.G.S. and J.R.K.S. designed the experiments. C.G.S. carried out most of the Drosophila crosses, dissections and immunofluorescence stainings. J.R.K.S. and F.K.T. acquired the majority of the confocal microscopy images, with T.R.H. and C.G.S. also contributing. F.K.T. made the dsRNA, carried out the bioinformatics analysis and carried out the RNA expression analysis. T.R.H. carried out the cell culture transfections and CN-PAGE analysis. T.R.H., F.K.T. and R.L. wrote the manuscript, with all authors approving the final version. B.C., J.B.P. and G.J.H. provided the initial list of genes required for oogenesis. R.L., T.R.H., F.K.T., C.G.S. and J.R.K.S. contributed to the discussion of the results.

Corresponding authors

Correspondence to Thomas R. Hurd or Ruth Lehmann.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Mitochondrial transcription, translation and protein import machinery components are required for germ cell differentiation.

The following VDRC UAS-RNAi strains were expressed in the germline: 105995/KK for mRpL18, 104412/KK for mRpL21, 101506/KK for mRpL27, 101442/KK for mRpL40, 101656/KK for mTTF, and 13794/GD for Blp. Knockdown germaria were immunostained with α-Vasa (green) and α-1B1 (red). Images are representative of at least 100 ovarioles analyzed per genotype. Scale bar, 20 μm.

Supplementary Figure 2 Transfection of S2R+ cells with dsRNA effectively silences the expression of target genes as measured by RT-qPCR.

The data are the means of three technical replicates.

Supplementary Figure 3 ATP synthase subunit β is up-regulated in differentiating cysts.

Germaria expressing a mitochondrially-targeted EYFP (mitoEYFP) were immunostained with α-mei-P26 (right), GFP, and ATP synthase β. The ratio of ATP synthase β to mitoEYFP is shown. Image is representative of at least 100 ovarioles analyzed per genotype. Scale bar, 20 μm.

Supplementary Figure 4 ATP synthase subunit g is not required for ATP synthase stability in S2R+ cells.

Immunofluorescence staining of S2R+ cells transfected with dsRNA that silences complex III, IV and ATP synthase subunits. Cells were immunostained with α-ATP synthase α (green top), α-ATP synthase β (green middle), and α-PDH E1α (green bottom). Images are representative of approximately 300 cells assessed from two fields. Scale bar, 100 μm.

Supplementary Figure 5 Uncropped images of electrophoretic separation techniques in Fig. 5.

(a) CN-PAGE of S2R+ cells treated with dsRNA targeting lacZ or ATP synthase subunits g, e, α or β. ATP synthase was detected by immunoblotting with α-ATP synthase β. Image is representative of 3 independent experiments. (b) SDS-PAGE of the same samples followed by immunoblotting with α-porin served as a sample processing control. (c) CN-PAGE of S2R+ cells treated with dsRNA targeting lacZ or ATP synthase subunits g, e, α or β. ATPase activity was measured in gel. Image is representative of 2 independent experiments.

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Teixeira, F., Sanchez, C., Hurd, T. et al. ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation. Nat Cell Biol 17, 689–696 (2015). https://doi.org/10.1038/ncb3165

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