The capacity of stem cells to self-renew or differentiate has been attributed to distinct metabolic states. A genetic screen targeting regulators of mitochondrial dynamics revealed that mitochondrial fusion is required for the maintenance of male germline stem cells (GSCs) in Drosophila melanogaster. Depletion of Mitofusin (dMfn) or Opa1 led to dysfunctional mitochondria, activation of Target of rapamycin (TOR) and a marked accumulation of lipid droplets. Enhancement of lipid utilization by the mitochondria attenuated TOR activation and rescued the loss of GSCs that was caused by inhibition of mitochondrial fusion. Moreover, constitutive activation of the TOR-pathway target and lipogenesis factor Sterol regulatory element binding protein (SREBP) also resulted in GSC loss, whereas inhibition of SREBP rescued GSC loss triggered by depletion of dMfn. Our findings highlight a critical role for mitochondrial fusion and lipid homeostasis in GSC maintenance, providing insight into the potential impact of mitochondrial and metabolic diseases on the function of stem and/or germ cells.
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We thank M. Guo, U. Banerjee, H. Jasper, H. Bellen, R. Gottlieb, R. Kühnlein, J. Chung, K. Mitra, M. Van Doren, R. Thakur, the Vienna Drosophila RNAi Center and the Bloomington Stock Center for reagents, M. Cilluffo from the BRI/UCLA EM Core Facility and J. Fitzpatrick from the Salk Institute for Biological Studies for EM processing, the BSCRC/MCDB Microscopy Core Facility at UCLA, members of the Walker and Shirihai laboratories for discussions and members of the Jones Laboratory for comments on the manuscript. This work was supported by the NIH (grant numbers AG028092, AG040288 and AG052732 to D.L.J.) and an Eli and Edythe Broad Center of Regenerative Medicine & Stem Cell Research Postdoctoral Fellowship (to R.S.D.).
The authors declare no competing interests.
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Integrated supplementary information
a) Quantification of GSCs per testes in 10 days old (do) nos-GAL4:VP16 animals expressing transgenes targeting the down regulation of dMfn, Opa1 and Drp1. Symbol colors refer to appropriate control. Dark-red symbols represent similar experiments done in 10do nos-Gal4; Gal80TS animals, in which the transgene expression was only induced after eclosion (see Methods). N = 45 testes for Controlw, n = 23 testes for dMfnRNAi-Guo, n = 19 testes for dMfnRNAi-Bellen, n = 21 testes for ControlTRiP, n = 28 testes for dMfnRNAi-TRiP, n = 32 testes for ControlKK, n = 27 testes for dMfnRNAi-KK, n = 24 testes for Controlw-TS, n = 29 testes for dMfnRNAi-GuoTS, n = 25 testes for Opa1RNAi-TRiP, n = 28 testes for Opa1RNAi-Guo, n = 17 testes for Opa1RNAi-GuoTS, n = 27 testes for Drp1RNAi-GD, n = 46 testes for Drp1DN, n = 25 testes for Drp1DN TS. Two-tailed t-test used. b) Representative IFs of testes stained with ApopTag. Arrowheads denote cells positive for ApopTag. Dotted circle in right panel denotes a dMfn- clone. 3 biological replicates. c) Quantification of ApopTag+ GSCs. Two-sided fisher’s exact test was used. N = 142 GSCs for controlRNAi, n = 103 GSCs for dMfnRNAi, n = 91 GSCs for controlclones, n = 40 GSCs for dMfn-, n = 58 GSCs for dMfn++. d) Quantification of GSC number per testis of the noted genotypes. Control and dMfnRNAi (+GFPmito) data reproduced from Fig. 6c for comparison. N = 20 testes for p35, n = 25 testes for DroncRNAi, n = 48 testes for dMfnRNAi+p35, n = 43 testes for dMfnRNAi+DroncRNAi. Two-tailed t-test used. e) Schematics and IF examples of the twin-spot clonal generation of dMfn loss-of-function clones. In representative IFs of testes with dMfn- and dMfn++ clones, arrowhead and trace colors are keyed at the bottom of the panel. 3 biological replicates. f-h) Clonal quantification. Quantification of the percentage of clonal GSCs per total GSCs in F (two-tailed t-test used), testes with early spermatogonial clones in G, and testes with early spermatocyte clones in H. Two-sided fisher’s exact test was used in G and H. n = 68 testes for all conditions. i) Representative IF of testes from 5do animals expressing a mitochondrially targeted GFP in early spermatogonia with the driver bam-GAL4:VP16. Note that when dMfn is depleted, the germline mitochondria of late spermatogonia/early spermatocytes do not fuse and fail to aggregate on one side of the cell. Representative of 30 testes per genotype, analyzed in 3 independent experiments. j) Quantification of the percentage of testes containing control GSC clones or GSCs homozygous for a null allele of Drp1. N = 102 testes for control 1-2dphs and n = 110 testes for control 7-8dphs; n = 67 testes for Drp1KG03815 1-2dphs and n = 101 for Drp1KG03815 7-8dphs. Two-sided fisher’s exact used. k) GSC quantification in animals expressing nos-GAL4:VP16>dMfnRNAi (+GFPmito), Drp1DN (+GFPmito), nos-GAL4:VP16>dMfnRNAi+Drp1DN and control. Two-tailed t-test was used. N = 24 testes for control, n = 25 testes for dMfnRNAi, n = 20 testes for Drp1DN, n = 27 testes for dMfnRNAi+Drp1DN. l) Quantification of STAT-GFP pixel intensity in nuclei of control or Opa1-depleted GSCs from 2do animals. N = 50 GSCs for control and n = 68 GSCs for Opa1RNAi. Two-tailed t-test used. In all figures, asterisk (*) denotes the hub; scale bar, 20 μm. Raw data and statistical detail for data plots in A, C, D, F-H, J-L available in Supplemental Table 2. Plots in A, D, F, K and L display mean with error bars displaying standard deviation. C, G, H and J display graphical representations of proportion.
Supplementary Figure 2 Block in mitochondrial fusion leads to loss of mitochondrial function and induction of mitophagy.
a) Representative images of GSCs expressing Timermito in either control or dMfnRNAi animals. Recently folded (‘young’) Timermito fluoresces green (Timermito(y)), but with maturation (‘old’), Timermito shifts fluorescence to red (Timermito(o)). Note the accumulation of mitochondria with primarily red (red arrowhead) or green (green arrowhead) Timermito signal (that is, no mixing) upon dMfn depletion. N = 30 testes per genotype, analyzed in 3 individual experiments. b-c) Images of testes from 5do animals of the noted genotypes also expressing UAS-LAMP1:GFP (B) or UAS-Ref(2)P:GFP (C) transgenes. Fluorescence intensity was calibrated using the maximal signal emitted from the dMfnRNAi samples for comparison. Pixel quantifications of LAMP1 (B) or Ref(2)P (C) signal in GSCs displayed to the right of each IF panel (two-tailed t-test used). For LAMP1, n = 81 GSCs in control, n = 71 GSCs in dMfnRNAi; for Ref(2)P, n = 75 GSCs for control, n = 74 GSCs for dMfnRNAi. d) Quantification of the percentage of GSCs with at least one mitochondrial unit associated with Ref(2)P aggregates (see Methods). In testes from both dMfnRNAi and Opa1RNAi, there was a significant increase in co-localization of the two signals in GSCs (p<0.0001 compared to controls). Two-sided fisher’s exact was used. N = 53 GSCs in control, n = 32 GSCs in dMfnRNAi, n = 42 GSCs in Opa1RNAi. In all figures, asterisk (*) denotes the hub; scale bar, 20μm. Raw data and statistical detail for data plots in B-D available in Supplemental Table 2. Plots in B and C display mean with error bars displaying standard deviation. D displays graphical representation of proportion.
a) DHE stains showed no difference in ROS levels between GSCs of control vs dMfn loss of function clones. Quantification shown in A’ (two-tailed t-test used). N = 10 clones for dMfn++, n = 9 clones for dMfn-. b) The reporter for antioxidant gene-regulation GstD1-GFP showed no difference between control and dMfn-depleted GSCs. Pixel intensity quantification is shown in B’. N = 45 GSCs for control, n = 26 GSCs for dMfnRNAi. Two-tailed t-test used. c) Quantification of GSCs in testes that expressed nos-GAL4:VP16>dMfnRNAi (+GFPmito) and were fed either a regular diet (n = 41 testes) or food containing 100mM of the antioxidant NAC (n = 43 testes) for 5 days; or that co-expressed dMfnRNAi with either the overexpression of the ROS-scavenger Sod2 (n = 33 testes) or the knockdown of the CnC/NRF2 inhibitor Keap1 (n = 36 testes) in the germline. No statistically significant difference was observed among any groups with dMfnRNAi. Two-tailed t-test was used. For comparison, nos-GAL4:VP16>Sod2OE (n = 29 testes) and nos-GAL4:VP16>Keap1RNAi (n = 21 testes) are also displayed (no statistically significant difference to control represented in Fig. 4c). d) Western blot analysis shows no change in pAMPK levels when dMfn was depleted in 2do animals. Experiment repeated 3x with similar trends. e) Quantification of GSCs per testes in 5do animals show that the co-expression of a dominant-negative TOR construct with dMfnRNAi rescued the GSC loss (two-tailed t-test used). N = 26 testes for dMfnRNAi and n = 30 testes for dMfnRNAi+TORDN. In all figures, asterisk (*) denotes the hub; scale bar, 20μm. Raw data and statistical detail for blots in D and data plots in A’, B’, C and E available in Supplemental Fig. 7 and Supplemental Table 2, respectively. Plots in A’, B’, C and E display mean with error bars displaying standard deviation. D displays graphical representation of pixel intensity ratio.
Supplementary Figure 4 FA utilization by mitochondria is disrupted when mitochondrial fusion is blocked.
a) Thin-layer chromatography blots for triglyceride level measurements displayed in Fig. 5b. Raw blots available in Supplementary Fig. 7. b) Quantification of the percentage of GSCs with at least one mitochondrial unit overlapping with BODIPY-C16 for Fig. 5c. For etomoxir feeding or dMfnRNAi, p<0.0001 compared to control; dMfnRNAi+L-Carnitine p<0.0001 compared to dMfnRNAi. Two-sided fisher’s exact was used. Plot display graphical representations of proportion. N = 79 GSCs for control, n = 61 GSCs for control+etomoxir, n = 45 GSCs for dMfnRNAi, n = 43 GSCs for dMfnRNAi+L-carnitine. Raw data and statistical detail available in Supplemental Table 2.
A) IFs of testes from 5do animals stained with the lipophilic dye BODIPY 493/503 of the noted genotypes. Compare to Fig. 6a. Representative of 30 testes per condition, analyzed in 3 individual experiments. A’) Plot representation of volume of individual LDs in tips of testes of the noted genotypes. n = 10 testes per condition. Experiment was repeated 3x with similar trends. Two-tailed t-test used. A”) Distribution of the total number of LDs of a given volume in the noted genotypes (see Methods for detail on quantification). N = 122 LDs for control, n = 881 LDs for dMfnRNAi, n = 391 LDs for dMfnRNAi + L-carnitine, n = 697 LDs for dMfnRNAi+coltOE, n = 540 LDs for dMfnRNAi+bmmOE. B) Quantification of GSCs from 5do animals expressing nos-GAL4:VP16>Opa1RNAi fed either a regular diet (n = 35 testes) or a diet supplemented with L-carnitine (n = 36 testes). Two-tailed t-test used. C) Representative images of GSCs expressing nos-GAL4:VP16>GFPmito and the described genotypes. Note that the mitochondrial network in rescue experiments remains similar to the one observed in dMfnRNAi alone. 30 testes per condition. Experiment was repeated 3x with similar trends. D) Quantification of GSCs from 10do w1118 (control) animals fed either a regular diet (n = 14 testes) or a diet supplemented with 100μM etomoxir (n = 19 testes) (two-tailed t-test used). N = 14 testes (control), n = 19 testes (etomoxir); 3 biological replicates. E-G) Depletion of lipid catabolism genes in GSCs. Representative examples of testes from either control or animals expressing bmmRNAi for 10 days in early germ cells (nos-GAL4:VP16) shows accumulation of LDs in the germline (E). Image representative of 30 testes, analyzed in 3 individual experiments. Quantification of LDs per GSCs shown in F, and GSCs per testes shown in G (two-tailed t-test used). For F, n = 63 GSCs for control, n = 81 GSCs for whdRNAi, n = 62 GSCs for coltRNAi, n = 65 GSCs for bmmRNAi, n = 76 GSCs for CG6178RNAi; for G, n = 25 testes for control, n = 28 testes for whdRNAi, n = 23 testes for coltRNAi, n = 30 testes for bmmRNAi, n = 43 testes for CG6178RNAi. H-H’) Depletion of CPT2 in early germ cells with nos-GAL4:VP16 leads to a significant decrease in GSC number in third instar larvae (H; two-tailed t-test used) and an increase in GSCs positive for p4E-BP (H’; two-sided fisher’s exact used). For H, n = 30 testes for control and n = 11 testes for CPT2RNAi; for H’, n = 60 GSCs for control and n = 38 GSCs for CPT2RNAi. I) Quantification of GSCs in testes from 5do animals expressing dMfnRNAi(+GFPmito) (reproduced from Supplementary Fig. 3e) (n = 26 testes) or dMfnRNAi+LPPOE (n = 28 testes) with nos-GAL4:VP16 (two-tailed t-test used). J) Quantification of p4E-BP+ GSCs per total GSCs (two-sided fisher’s exact used). N = 49 GSCs for dMfnRNAi, n = 69 GSCs for dMfnRNAi+LPPOE. K) Quantification of LDs per GSCs in controls (n = 56 GSCs) or flies expressing nos-GAL4:VP16>TSC2RNAi (n = 58 GSCs) for 10 days (two-tailed t-test used). In all figures, asterisk (*) denotes the hub; scale bar, 20 μm. Raw data and statistical detail for data plots in A-B, D, F-K available in Supplemental Table 2. Plots in A’, B, D, F-H, I, K display mean with error bars displaying standard deviation. H’, J display graphical representations of proportion. A” displays absolute number of LDs across binning volumes of 3 μm3.
Supplementary Figure 6 Mitochondrial fusion influences lipid homeostasis to regulate GSC maintenance.
Schematic showing that disruption of mitochondrial fusion leads to loss of GSCs in Drosophila males.
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Sênos Demarco, R., Uyemura, B.S., D’Alterio, C. et al. Mitochondrial fusion regulates lipid homeostasis and stem cell maintenance in the Drosophila testis. Nat Cell Biol 21, 710–720 (2019). https://doi.org/10.1038/s41556-019-0332-3
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