Olivia Jones’s research while affiliated with Memorial Sloan Kettering Cancer Center and other places

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Publications (3)


Increasing OXPHOS does not alter proline synthesis in proliferating cells
a, Schematic representation of cellular energy metabolism. Cells cultured in a glucose-rich environment produce ATP through both glycolysis and mitochondrial respiration (left panel). Substituting glucose with galactose or depriving glucose compels cells to primarily generate ATP through glutamine dependent OXPHOS (right panel). b, Steady-state intracellular glutamate and proline level measured by GC–MS in 10T1/2, HCT116 and PANC-1 cells cultured in glucose medium (Glc) or galactose medium (Gal) for 8 h. c, Fractional labelling of indicated metabolites from [U-¹³C] glutamine. MEFs were cultured in medium containing glucose or galactose or medium without glucose for 8 h. Isotope-labelled glutamine was added in the last 4 h of the experiments. Note that these labelling are data from Fig. 1g,h replotted as percentages of all isotopologue distributions. αKG, α-ketoglutarate; Glc, glucose; Gal, galactose; – Glc, glucose deficient. d, OCR measured by Seahorse analyzer in MEFs either treated with PBS (Con) or 20 mM D-lactate (D-lac) for 2 h prior to the measurement (mean ± s.d. n = 5 independent replicates). Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot, rotenone; AA, antimycin A. e, Steady-state proline level measured by GC–MS in MEF treated with PBS (Con) or 20 mM D-lactate (D-lac) for 8 h. f, OCR measured by Seahorse analyzer in MEFs treated with vehicle (DMSO) or 1μM FCCP (mean ± s.d. n = 4 independent replicates). Rot, rotenone; AA, antimycin A. g, Intracellular proline level measured by GC–MS in MEFs treated with vehicle (–) or increasing amount of FCCP (0.5 and 1 μM, respectively) for 8 h. h, Schematic depicting the reaction catalysed by LbNOX (top) and confirmation of LbNOX expression in U2OS cells via western blot. LbNOX expression is detected using an anti-Flag tag antibody. Vinculin is used as a loading control. Cells were induced to express LbNOX by treatment with doxycycline (500 ng/mL) for 24 h. i, NADH/NAD⁺ ratio in U2OS cells expressing LbNOX. Cells were treated with doxycycline (500 ng/mL) for 48 h. j, Steady-state proline level in U2OS expressing LbNOX measured by GC–MS. Cells were treated with doxycycline (500 ng/mL) for 48 h. Data are shown as mean ± s.d. n = 3 independent replicates unless otherwise noted. Significance was assessed using two-way ANOVA (b) with Sidak’s multiple comparisons post-test relative to the controls, two-tailed t-tests (e,i,j) and one-way ANOVA (g) with Tukey’s multiple comparisons test.
Source Data
P5CS is an essential enzyme for proline biosynthesis
a, Western blot of MEF (left panel) and U2OS cells (right panel) in which CRISPR/Cas9-mediated editing was used to target Aldh18a1 (gene name for P5CS) and data for 2 edited clones shown, (hereafter sgP5CS-1 and sgP5CS-2). ROSA26 locus (sgCon for MEF) is used as control (Con) or silent gene PRM1 (sgCon for U2OS) were used as control (Con). Vinculin and Actin are used as loading controls. b,c, Steady-state level of intracellular glutamate and proline measured by GC–MS in MEF (b) and U2OS (c) cells expressing sgCon, sgP5CS-1, and sgP5CS-2. d, Cell proliferation of indicated U2OS cells after 4 days of culture. All cells were cultured in DMEM, which lacks proline (–Pro) or DMEM supplemented with 1 mM proline (+Pro). Proliferation rate was measured as population doublings per day. e–g, Western blot (e), steady-state proline level measured by GC–MS (f), and cell proliferation (g) of U2OS cells expressing sgCon, sgP5CS-1 or sgP5CS-1 with an ectopically expressing flag-tagged P5CS cDNA. The P5CS cDNA is resistant to sgP5CS-1-mediated CRISPR–Cas9 genome editing. For proliferation in f, cells were cultured in DMEM (–Pro) or DMEM supplemented with 1 mM proline (+Pro) for 4 days. Actin is used as a loading control in e. Data are shown as mean ± s.d. n = 3 independent replicates. Significance was assessed using one-way ANOVA (b,c,f) with Tukey’s multiple comparisons test.
Source Data
Reversible clustering of P5CS in mitochondria occurs in cells with an increased demand for OXPHOS
a, Representative immunostained images of endogenous P5CS and TOM20 in indicated cell lines cultured in galactose medium for 8 h. b, Western blot analysis of whole cell (input) or anti-HA immunopurified mitochondria (Mito-IP) of 293 T cells expressing HA-tagged OMP25 or the Myc-tagged OMP25 as control. All cells are cultured in glucose or galactose containing media for 8 h. In addition to immunoblotting for P5CS, immunoblotting was performed for Lamin A as a representative nuclear protein, β-Tubulin as a representative cytosolic protein, citrate synthase (CS) as a representative mitochondrial protein, GOLGA1 as a representative Golgi protein, cathepsin C (CTSC) as a representative lysosomal protein, and calreticulin (CALR) as a representative ER protein. ER, endoplasmic reticulum. c, Representative immunostaining of endogenous P5CS with mitochondrial inner membrane protein TIM23. MEFs were cultured in galactose for 8 h. The 3D reconstructed view of the gated area is shown to the right of the enlarged image. d,e, Representative images of MEFs stained for endogenous P5CS and intermembrane space protein adenylate kinase 2 (AK2) (d) matrix-localized enzymes citrate synthase (CS) and glutaminase 1 (GLS1) (e) and P5CS. MEFs were cultured in galactose for 8 h. f, Representative immunostaining of endogenous P5CS and TOM20 in MEFs treated with 20 mM D-lactate or 1 μM FCCP for 8 h. g, Representative immunofluorescence images of endogenous P5CS and TOM20 in MEFs expressing tamoxifen-inducible MYC (MYC-ER). MEFs were serum starved for 48 h and treated with 200 nM 4-hydroxytamoxifen (4-OHT) for 8 h. h, Immunostaining of endogenous P5CS and TOM20 in U2OS cells expressing LbNOX. Cells were treated with doxycycline (500 ng/mL) for 24 h prior to fixation. i, Time-lapse imaging of P5CS–GFP in MEF upon serum activation. MEFs were cultured in serum-free media overnight and then maintained in media containing 1% FBS for an additional 24 h to induce quiescence. For serum stimulation, a final concentration of 10% FBS was added to quiescent MEFs immediately before live-cell imaging to track the clustering of P5CS–GFP. Mitochondria were labelled with the mitochondria-targeted mScarlet protein (Mito). j, Western blot showing the expression of mutant P5CS in MEFs. The presence of the Flag tag confirms the expression of the mutant protein. Vinculin is used as a loading control. k, Representative live-cell images of P5CS–GFP in MEFs cultured in galactose. Shown is the reduction in P5CS–GFP filaments over a 4-hour time-lapse movie upon addition of 5 mM ornithine and 5 mM proline. For all immunostaining, DAPI was used to stain the nucleus. Scale bars, 5 μm for all images and 2 μm for all insets.
P5CS forms filamentous structures in mitochondria in vivo
a, Representative haematoxylin and eosin (H&E) staining along with cytokeratin (CK) and P5CS immunostaining, showing normal and tumour areas from a surgical specimen. b, Representative H&E staining along with P5CS and TOM20 immunostaining of two different areas from a surgical specimen, the tumour (area 1) and the surrounding normal tissue (area 2) are shown at different magnifications as indicated. The nuclei were stained with DAPI. Scale bars, 20 μm (a, left four panels and b, four right panels), 10 μm (a, four right panels) and 200 μm (b, left H&E panel).
P5CS-containing mitochondria exhibit distinct molecular features
a, Representative immunostained images of endogenous P5CS, ATP synthase (ATP5B), and TOM20 in 10T1/2 (left panel) and U2OS (right panel). All cells were cultured in galactose media for 8 h. Corresponding fluorescence pixel intensity plots for the orange lines in gated images are shown for each cell line. b, Representative three-dimensional reconstruction of z-stack images showing immunofluorescence staining for P5CS and ATP5B in MEFs cultured in galactose medium for 8 h. TIM23 was used to label the mitochondrial inner membrane. c,d, Representative images of endogenous P5CS, ATP5B and TOM20 in MEFs treated with 20 mM D-lactate (c) or 1 μM FCCP (d) for 8 h. Fluorescence pixel intensity for the orange lines in gated areas are plotted for each condition. e, Representative three-dimensional reconstruction of z-stack images in MEFs treated with 1 μM FCCP for 8 h using Imaris software showing endogenous P5CS, ATP5B, and TOM20 to label the mitochondrial outer membrane. f,g, Representative immunostained images of P5CS, ATP5B, and TOM20 in U2OS cells expressing LbNOX (f) or MEFs expressing MYC-ER (g). Cells were treated with doxycycline (500 ng/ml) for 24 h to induce expression of LbNOX (f) or with 200 nM 4-hydroxytamoxifen for 8 h to activate MYC (g). Corresponding fluorescence pixel intensity plots for the orange lines in the gated images are shown. h, Immunostaining of P5CS, ATP5A1, and TOM70 in a human PDAC specimen. Enlarged views of the boxed area are shown below. Fluorescence pixel intensity for the orange line in the boxed area is plotted on the right, next to the enlarged images. i, Representative immunostained images of P5CS with ETC complex (complex I) or ETC complex subunits (SDHA for complex II, MT-COI for complex IV) in MEFs cultured in galactose medium for 8 h. j, Western blot analysis of whole-cell lysates (Input) or streptavidin pull-down proteins from MEF expressing V5-tagged OSCP-TurboID. Cells were cultured in media with or without 5 mM ornithine and proline, as described in Fig. 3e,f and biotinylation activity was analysed by streptavidin–HRP blotting. MEFs omitting biotin (–Bio) were used as a negative control. Molecular weights are indicated. k, TMRE signal intensity distribution of mitochondria isolated from MEFs expressing P5CS–GFP. GFP signal was used to distinguish mitochondria containing P5CS (P5CS–GFP⁺) from mitochondria lacking P5CS (P5CS–GFP–). To confirm membrane potential dependent enrichment of TMRE in isolated mitochondria, mitochondria were treated with 20 μM FCCP for 30 min as an additional control. Scale bars, 5 μm for all images and 1 μm for all insets.
Source Data

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Cellular ATP demand creates metabolically distinct subpopulations of mitochondria
  • Article
  • Publisher preview available

November 2024

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785 Reads

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19 Citations

Nature

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Tak Shun Fung

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Daphne C. Baker

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[...]

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Craig B. Thompson

Mitochondria serve a crucial role in cell growth and proliferation by supporting both ATP synthesis and the production of macromolecular precursors. Whereas oxidative phosphorylation (OXPHOS) depends mainly on the oxidation of intermediates from the tricarboxylic acid cycle, the mitochondrial production of proline and ornithine relies on reductive synthesis¹. How these competing metabolic pathways take place in the same organelle is not clear. Here we show that when cellular dependence on OXPHOS increases, pyrroline-5-carboxylate synthase (P5CS)—the rate-limiting enzyme in the reductive synthesis of proline and ornithine—becomes sequestered in a subset of mitochondria that lack cristae and ATP synthase. This sequestration is driven by both the intrinsic ability of P5CS to form filaments and the mitochondrial fusion and fission cycle. Disruption of mitochondrial dynamics, by impeding mitofusin-mediated fusion or dynamin-like-protein-1-mediated fission, impairs the separation of P5CS-containing mitochondria from mitochondria that are enriched in cristae and ATP synthase. Failure to segregate these metabolic pathways through mitochondrial fusion and fission results in cells either sacrificing the capacity for OXPHOS while sustaining the reductive synthesis of proline, or foregoing proline synthesis while preserving adaptive OXPHOS. These findings provide evidence of the key role of mitochondrial fission and fusion in maintaining both oxidative and reductive biosyntheses in response to changing nutrient availability and bioenergetic demand.

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Lactate activates the mitochondrial electron transport chain independent of its metabolism

August 2023

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78 Reads

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4 Citations

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.

Citations (3)


... In a recent study published in Nature, Ryu et al. 1 demonstrate the presence of metabolically distinct mitochondrial subpopulations within one cell. One mitochondrial subpopulation contains the F 1 F O -ATP synthase for oxidative phosphorylation (OXPHOS), while a second population performs reductive biosynthesis of proline and ornithine. ...

Reference:

Mitochondrial heterogeneity: subpopulations with distinct metabolic activities
Cellular ATP demand creates metabolically distinct subpopulations of mitochondria

Nature

... The regulation of metabolic pathways within the TME also affects the efficacy of immunotherapy. Studies have found that metabolic competition in the immunosuppressive TME, such as lactate accumulation and glucose depletion, inhibits the function of effector T cells [213][214][215]. By using metabolic modulators, such as inhibitors of lactate dehydrogenase or glucose transporter protein, the metabolic state of the TME can be reprogrammed, restoring T-cell antitumor activity [216,217]. ...

Lactate activates the mitochondrial electron transport chain independently of its metabolism
  • Citing Article
  • October 2023

Molecular Cell

... In the cell culture media, lactate, a byproduct of glucose metabolism as well as a stimulator of mitochondrial electron transport chain (ETC) activity [23], showed significant decline in senescent cells' media. Senescent HBMECs display significantly lower release of another four metabolites by t-test only, including guanine, hypoxanthine (both purine bases), 2 ′ -deoxycytidine, and O-succinyl-homoserine, and increased production of L-thyronine, 3-or 4-methyl-2-oxopentanoate, and 3-methyl-2-oxobutanoate, with the latter two being the first intermediates of branched-chain amino acid (BCAA) degradation ( Table 2). ...

Lactate activates the mitochondrial electron transport chain independent of its metabolism
  • Citing Preprint
  • August 2023