Measurements of glycolysis and mitochondrial function are required to quantify energy metabolism in a wide variety of cellular contexts. In human pluripotent stem cells (hPSCs) and their ...differentiated progeny, this analysis can be challenging because of the unique cell properties, growth conditions and expense required to maintain these cell types. Here we provide protocols for analyzing energy metabolism in hPSCs and their early differentiated progenies that are generally applicable to mature cell types as well. Our approach has revealed distinct energy metabolism profiles used by hPSCs, differentiated cells, a variety of cancer cells and Rho-null cells. The protocols measure or estimate glycolysis on the basis of the extracellular acidification rate, and they measure or estimate oxidative phosphorylation on the basis of the oxygen consumption rate. Assays typically require 3 h after overnight sample preparation. Companion methods are also discussed and provided to aid researchers in developing more sophisticated experimental regimens for extended analyses of cellular bioenergetics.
Small, rapidly dividing pluripotent stem cells (PSCs) have unique energetic and biosynthetic demands compared with typically larger, quiescent differentiated cells. Shifts between glycolysis and ...oxidative phosphorylation with PSC differentiation or reprogramming to pluripotency are accompanied by changes in cell cycle, biomass, metabolite levels, and redox state. PSC and cancer cell metabolism are overtly similar, with metabolite levels influencing epigenetic/genetic programs. Here, we discuss the emerging roles for metabolism in PSC self-renewal, differentiation, and reprogramming.
It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells ...harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F1F0 ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.
While studying metabolic fluxes in human pluripotent stem cells, this paper reveals UCP2 as metabolic switch from glycolysis to OXPHOS, facilitating early differentiation events.
Peroxisomal biogenesis disorders (PBDs) are genetic disorders of peroxisome biogenesis and metabolism that are characterized by profound developmental and neurological phenotypes. The most severe ...class of PBDs—Zellweger spectrum disorder (ZSD)—is caused by mutations in peroxin genes that result in both non‐functional peroxisomes and mitochondrial dysfunction. It is unclear, however, how defective peroxisomes contribute to mitochondrial impairment. In order to understand the molecular basis of this inter‐organellar relationship, we investigated the fate of peroxisomal mRNAs and proteins in ZSD model systems. We found that peroxins were still expressed and a subset of them accumulated on the mitochondrial membrane, which resulted in gross mitochondrial abnormalities and impaired mitochondrial metabolic function. We showed that overexpression of ATAD1, a mitochondrial quality control factor, was sufficient to rescue several aspects of mitochondrial function in human ZSD fibroblasts. Together, these data suggest that aberrant peroxisomal protein localization is necessary and sufficient for the devastating mitochondrial morphological and metabolic phenotypes in ZSDs.
Synopsis
How peroxisomal biogenesis disorders lead to mitochondrial dysfunction is not well understood. This study reveals that peroxisomal proteins mislocalize to the mitochondria, thereby disrupting mitochondrial function. The mitochondrial quality control protein ATAD1 can rescue this defect.
Peroxin transcription and translation is unperturbed by loss of peroxisomes.
Several peroxins (Pex13, Pex11, Pex14, Pex2, Pex17, Pex25) mislocalize to the mitochondrial outer membrane.
Mislocalized peroxins are able to form a subassembly of the peroxisomal importomer on mitochondria, which interferes with mitochondrial function.
The mitochondrial extractase ATAD1 can remove peroxins from the mitochondrial membrane, which is sufficient to restore mitochondrial morphology, respiration, and metabolism in cells derived from ZSD patients.
How peroxisomal biogenesis disorders lead to mitochondrial dysfunction is not well understood. This study reveals that peroxisomal proteins mislocalize to the mitochondria, thereby disrupting mitochondrial function. The mitochondrial quality control protein ATAD1 can rescue this defect.
The mitochondrial intermembrane space (IMS) is involved in protein transport, lipid homeostasis and metal ion exchange, while further acting in signalling pathways such as apoptosis. Regulation of ...these processes involves protein modifications, as well as stress-induced import or release of proteins and other signalling molecules. Even though the IMS is the smallest sub-compartment of mitochondria, its redox state seems to be tightly regulated. However, the way in which this compartment participates in the cross-talk between the multiple organelles and the cytosol is far from understood. Here we focus on newly identified IMS proteins that may represent future challenges in mitochondrial research. We present an overview of the import pathways, the recently discovered new components of the IMS proteome and how these relate to key aspects of cell signalling and progress made in stem cell and cancer research.
Display omitted
•A brief overview of the classic mitochondrial import pathways is featured•Recent studies assigning a number of new proteins to the mitochondrial IMS are discussed•Analysis of the expanded IMS proteomes can provide insights into organelle cross-talk and signalling pathways
Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an ...AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.
Display omitted
•AIF interacts with CHCHD4, a regulator of the intermembrane space import machinery•AIF regulates specific respiratory chain complexes by acting upstream of CHCHD4•AIF is indispensable for translation-coupled mitochondrial import of CHCHD4•Restoring CHCHD4 reverses the metabolic and cell death phenotypes of Aif−/y ESCs
Hangen et al. show that the mitochondrial protein AIF regulates the biogenesis of respiratory chain complexes by interacting with, and by controlling the mitochondrial import of the mammalian homolog of yeast MIA40, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery.
Abstract
Context
The etiology of primary ovarian insufficiency (POI) remains unknown in most cases.
Objective
We sought to identify the genes causing POI.
Design
The study was a familial genetic ...study.
Setting
The study was performed at two academic institutions.
Patients
We identified a consanguineous Yemeni family in which four daughters had POI. A brother had azoospermia.
Intervention
DNA was subjected to whole genome sequencing. Shared regions of homozygosity were identified using Truploidy and prioritized using the Variant Annotation, Analysis, and Search Tool with control data from 387 healthy subjects. Imaging and quantification of protein localization and mitochondrial function were examined in cell lines.
Main Outcome
Homozygous recessive gene variants shared by the four sisters.
Results
The sisters shared a homozygous stop gain mutation in exon 6 of PSMC3IP (c.489 C>G, p.Tyr163Ter) and a missense variant in exon 1 of CLPP (c.100C>T, p.Pro34Ser). The affected brother also carried the homozygous PSMC3IP mutation. Functional studies demonstrated mitochondrial fragmentation in cells infected with the CLPP mutation. However, no abnormality was found in mitochondrial targeting or respiration.
Conclusions
The PSMC3IP mutation provides additional evidence that mutations in meiotic homologous recombination and DNA repair genes result in distinct female and male reproductive phenotypes, including delayed puberty and primary amenorrhea caused by POI (XX gonadal dysgenesis) in females but isolated azoospermia with normal pubertal development in males. The findings also suggest that the N-terminal missense mutation in CLPP does not cause substantial mitochondrial dysfunction or contribute to ovarian insufficiency in an oligogenic manner.
Whole genome sequencing identified a stop gain mutation in PSMC3IP in a family in which four daughters had delayed puberty and ovarian insufficiency and one brother had normal puberty and azoospermia.
Mitochondria of the strictly aerobic yeast Yarrowia lipolytica contain respiratory complex I with close functional and structural similarity to the mammalian enzyme. Unlike mammalian mitochondria, ...however, Yarrowia mitochondria have been thought not to contain supercomplexes. Here, we identify respiratory supercomplexes composed of complexes I, III and IV also in Y. lipolytica. Evidence for dimeric complex I suggests further association of respiratory supercomplexes into respiratory strings or patches. Similar supercomplex organization in Yarrowia and mammalian mitochondria further makes this aerobic yeast a useful model for the human oxidative phosphorylation system. The analysis of supercomplexes and their constituent complexes was made possible by 2-D native electrophoresis, i.e. by using native electrophoresis for both dimensions. Digitonin and blue-native electrophoresis were generally applied for the initial separation of supercomplexes followed by less mild native electrophoresis variants in the second dimension to release the individual complexes from the supercomplexes. Such 2-D native systems are useful means to identify the constituent proteins and their copy numbers in detergent-labile physiological assemblies, since they can reduce the complexity of supramolecular systems to the level of individual complexes.
The large membrane protein complexes of mitochondrial oxidative phosphorylation are composed of central subunits that are essential for their bioenergetic core function and accessory subunits that ...may assist in regulation, assembly or stabilization. Although sequence conservation is low, a significant proportion of the accessory subunits is characterized by a common single transmembrane (STMD) topology. The STMD signature is also found in subunits of other membrane protein complexes. We hypothesize that the general function of STMD subunits is to organize the hydrophobic subunits of large membrane protein complexes in specialized environments like the inner mitochondrial membrane.
Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into ...the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na(+)/H(+) antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
