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Mitochondrial structure and function are disrupted by standard isolation methods.

ABSTRACT: Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca(2+) handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca(2+) challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H(2)O(2) production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.

SUBMITTER: Picard M

PROVIDER: S-EPMC3065478 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Mitochondrial structure and function are disrupted by standard isolation methods.

Picard Martin M Taivassalo Tanja T Ritchie Darmyn D Wright Kathryn J KJ Thomas Melissa M MM Romestaing Caroline C Hepple Russell T RT

PloS one 20110328 3

Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca(2+) handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intra ...[more]

PMID: 21512578

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Project description:Algae with potential biotechnological applications in different industries are commonly isolated from the environment in order to obtain pure (axenic) stocks that can be safely stored for long periods of time. To obtain axenic cultures, antibiotics are frequently employed, and cryopreservation is applied to preserve standing stocks. However, many of these now standard methods were developed using strains derived from pristine to near-pristine environments and cold to temperate regions. The potential effect of the said methods on the life cycle and biochemical profile of algae isolates from hyper-eutrophic and constant high-temperature tropical regions is not well understood. These effects could potentially render them unsuitable for their intended biotechnological application. In this study, we conducted a genetic characterization (18S rRNA) and evaluated the effect of purification (the use of the antibiotic chloramphenicol, CAP) and cryopreservation (dimethyl sulfoxide; DMSO-sucrose mix and glycerol) on the growth rate and lipid content of three new tropical freshwater algal isolates: Chorella sp. M2, Chlorella sp. M6, and Scenedesmus sp. R3, obtained from the Ecuadorian coast. The genetic and morphological characterization revealed a clear discrimination between these strains. All strains cultured with CAP exhibited a lower growth rate. Subsequent to cryopreservation, Chorella sp. M2, Chlorella sp. M6, and Scenedesmus sp. R3 presented no significant difference in growth rate between the cryopreservants. Further, a significantly higher lipid content was observed in the biomass cryopreserved with glycerol in relation to the DMSO-sucrose, with Chorella sp. M2 and Chlorella sp. M6 having twice as much as they had in the first treatment. These results highlight the relevance of selecting an appropriate method for storage, as the materials used can affect the biological performance of different tropical species, although it is still to be determined if the effects observed in this study are long lasting in subsequent cultures of these algae.

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Mitochondrial structure and function are disrupted by standard isolation methods.

Publications

Mitochondrial structure and function are disrupted by standard isolation methods.

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