Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study

One of the most crucial steps in mitochondrial isolation is disruption of intact cells to denude intracellular organelles, but the yield and purity of different disruption protocols have not been well addressed. In the present study, MDCK cells were disrupted by mechanical (sonication and homogenization), physical (repeated freeze/thaw cycles and hypoosmotic burst), and chemical (using Triton X-100, NP-40, or CHAPS) methods. Efficacy of cell disruption was evaluated by trypan blue staining and mitochondria were subsequently isolated by standardized differential centrifugation. The yield of isolation was also determined by measuring protein concentrations, whereas the purity was examined by Janus green B staining, Western blot analyses of markers for mitochondria (COX-4) and other subcellular organelles/locales (i.e., nucleus, cytoplasm, endoplasmic reticulum, and lysosome), transmission electron microscopy, two-dimensional electrophoresis, and Q-TOF MS and/or MS/MS analyses. Our data demonstrated that sonication is the method of choice for disruption of cells prior to mitochondrial isolation for proteome analysis.     

Murine leukemic lymphoblasts: homogenization and fractionation procedures for plasma membrane vesicles isolation

Plasma membrane vesicles were isolated from murine leukemic lymphoblasts L5178Y. The isolation procedure selected involved a method of mechanical disruption in a hypoosmotic-buffered solution and the separation of plasma membrane vesicles by an adaptation of the fractionation method described by D. W. McKeel and L. Jarett for fat cells (J. Cell Biol., 44, 417, 1970). In order to select the homogenization method we took into account several parameters: the extent of cell and nuclear disruption, the integrity of the nuclear membrane, the 5′-nucleotidase activity recovered at the first step of fractionation and the mitochondrial rupture. The homogenization method finally used yielded 89% of cellular rupture with only 9% of nuclear damage. The isolation procedure showed an overall yield of 70–90%. A plasma membrane fraction was isolated with an enrichment in 5′-nucleotidase and ouabain-sensitive (Na⁺K⁺)-ATPase specific activities of 15- and 13-fold, respectively, and essentially free of mitochondrial, lysosomal, and endoplasmic reticulum contamination. The electron microscopy demonstrated that the plasma membrane fraction essentially consisted of smooth vesicles of several sizes.     

Isolation of functional pure mitochondria by superparamagnetic microbeads

Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2 h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays.     

A simple and efficient procedure for the rapid homogenization of cultured animal cells grown in monolayer

Cell swelling takes place rapidly when animal cells in monolayer culture are treated with hypotonic buffer in situ; scraping of the swollen cells causes virtually 100% cell lysis. Because this procedure avoids the use of scraping and centrifugation to collect the cells and the use of Dounce homogenizers for cell disruption, recovery of the cell extract is very high. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activities of cell extracts prepared by this method are virtually identical to those prepared by the conventional procedure involving Dounce homogenization.     

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator’s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10¹⁰ viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.     

Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.     

A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system

Summary A dextran-polythylene glycol aqueous two-phase system has been used to separate cell surface membranes from other cellular organelles. The surface membranes have been identified on the basis of morphology, content of Na⁺, K⁺-ATPase, and presence of surface antigen as detected by a⁵¹Cr release method. Contamination of the surface membrane preparations by smooth endoplasmic reticulum, mitochondria, and nuclei has been found to be minimal. An average of 6.5% of the total protein was found in the membrane fraction. Less than two hours is required to isolate the membrane fraction after preparation of a Dounce homogenate. Fractionation by aqueous two-phase polymer systems appears to be a rapid and effective method for the isolation of surface membranes.     

Protein synthesis in mitochondria. 4. Preparation and properties of mitochondria from Krebs II mouse ascites-tumour cells

1. Methods of disrupting Krebs II mouse ascites-tumour cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. 2. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0·3 m-sucrose. 3. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succin-oxidase was measured in the various cell fractions after separation by differential centrifuging. 4. The mitochondrial fraction sedimented at 9500g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500g pellet contains other material besides mitochondria. 5. Krebs-cell mitochondria contain up to five times as much RNA as do liver mitochondria. 6. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.     

Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis

Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.     

Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: An old tool for a new application

The nematode Caenorhabditis elegans is a model organism best known for its powerful genetics. There is an increasing need in the worm community to couple genetics with biochemistry. Isolation of functionally active proteins or nucleic acids without the use of strong oxidizing denaturants or of subcellular compartments from C. elegans has, however, been challenging because of the worms’ thick surrounding cuticle. The Balch homogenizer is a tool that has found much use in mammalian cell culture biology. The interchangeable single ball-bearing design of this instrument permits rapid permeabilization, or homogenization, of cells. Here we demonstrate the utility of the Balch homogenizer for studies with C. elegans. We describe procedures for the efficient breakage and homogenization of every larval stage, including dauers, and show that the Balch homogenizer can be used to extract functionally active proteins. Enzymatic assays for catalase and dihydrolipoamide dehydrogenase show that sample preparation using the Balch homogenizer equals or outperforms conventional methods employing boiling, sonication, or Dounce homogenization. We also describe phenol-free techniques for isolation of genomic DNA and RNA. Finally, we used the tool to isolate coupled mitochondria and polysomes. The reusable Balch homogenizer represents a quick and convenient solution for undertaking biochemical studies on C. elegans.     

Tissue-culture fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients.

1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.     

Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes

Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are "immature," in contrast to "mature" mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 microM for 15 min) hydrogen peroxide (H(2)O(2)) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H(2)O(2) induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.     

Improve method for isolation of rat liver plasma membrane

An improved method for the isolation of plasma membrane from rat liver is presented.Gentle homogenization of perfused livers in buffered isotonic KCI, followed by direct flotation of a low-speed nuclear pellet through a discontinuous sucrose density gradient results in a 32% yield, and 25-fold enrichment for the plasma membrane marker, phosphodiesterase I, in a crude plasma membrane fraction. This fraction contains less than 1% of the mitochondria, and endoplasmic reticulum present in the original homogenate, but is more heavily contaminated with lysosomes and Golgi membrane.Vigorous mechanical disruption of this material, followed by a second discontinuous sucrose density gradient, gives a light plasma membrane fraction with an 80-fold purification and 20% yield of phosphodiesterase I over the original homogete (with further reduction of contaminants).     

Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria.

Because of its importance in the chemiosmotic theory, mitochondrial membrane potential has been the object of many investigations. Significantly, however, quantitative data on how energy transduction might be regulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have not been possible because of their small size, their intrinsic motility, and the absence of appropriate analytical reagents. In this article, we combine techniques for rapid, high resolution, quantitative three-dimensional imaging microscopy and mathematical modeling to determine accurate distributions of a potentiometric fluorescent probe between the cytosol and individual mitochondria inside a living cell. Analysis of this distribution via the Nernst equation permits assignment of potentials to each of the imaged mitochondrial membranes. The mitochondrial membrane potentials are distributed over a narrow range centered at -150 mV within the neurites of differentiated neuroblastoma cells. We find that the membrane potential of a single mitochondrion is generally remarkably stable over times of 40-80 s, but significant fluctuations can occasionally be seen. The motility of individual mitochondria is not directly correlated to membrane potential, but mitochondria do become immobile after prolonged treatment with respiratory inhibitors or uncouplers. Thus, three spatial dimensions, a key physiological parameter, and their changes over time are all quantitated for objects at the resolution limit of light microscopy. The methods described may be readily extended to permit investigations of how mitochondrial function is integrated with other processes in the intact cell.     

Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria

Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in “reverse” mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.     

The preparation of rat-liver mitochondria by conventional differential centrifugation and by zonal Centrifugation,after various disruption procedures: A comparative study

Rat liver was subjected to three different, disruption procedures (homogenization, explosive decompression, and Chaikoff press) and mitochondria were subsequently isolated by conventional differential Centrifugation and by zonal Centrifugation. The properties of these mitochondria were investigated by polarographic measurement of oxygen uptake and they were examined by electron microscopy. All three methods of disruption gave mitochondria which showed respiratory control. Nitrogen cavitation gave the most reproducible conditions for cell breakage and zonal Centrifugation gave good separation of subcellular organdies in extracts produced by this method. Some separation of the heterogenous mitochondrial populations was achieved by zonal Centrifugation.     

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.     

A rapid procedure for isolating hemopoietic cell nuclei

A new method for isolating cell nuclei is described which involves freezing and thawing cells in 2% Tween 40, then gentle homogenization to release nuclei, followed by immediate microcentrifugation through 50% sucrose. Purified nuclei were obtained in 3 min and yields of 78-95% were obtained from a variety of human hemopoietic cells. Electron microscope analysis of nuclei obtained from HL60 cells showed that 89% of the nuclei were intact and have an appropriate morphology. A low level of contamination with other organelles was revealed by electron microscopy and by using specific assays for plasma membrane, mitochondria, lysosomes, Golgi membrane, and endoplasmic reticulum (0.5-5.5%). The value of the technique is that nuclear proteins and small metabolites which might be lost by rapid leakage from isolated nuclei and the possibility of biochemical modification of cellular constituents are minimized by using a rapid isolation procedure.     

Preparation of subcellular fractions from rat liver: comparison of the Polytron with the Dounce homogenizer.

The PolytronR and Dounce homogenizers have been evaluated for preparation of homogenates of rat liver prior to isolation of subcellular fractions by differential centrifugation. Marker enzymes used to evaluate the subcellular fractions included cytochrome oxidase, monoamine oxidase, D-amino acid oxidase, acid phosphatase, glucose-6-phosphatase, ethyl morphine demethylase, and lactate dehydrogenase. No significant difference in the distribution of enzymes (percent recovery or specific activity) was observed between the two methods of homogenization. In addition, there were no significant differences in the ultrastructural appearances and respiratory control ratios of the mitochondrial fractions prepared by the two methods of homogenization.     

Mitochondria in apoptosis and human disease

Apoptosis is a process of cell suicide whereby individual cells are destroyed while preserving the integrity and architecture of surrounding tissue. This targeted cell destruction is critical both in physiological contexts as well as pathological states. It seems increasingly evident that mitochondria play an important role in the regulation of programmed cell death via release of proapoptotic agents and/or disruption of cellular energy metabolism. The mechanisms of mitochondrial involvement are beginning to be elucidated, and may involve the participation of bcl-2 family members, reactive oxygen species, and caspases. As part of a central mechanism of amplification of the apoptotic signal, mitochondria may be an appropriate target for therapeutic agents designed to modulate apoptosis. This review focuses on recent advances in understanding mitochondrial mechanisms in apoptosis and the involvement of these pathways in human disease.