Staining of cellular mitochondria with LDS-751 (original) (raw)
Related papers
Localization of mitochondria in living cells with rhodamine 123
Proceedings of the National Academy of Sciences, 1980
The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment.
Journal of Microscopy, 1991
Cultured rat fibroblasts were exposed to millimolar concentrations of forty-four noncationic fluorescent probes, of very varied physico-chemical properties. Mitochondria1 staining occurred with nineteen of these probes, nine of which were nominally anionic and ten nominally non-ionic. All nineteen were in fact lipophilic weak acids. Using structural parameters these could be specified numerically as follows: electric charge < 0; log P(1ess-ionized form) < 0; and pKa x 7. In addition to these structural variables, dye concentration and the time of exposure of cells to probes were significant factors for the staining of mitochondria. Accumulation of these compounds can be understood in terms of ion-trapping of hydrophilic salts of lipophilic weak acids, due to the internal pH of respiring mitochondria being higher than the cytosolic pH. As a case example of the application of this approach, the mode of action of many inhibitors of mitochondrial anabolism is discussed in terms of the mechanisms introduced here.
Cancer Research, 1980
Mitochondria were isolated from whole homogenates of nor mal liver and Novikoff hepatomas using reorienting rate zonal centrifugation on sucrose gradients. The activities of several mitochondrial-specific enzymes and ultrastructure were com pared in the two tissues. Our results indicate that cytochrome oxidase, lipoamide dehydrogenase, malate dehydrogenase, and Buccinate dehydrogenase activities are all higher in liver homogenates than in Novikoff hepatoma homogenates. Mitochondrial hexokinase, however, is much greater in the hepa toma than in liver. The activity of these enzymes in isolated mitochondria displayed a much different pattern. Both cyto chrome oxidase and succinate dehydrogenase activities were higher in hepatoma mitochondria than in liver mitochondria. Lipoamide dehydrogenase and malate dehydrogenase, con versely, were higher in liver mitochondria. Hexokinase was found to be virtually absent in liver mitochondria but plentiful in hepatoma mitochondria. Ultrastructural studies have shown that the hepatoma mitochondria are much smaller in size, which results in a decreased rate of migration into the gradient. These studies have also shown that normal liver consists of predom inantly "condensed" forms of mitochondria, whereas hepa toma contained a majority of "twisted" species. Experiments using 1% bovine serum albumin in the homogenization proce dures and in the gradient have confirmed earlier observations that bovine serum albumin is essential for optimal isolation of neoplastic mitochondria.
Acta Histochemica, 2020
Mitochondria are highly dynamic intracellular organelles with ultrastructural heterogeneity reflecting the behaviour and functions of the cells. The ultrastructural remodelling, performed by the counteracting active processes of mitochondrial fusion and fission, enables the organelles to respond to diverse cellular requirements and cues. It is also an important part of mechanisms underlying adaptation of mitochondria to pathophysiological conditions that challenge the cell homeostasis. However, if the stressor is constantly acting, the adaptive capacity of the cell can be exceeded and defective changes in mitochondrial morphology (indicating the insufficient functionality of mitochondria or development of mitochondrial disorders) may appear. Beside qualitative description of mitochondrial ultrastructure, stereological principles concerning the estimation of alterations in mitochondrial volume density or surface density are invaluable approaches for unbiased quantification of cells under physiological or pathophysiological conditions. In order to improve our understanding of cellular functions and dysfunctions, transmission electron microscopy (TEM) still remains a gold standard for qualitative and quantitative ultrastructural examination of mitochondria from various cell types, as well as from those experienced to different stimuli or toxicity-inducing factors. In the current study, general morphological and functional features of mitochondria, and their ultrastructural heterogeneity related to physiological and pathophysiological states of the cells are reviewed. Moreover, stereological approaches for accurate quantification of mitochondrial ultrastructure from electron micrographs taken from TEM are described in detail.
Experimental Cell Research, 1980
Synthesis of mitochondrial proteins was studied in a monkey kidney epithelium cell line by electron microscope radioautography after pulse label for 1 h with [3H]leucine in the presence of cycloheximide followed by chase in the absence of the antibiotic. At all chase times tested the highest specific activities were found over mitochondria and lysosomes (dense bodies). Based on regression analysis of the decay curve of mitochondrial labeling, there appeared to be two main mitochondrial populations with apparent half-lives of l-2 h and 6 days. In parallel experiments, cells were labelled with [3H]leucine in the presence of cycloheximide plus chloramphenicol. Chloramphenicol markedly inhibited labelling over mitochondria and dense bodies, whereas it had no significant effect on labelling of other cell compartments (nucleus, endoplasmic reticulum, remainder cytoplasm). The significance of the cycloheximide-insensitive and chloramphenicolsensitive lysosomal labelling is discussed. The data suggest that in cultured cells lysosomes are involved in the degrading of mitochondrial proteins.
Pfl�gers Archiv European Journal of Physiology, 2000
The mitochondrial membrane potential (Ψ mito ) is an important parameter not only of mitochondrial but also of cellular status. Prolonged mitochondrial depolarization is associated with various forms of neuronal death. Assessment of mitochondrial depolarization can take advantage of the specific properties of the lipophilic dyes that distribute in a potentiometrically determined ratio across membranes. Using conventional imaging, we showed that rhodamine 123 accumulated in the mitochondria, generating a highly heterogeneous pattern of spatial distribution of fluorescence across the cell body. Collapse of the Ψ mito following exposure to a protonophore, carbonylcyanide p-chloromethoxyphenylhydrazone (CCCP), released rhodamine 123 from mitochondria into the cytosol. Under acutely changed conditions, this increased the overall intensity of the fluorescence signal and significantly decreased the degree of spatial heterogeneity of the signal. If mitochondrial depolarization was sustained chronically, the intensity of the signal decreased, but the increase in the spatial homogeneity of the fluorescent signal was maintained. Image analysis showed that the level of spatial heterogeneity of the signal can be assessed by calculating, for each individual neurone, the spread of pixel intensities values around the mean. This spread is defined by the coefficient of variation (CV), which is a measure of the standard deviation normalized to the average, and was inversely related to mitochondrial depolarization measured under different conditions. Thus, the degree of spatial heterogeneity of the rhodamine 123 signal measured from a neurone is a reliable indicator for the assessment of mitochondrial depolarization and can be used in experiments to monitor Ψ mito over shorter or longer periods.
Analysis of Mitochondria Isolated From Single Cells
Analytical and …, 2007
Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrionspecific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.
The Journal of Cell Biology
The buoyant densities of the nuclear and mitochondrial DNA from the thoracic muscles of Sch~stocerca gregarm ,,-ere found to be 1 702 and 1.689 g/cm s, respectively, corresponding to guanine plus cytosine (G + C) content of 42.2 and 30% A prehminary treatment of the mitochondrial pellet with DNase (25°C, 20 min) is necessary to eliminate the contaminating nuclear DNA. The mitochondrml DNA renatures readily after heat denaturation and incubation at 65°C. The DNA released from the mltochondrial pellet by osmotic shock consists of circular open and closed molecules with a contour length around 5/~ The instability of insect mitochondrla in in vitro preparations is discussed.
Mitochondria are morphologically and functionally heterogeneous within cells
The EMBO journal, 2002
We investigated whether mitochondria represent morphologically continuous and functionally homogenous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or calcein. The mitochondria of HeLa, PAEC, COS-7, HUVEC, hepatocytes, cortical astrocytes and neuronal cells all displayed heterogeneous distributions and were of varying sizes. There was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could be seen clearly. Using fluorescence-recovery after photobleaching, we observed that DsRed1 and calcein were highly mobile within the matrix of individual mitochondria, and that mitochondria within a cell were not lumenally continuous. Mitochondria were not electrically coupled, since only individual mitochondria were observed to depolarize following irradiation of TMRE-loaded cells. Function...
European journal of biochemistry, 1990
Washed and purified rat-or mouse-liver mitochondria exhibiting high membrane integrity and metabolic activity were studied by flow cytometry. The electrophoretic accumulation/redistribution of cationic lipophilic probes, rhodamine 123, safranine 0 and a cyanine derivative, 3,3'-dihexyloxadicarbocyanine iodide, during the energization process was studied and was consistent with the generation of a negative internal membrane potential. An exception to this was nonylacridine orange which spontaneously bound to the mitochondrial membrane by hydrophobic interactions via its hydrocarbon chain. Energized purified mitochondria stained with potentiometric dyes exhibited both higher fluorescence and population homogeneity than the non-energized or deenergized (nigericin plus valinomycin) mitochondria. By contrast, under non-energized or deenergized conditions, the mitochondrial population exhibited fluorescence intensity heterogeneity related to the residual membrane potential; two subpopulations were evident, one of low fluorescence which may be related to the autofluorescence of the mitochondria (plus non-specific dye binding) and a second population which exhibited high fluorescence. Flow cytometry of the unpurified, simply washed, rat-liver mitochondria stained with rhodamine 123, a classically used dye, provided evidence of their heterogeneity in terms of light-scattering properties and membranepotential-related fluorescence. One third of the washed mitochondria were found to be non-functional by such assays. The fluorescence of purified rat-liver mitochondria due to the membrane potential built up by endogenous substrates indicates heterogeneity of the mitochondrial population with respect to levels of endogeneous substrates. The low-angle light scattering increases upon energization and provides some original information about the shape and modification of the inner mitochondrial conformation accompanying the energization. The heterogeneity of the rat liver mitochondrial population, from a structural, metabolic (existence of endogenous substrates) and functional (active and non-active mitochondrial population dispersion) point of view could thus be demonstrated by flow-cytometry analysis. Two animal models were examined with regard to the alteration of the mitochondrial membrane potential under the effects of drugs (rat-liver mitochondria), and the effects of ammonium toxicity (mouse-liver mitochondria). These results are promising and open new perspectives in the study of mitochondriopathies. Energy-transducing organelles, such as mitochondria, generate a high proton electrochemical gradient, as predicted by Mitchell's chemiosmotic hypothesis [l]. This proton gradient (dpH ') comprises two components : a membrane potential difference (Ay), negative interior, and a pH difference (ApH). In mitochondria, the membrane potential predominates over the dpH component.