Analysis of freeze-fracture electron micrographs by a computer-based technique

Mehlhorn, Rolf J.; Packer, Lester

doi:10.1016/s0006-3495(76)85716-5

Cited by 19 publications

(9 citation statements)

References 7 publications

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“…As an additional index of IMP distribution, we also studied the extent of aggregation by comparison with Poisson expectations. This approach has often been used by other investigators (1,36,43). Table I shows the results of Chi-square analysis based on differences betwen Poisson expectations and the observed number of IMP per quadrat.…”

Section: Discussionmentioning

confidence: 99%

“…Several types of measurements have been applied to the analysis of particle distributions in membranes. Techniques of radial distribution analysis (18,34,45) allow accurate modelling of IMP aggregation in one or a few micrographs (22,36,43). These techniques involve the selection of a representative micrograph and calculation of relative frequencies over a range of interparticle distances.…”

Section: Statistical Analysis Of Particle Distributionsmentioning

confidence: 99%

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Cell fusion and intramembrane particle distribution in polyethylene glycol-resistant cells.

Roos¹,

Robinson²,

Davidson³

1983

The Journal of Cell Biology

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The distribution of intramembrane particles (IMP) as revealed by freeze-fracture electron microscopy has been analyzed following treatment of mouse L cells and fusiondeficient L cell derivatives with several concentrations of polyethylene glycol (PEG). In cell cultures treated with concentrations of PEG below the critical level for fusion, no aggregation of IMP was observed. When confluent cultures of the parental cells are treated with 50% PEG, >90% of the cells fuse, and cold-induced IMP aggregation is extensive. In contrast, identical treatment of fusion-deficient cell lines shows neither extensive fusion nor IMP redistribution. At higher concentrations of PEG, however, the PEG-resistant cells fuse extensively and IMP aggregation is evident. Thus the decreased ability of the fusion-deficient cells to fuse after treatment with PEG is correlated with the failure of IMP aggregation to occur. A technique for quantifying particle distribution was developed that is practical for the accurate analysis of a large number of micrographs. The variance from the mean number of particles in randomly chosen areas of fixed size was calculated for each cell line at each concentration of PEG. Statistical analysis confirms visual observation of highly aggregated IMP, and allows detection of low levels of aggregation in parental cells that were less extensively fused by exposure to lower concentrations of PEG. When low levels of fusion were induced in fusion-deficient cells, however, no IMP aggregation could be detected.

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Section: Discussionmentioning

confidence: 99%

Section: Statistical Analysis Of Particle Distributionsmentioning

confidence: 99%

Cell fusion and intramembrane particle distribution in polyethylene glycol-resistant cells.

Roos¹,

Robinson²,

Davidson³

1983

The Journal of Cell Biology

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“…CDTAB can flip-flop in certain systems, e.g., by using the assay method essentially of Kornberg and McConnell (17) it was found by Mehlhorn and Packer (20) Our results of CDTAB spin reduction in mitoplasts, SMP, and by the semiquinone free radical, similar inhibitor sensitivity, midpoint potential range and a certain degree of structural similarity, suggest that the piperidine label on CDTAB (+60 mV) can accept reducing equivalents from electron transport in the ubiquinone region at each membrane interface, The physical properties expected from the structure of CDTAB suggest that its hydrophobic moiety will locate inside the membrane with the cationic moiety directed toward either surface. In terms of the strategy outlined above, we are led to suggest that ubiquinone must reside wherever the nitroxyl of CDTAB resides, namely, toward either interface.…”

Section: Discussionmentioning

confidence: 99%

Surface localization of sites of reduction of nitroxide spin-labeled molecules in mitochondria.

Quintanilha¹,

Packer²

1977

Proc. Natl. Acad. Sci. U.S.A.

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The relative rates of reduction of several spin-labeled molecules that partition differently across the hydrophobic-interface of inner membranes from rat liver mitochondria were investigated. Spin labels localized either deep in the hydrophobic region or in the aqueous phase are only slowly reduced; however, a spin-labeled analogue of the cationic detergent cetyltrimethylammonium bromide that partitions at the interface is rapidly reduced by coupled electron transport. Chemical studies on the reduction and oxidation of the spin label show that loss of signal is due to reduction and not destruction ofthe label. No evidence was found for flip-flop of the label in submitochondrial preparations.Spin reduction of respiring mitochondria, mitoplasts, or in- (4)(5)(6)(7)(8). By manipulation of available 02, with inhibitors specific for particular "sites" and uncouplers of respiration, it is sometimes possible to deduce the particular "site" at which the substance is being reduced. On the other hand, the test substance, e.g., a spin label, may have physicochemical properties ensuring that it will come to rest in a particular region of the mitochondrial structure. When both of these circumstances are realized, one can logically conclude that a transfer of reducing equivalents from the electron transport chain to the spin label goes on in the particular region of the mitochondrial structure where the spin-labeled molecule is localized. Our paper illustrates the foregoing strategy. To apply this strategy, we had to find a spin-labeled substance with the right properties (an analogue of cetyltrimethylammonium bromide, CDTAB) to show that after it was intercalated into the mitochondrial structure its signal decayed due to respiratory chain activity, and finally to show by manipulation of this activity that the reductant in the chain most likely to be affecting the spin label was ubiquinone.MATERIALS AND METHODS Preparations. Rat liver mitochondria were prepared in a 0.25 M sucrose, 1 mM Tris-HCl and 1 mM EDTA medium at pH 7.4 as described (9). Mitoplasts were also prepared (10). Submitochondrial preparations (SMP) were made following a method described in ref. 11. Protein concentrations were determined by a standard method (12).Spin Labels. The nitroxide spin-labeled molecules were provided by R. J. Mehlhorn (of this laboratory) and have the structures shown in Fig. 2 RESULTSChoosing an appropriate spin label We first surveyed the behavior of various classes of spin labels (Fig. 2) toward the respiratory chain activity. CDTAB, a cation 570 Abbreviations: CDTAB, spin-labeled analogue of cetyltrimethylammonium bromide; SMP, submitochondrial preparation.

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“…Similar methods have been used to describe aggregations (2,3). This method involves the coarse digitization of the data.…”

Section: Boxing Methodsmentioning

confidence: 99%

“…To understand better the mechanism of the clustering of the particles, one needs a (preferably simple) measure of the amount of aggregation in the sample electron micrographs under investigation. Several techniques for the comparison of states of aggregation have been attempted (2)(3)(4)(5)(6)(7)(8). A sample of tracings of electron micrographs showing particle aggregations, is shown in Fig.…”

Section: Introductionmentioning

confidence: 99%

Testing of aggregation measurement techniques for intramembranous particles

Donnell¹,

Finegold²

1981

Biophysical Journal

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Under various physiological and nonphysiological conditions, the intramembranous particles, as seen by freeze-fracture electron microscopy, may be in various degrees of aggregation. To compare various schemes for the measurement of the degree of aggregation, a computer program has been used to generate simulated aggregations. A simple and adequate technique for quantifying the degree of aggregation, which is practical for the electron microscopist, is presented.

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Analysis of freeze-fracture electron micrographs by a computer-based technique

Cited by 19 publications

References 7 publications

Cell fusion and intramembrane particle distribution in polyethylene glycol-resistant cells.

Cell fusion and intramembrane particle distribution in polyethylene glycol-resistant cells.

Surface localization of sites of reduction of nitroxide spin-labeled molecules in mitochondria.

Testing of aggregation measurement techniques for intramembranous particles

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