Studies Of Peroxisomes Research Articles

Super-resolution microscopy and studies of peroxisomes.

Fluorescence microscopy is an important tool forstudying cellular structures such as organelles. Unfortunately, many details in the corresponding images are hidden due to the resolution limit of conventional lens-based far-field microscopy. An example is the study of peroxisomes, where important processes such as molecular organization during protein important can simply not be studied with conventional far-field microscopy methods. A remedy is super-resolution fluorescence microscopy, which is nowadays a well-established technique for the investigation of inner-cellular structures but has so far to a lesser extent been applied to the study of peroxisomes. To help advancing the latter, we here give an overview over the different super-resolution microscopy approaches and their potentials and challenges in cell-biological research, including labelling issues and a focus on studies on peroxisomes. Here, we alsohighlight experiments beyond simple imaging such as observations of diffusion dynamics of peroxisomal proteins.

Rosy Beginnings: Studying Peroxisomes in Drosophila.

Research using the fruit fly Drosophila melanogaster has traditionally focused on understanding how mutations affecting gene regulation or function affect processes linked to animal development. Accordingly, flies have become an essential foundation of modern medical research through repeated contributions to our fundamental understanding of how their homologs of human genes function. Peroxisomes are organelles that metabolize lipids and reactive oxygen species like peroxides. However, despite clear linkage of mutations in human genes affecting peroxisomes to developmental defects, for many years fly models were conspicuously absent from the study of peroxisomes. Now, the few early studies linking the Rosy eye color phenotype to peroxisomes in flies have been joined by a growing body of research establishing novel roles for peroxisomes during the development or function of specific tissues or cell types. Similarly, unique properties of cultured fly Schneider 2 cells have advanced our understanding of how peroxisomes move on the cytoskeleton. Here, we profile how those past and more recent Drosophila studies started to link specific effects of peroxisome dysfunction to organ development and highlight the utility of flies as a model for human peroxisomal diseases. We also identify key differences in the function and proliferation of fly peroxisomes compared to yeast or mammals. Finally, we discuss the future of the fly model system for peroxisome research including new techniques that should support identification of additional tissue specific regulation of and roles for peroxisomes.

Using Co-Expression Analysis and Stress-Based Screens to Uncover Arabidopsis Peroxisomal Proteins Involved in Drought Response.

Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Using microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their co-expression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to environmental stresses.

MRI as diagnostic tool in early-onset peroxisomal disorders

Peroxisomal blood tests are generally considered to be conclusive. We observed several patients with a clinical and MRI phenotype suggestive of an infantile onset peroxisomal defect, but no convincing abnormalities in initial peroxisomal blood tests. Brain MRI showed typical abnormalities as observed in the neonatal adrenoleukodystrophy variant of infantile peroxisomal disorders. Our aim was to evaluate the accuracy of this MRI diagnosis with further peroxisomal testing. We searched our database of unclassified leukoencephalopathies and found 6 such patients. We collected clinical data and scored available MRIs of these patients. We performed further peroxisomal studies in fibroblasts, including immunofluorescence microscopy analysis with antibodies against catalase, a peroxisomal matrix enzyme. We performed complementation analysis and analyzed the suspected genes. We confirmed the diagnosis of Zellweger spectrum disorder in 3 patients and D-bifunctional protein deficiency in the others. The clinical findings were within the spectrum known for these diagnoses. Sequential MRIs showed that the abnormalities started in the hilus of the dentate nucleus and superior cerebellar peduncles. Subsequently, the cerebellar white matter and brainstem tracts were affected, followed by the parieto-occipital white matter, splenium of the corpus callosum, and posterior limb of the internal capsule. Eventually, all cerebral white matter became abnormal. The thalamus was typically affected as well. If MRI reveals abnormalities suggestive of infantile onset peroxisomal defects, negative peroxisomal blood tests do not exclude the diagnosis. Further tests in fibroblasts should be performed, most importantly immunofluorescence microscopy analysis with antibodies against catalase to stain peroxisomes.

Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system

The immunofluorescence technique has become an important tool for the investigation of peroxisomes in cell culture. We have used this method for the study of peroxisomes in the human hepatoblastoma cell line HepG2. A marked heterogeneity of peroxisomal forms was detected. Besides spherical (about 100 nm) and rod-shaped structures (about 300 nm) many elongated, undulating tubular forms (up to 5 microns) were found. Further observations indicate that the appearance of the peroxisomal forms in immunofluorescence is dependent on the fixation procedure used. Whereas the fixation with methanol-acetone (-20 degrees C) or ethanol results in a punctate pattern with spherical particles, the use of formaldehyde/Triton X-100 fixation shows well-preserved tubules and rods. These observations may be of special importance for studies on the biogenesis of peroxisomes.

Three-dimensional and histochemical studies of peroxisomes in cultured hepatocytes by quickfreezing and deep-etching method

Primary cultured mouse hepatocytes were treated with clofibric acid to induce peroxisome proliferation. They were briefly fixed with paraformaldehyde and centrifuged to prepare pellets. The hepatocytes were split open to remove cytoplasmic soluble proteins and fixed with glutaraldehyde. They were routinely incubated for catalase enzyme cytochemistry and fixed in osmium tetroxide. The specimens were quickly frozen, deeply etched and rotary shadowed by platinum and carbon. Peroxisomes were identified as osmium reaction products on replica membranes. In treated hepatocytes, the number of peroxisomes was considerably increased and smooth membranous structures resembling sER (referred to as 'peroxisome-forming sheets') showed hypertrophy. The rER associated with intermediate filaments was significantly decreased and 'peroxisome-forming sheets' appeared, being accompanied with budding and fragmentation of peroxisomes.

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase. II. Light-microscopic application.

The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semithin (0.25 and 1 micron) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for light-microscopic analysis may account for the differences. The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

The feasibility of the application of an electronic image analyzer, the Texture Analysis System (TAS) (Leitz Wetzlar, FRG), for fast automatic ultrastructural morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained selectively with the alkaline DAB procedure for localization of catalase in order to obtain sufficient contrast for automatic detection by TAS. Electron micrographs of ultrathin sections from this material were analyzed both automatically by TAS and manually by using a digitizer tablet connected with an Apple IIe microcomputer. The results showed negligible differences. As far as the speed of the operation is concerned, the image analysis was 4-5 times faster than the manual technique. In further studies, the importance of using DAB-stained sections for accurate morphometric studies of peroxisomes was demonstrated by comparing the results of such DAB-stained preparations with unstained material. This revealed that the numerical density was lower and the average profile diameter higher in unstained sections. The value for volume density was also affected, being about 30% lower in such preparations. It is likely that in unstained preparations small peroxisomes without crystalline nucleoids were frequently not identified as such and were not taken into account in morphometric calculations. These observations establish that computer-controlled electronic image analysis in conjunction with selective cytochemical staining of peroxisomes for catalase provides a fast, accurate and reliable method for ultrastructural morphometric studies of this organelle in rat liver.

Ultrastructural Study of Peroxisomes in Human Hepatocytes of a Chronic Alcoholic

Peroxisomes in normal human hepatocytes are catalase-containing rounded or oval organelles with an average diameter of 0.63 μm (l). They are usually surrounded by single tripartite membranes. Their presence can be easily demonstrated by the use of 3,3' diaminobenzidine (DAB) stain in the presence of H202 (2). This paper represents an ultrastructural study of peroxisomes in tne liver biopsy of an alcoholic patient with preoperative diagnosis of fatty infiltration.Biopsy tissues were fixed in 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 1% OsO4 and embedded in epoxy resin. Thin sections were stained with lead citrate and uranyl acetate and examined with a Zeiss EM 9 S-2.Light microscopic study of the liver biopsy reveals an intact hepatic architecture. The hepatocytes appear slightly edematous and possess many eosinophilic granules.Electron microscopy (Fig l) reveals that there is a marked increase in the number of randomly distributed peroxisomes in alcoholic human hepatocytes.