Degradation of normal and proliferated peroxisomes in rat hepatocytes: regulation of peroxisomes quantity in cells

Degradation and turnover of peroxisomes is reviewed. First, we describe the historical aspects of peroxisome degradation research and the two major concepts for breakdown of peroxisomes, i.e., autophagy and autolysis. Next, the comprehensive knowledge on autophagy of peroxisomes in mammalian and yeast cells is reviewed. It has been shown that proliferated peroxisomes are degraded by selective autophagy, and studies using yeast cells have been especially helpful in shedding light on the molecular mechanisms of this process. The degradation of extraperoxisomal urate oxidase crystalloid is noted. Overexpressed wild-type urate oxidase in cultured cells has been shown to be degraded through an unknown proteolytic pathway distinct from the lysosomal system including autophagy or the ubiquitin-proteasome system. Finally, peroxisome autolysis mediated by 15-lipoxygenase (15-LOX) is described. 15-LOX is integrated into the peroxisome membrane causing focal membrane disruptions. The content of the peroxisomes is then exposed to cytosol proteases and seems to be digested quickly. In conclusion, the number of peroxisomes appears to be regulated by two selective pathways, autophagy, including macro- and microautophagy, and 15-LOX-mediated autolysis.     

Selective degradation of peroxisomes in yeasts

In the last two decades, much progress has been made in understanding the process of induction and biogenesis of peroxisomes, essential organelles in all eukaryotes. Only relatively recently, the first molecular studies on the selective degradation of this important organelle-a process known as pexophagy, which occurs when the organelles have become redundant-have been performed, especially using methylotrophic yeasts. The finding that pexophagy and other transport pathways to the vacuole (vacuolar protein sorting, autophagy, cytoplasm-to-vacuole-targeting and endocytosis) utilize common but also unique genes has placed pexophagy in the heart of the machinery that recycles cellular material. The quest is now on to understand how peroxisome degradation has become such a highly selective process and what the signals are that trigger it. In addition, because the prime determinant of pexophagy is located on the peroxisome itself, it has become essential to study the role of peroxisomal membrane proteins in the degradation process in detail. This review highlights the main achievements of the last years.     

Human peroxisomal disorders

Peroxisomes are single membrane-bound cell organelles performing numerous metabolic functions. The present article aims to give an overview of our current knowledge about inherited peroxisomal disorders in which these organelles are lacking or one or more of their functions are impaired. They are multiorgan disorders and the nervous system is implicated in most. After a summary of the historical names and categories, each having distinct symptoms and prognosis, microscopic pathology is reviewed in detail. Data from the literature are added to experience in the authors' laboratory with 167 liver biopsy and autopsy samples from peroxisomal patients, and with a smaller number of chorion samples for prenatal diagnosis, adrenal-, kidney-, and brain samples. Various light and electron microscopic methods are used including enzyme- and immunocytochemistry, polarizing microscopy, and morphometry. Together with other laboratory investigations and clinical data, this approach continues to contribute to the diagnosis and further characterization of peroxisomal disorders, and the discovery of novel variants. When liver specimens are examined, three main groups including 9 novel variants (33 patients) are distinguished: (1) absence or (2) presence of peroxisomes, and (3) mosaic distribution of cells with and without peroxisomes (10 patients). Renal microcysts, polarizing trilamellar inclusions, and insoluble lipid in macrophages in liver, adrenal cortex, brain, and in interstitial cells of kidney are also valuable for classification. On a genetic basis, complementation of fibroblasts has classified peroxisome biogenesis disorders into 12 complementation groups. Peroxisome biogenesis genes (PEX), knock-out-mice, and induction of redundant genes are briefly reviewed, including some recent results with 4-phenylbutyrate. Finally, regulation of peroxisome expression during development and in cell cultures, and by physiological factors is discussed.     

Application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver in conjunction with cytochemical staining with 3-3'-diaminobenzidine and immunocytochemistry.

We describe the application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver. For automatic detection by light and electron microscopy peroxisomes must be stained with the alkaline DAB procedure for catalase. There is a good agreement between the results obtained by conventional morphometric techniques and by automatic image analysis of DAB-stained electron microscopic preparations. Moreover, the image analyzer may be used in conjunction with a light microscope for evaluation of semithin sections (1-0.25 microns), provided the section thickness factor is taken into consideration. This latter approach has proven highly efficient in estimation of peroxisome proliferation. The limitations of this method and the relevance of volume density as a reliable morphometric parameter for evaluation of peroxisome proliferation are discussed. In the second part of this study we present the application of image analysis for quantitation of alterations of individual peroxisomal enzyme proteins after treatment with bezafibrate in immunogold stained ultrathin sections. There is good agreement between the results of quantitative immunocytochemistry and Western (immuno) blot analysis of highly purified peroxisomal fractions. In our experience quantitative immunoelectron microscopy provides a versatile, highly sensitive, and efficient method for detection of modulations of various proteins in peroxisomes. Finally the limitations and prospects of quantitative immunocytochemistry for investigation of peroxisomal proteins are discussed.     

A review of morphological techniques for detection of peroxisomal (and mitochondrial) proteins and their corresponding mRNAs during ontogenesis in mice: application to the PEX5-knockout mouse with Zellweger syndrome

In the era of application of molecular biological gene-targeting technology for the generation of knockout mouse models to study human genetic diseases, the availability of highly sensitive and reliable methods for the morphological characterization of the specific phenotypes of these mice is of great importance. In the first part of this report, the role of morphological techniques for studying the biology and pathology of peroxisomes is reviewed, and the techniques established in our laboratories for the localization of peroxisomal proteins and corresponding mRNAs in fetal and newborn mice are presented and discussed in the context of the international literature. In the second part, the literature on the ontogenetic development of the peroxisomal compartment in mice, with special emphasis on liver and intestine is reviewed and compared with our own data reported recently. In addition, some recent data on the pathological alterations in the liver of the PEX5(-/-) mouse with a peroxisomal biogenesis defect are briefly discussed. Finally, the methods developed during these studies for the localization of mitochondrial proteins (respiratory chain complexes and MnSOD) are presented and their advantages and pitfalls discussed. With the help of these techniques, it is now possible to identify and distinguish unequivocally peroxisomes from mitochondria, two classes of cell organelles giving by light microscopy a punctate staining pattern in microscopical immunohistochemical preparations of paraffin-embedded mouse tissues.     

Peroxisome assembly in yeast

Peroxisomes are essential organelles that may be involved in various functions, dependent on organism, cell type, developmental stage of the cell, and the environment. Until recently, peroxisomes were viewed as a class of static organelles that developed by growth and fission from pre-existing organelles. Recent observations have challenged this view by providing evidence that peroxisomes may be part of the endomembrane system and constitute a highly dynamic population of organelles that arises and is removed upon environmental demands. Additionally, evidence is now accumulating that peroxisomes may arise by alternative methods. This review summarizes relevant recent data on this subject. In addition, the progress in the understanding of the principles of the peroxisomal matrix protein import machinery is discussed.     

Deciphering the human nucleolar proteome

Nucleoli are plurifunctional nuclear domains involved in the regulation of several major cellular processes such as ribosome biogenesis, the biogenesis of non-ribosomal ribonucleoprotein complexes, cell cycle, and cellular aging. Until recently, the protein content of nucleoli was poorly described. Several proteomic analyses have been undertaken to discover the molecular bases of the biological roles fulfilled by nucleoli. These studies have led to the identification of more than 700 proteins. Extensive bibliographic and bioinformatic analyses allowed the classification of the identified proteins into functional groups and suggested potential functions of 150 human proteins previously uncharacterized. The combination of improvements in mass spectrometry technologies, the characterization of protein complexes, and data mining will assist in furthering our understanding of the role of nucleoli in different physiological and pathological cell states.     

Peroxisome proliferation as a biomarker in environmental pollution assessment

Peroxisome proliferators comprise a heterogeneous group of compounds known for their ability to cause massive proliferation of peroxisomes and liver carcinogenesis in rodents. In recent years it has become evident that other animals may be threatened by peroxisome proliferators, in particular aquatic organisms living in coastal and estuarine areas. These animals are exposed to a variety of pollutants of industrial, agricultural and urban origin which are potential peroxisome proliferators. Both laboratory and field studies have shown that phthalate ester plasticizers, PAHs and oil derivatives, PCBs, certain pesticides, bleached kraft pulp and paper mill effluents, alkylphenols and estrogens provoke peroxisome proliferation in different fish or bivalve mollusc species. The response appears to be mediated by peroxisome-proliferator activated receptors, members of the nuclear receptor family, recently cloned in fish. Based on these results it is proposed that peroxisome proliferation could be used as a biomarker of exposure to a variety of pollutants in environmental pollution assessment. This is illustrated by a case study in which mussels, used worldwide as sentinels of environmental pollution, were transplanted from reference to contaminated areas and vice versa. In mussels native to an area polluted with PAHs and PCBs, peroxisomal acyl-CoA oxidase (AOX) activity and peroxisomal volume density were 2-3 fold and 5-fold higher, respectively, compared to the reference site. When animals were transplanted to the polluted station, with increased concentration of organic xenobiotics, a concomitant significant increase of AOX was recorded. Conversely, in animals transplanted to the cleaner station, AOX activity and peroxisomal volume density decreased significantly. These results indicate that peroxisome proliferation is a rapid (i.e., two days) and reversible response to pollution in mussels. Before peroxisome proliferation can be implemented as a biomarker in biomonitoring programs, a well-defined protocol should be established and validated in intercalibration and quality assurance programmes. Furthermore, the influence of biotic and abiotic factors, some of which are known to affect peroxisome proliferation (season, tide level, interpopulation and interindividual variability), should be taken into consideration. The possible hepatocarcinogenic effects as well as the potential adverse effects on reproduction, development, and growth of peroxisome proliferators are unknown in aquatic organisms, thus providing a challenge for future investigations.     

Peroxisomal motility and interaction with microtubules

Recent in vivo observations have revealed that peroxisomes are more dynamic and interactive than previously assumed. The growing recognition of the tubular and reticular morphology of peroxisomes in living cells, their association with microtubules, and the dynamic movements of peroxisomes in vivo and in vitro have inspired the query into the investigation of the cellular machinery that mediates such a complex behaviour. The characterisation of the underlying molecular components of this machinery is providing insight into the mechanisms regulating peroxisomal morphology and intracellular distribution.     

Functional morphology of the secretory pathway organelles in yeast

The glycoprotein secretory pathway of yeast serves mainly for cell surface growth and cell division. It involves a centrifugal transport of transit macromolecules among organelles, whose membranes contain resident proteins needed for driving the transport. These resident membrane proteins return by retrograde vesicular transport. Apart from this, the pathway involves endocytosis. The model yeast Saccharomyces cerevisiae and vertebrate cells were found to contain very similar gene products regulating the molecular mechanism of glycoprotein transport, and the cellular mechanism of their secretion pathways was therefore also presumed to be identical. Biochemists have postulated that, in S. cerevisiae, the translocation of peptides through the endoplasmic reticulum membranes into the lumen of ER cisternae and the core glycosylation is followed by a vector-mediated transport into the functional cascade of the Golgi system cisternae and between them. This is the site of maturation and sorting of glycoproteins, before the ultimate transport by other vectors involving either secretion from the cells (exocytosis across the plasmalemma into the cell wall) or transport into the lysosome-like vacuole via a prevacuolar compartment, which serves at the same time as a primary endosome. The established cellular model of secretion deals with budding yeast; interphase yeast cells, in which the secretion is limited and which predominate in exponential cultures, have not been taken into consideration. The quality of organelle imaging in S. cerevisiae ultra-thin sections depends on the fixation technique used and on specimen contrasting by metals. The results achieved by combinations of different techniques differ mostly in the imaging of bilayers of membrane interfaces and the transparence of the matrix phase. Fixation procedures are decisive for the results of topochemical localisations of cellular antigenic components or enzyme activities, which form the basis of the following survey of functional morphology of organelles involved in the yeast secretory pathway. The existing results of these studies do not confirm all aspects of the vertebrate model of the Golgi apparatus proposed by molecular geneticists to hold for S. cerevisiae, and alternative models of the cellular mechanism of secretion in this yeast are, therefore, also discussed.     

Molecular approach to echinoderm regeneration.

Until very recently echinoderm regeneration research and indeed echinoderm research in general has suffered because of the lack of critical mass. In terms of molecular studies of regeneration, echinoderms in particular have lagged behind other groups in this respect. This is in sharp contrast to the major advances achieved with molecular and genetic techniques in the study of embryonic development in echinoderms. The aim of our studies has been to identify genes involved in the process of regeneration and in particular neural regeneration in different echinoderm species. Our survey included the asteroid Asterias rubens and provided evidence for the expression of Hox gene homologues in regenerating radial nerve cords. Present evidence suggests: 1) ArHox1 expression is maintained in intact radial nerve cord and may be upregulated during regeneration. 2) ArHox1 expression may contribute to the dedifferentiation and/or cell proliferation process during epimorphic regeneration. From the crinoid Antedon bifida, we have been successful in cloning a fragment of a BMP2/4 homologue (AnBMP2/4) and analysing its expression during arm regeneration. Here, we discuss the importance of this family of growth factors in several regulatory spheres, including maintaining the identity of pluripotent blastemal cells or as a classic skeletal morphogenic regulator. There is clearly substantial scope for future echinoderm research in the area of molecular biology and certain aspects are discussed in this review.     

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species.

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.     

Meta-analysis of the cell cycle related <i>C12orf48</i>

The cell cycle is a conserved process from yeast to mammals and focuses on mechanisms that regulate the timing and frequency of DNA replication and cell division. The temporal and spatial expression of the genes is tightly regulated to ensure accurate replication and transmission of DNA to daughter cells during the cycle. Although the genes involved in interphase are well studied, most of the genes which are involved in mitotic events still remain unidentified. Since, the discovery of mitosis related genes is still incomplete, we performed a co-expression and gene ontology analysis for revealing novel mitosis regulated genes. In this study, we showed that C12orf48 is co-expressed with well-known mitotic genes. Moreover, it is also co-expressed with the genes that have roles in interphase such as DNA replication. Furthermore, our results showed that C12orf48 is also differentially expressed in various cancers. Therefore, the results presented in this study suggest that C12orf48 may be an important molecule for both interphase and mitosis. Since, the molecules involved in these mechanisms are crucial for proliferation as well as in carcinogenesis, C12orf48 should be considered as a novel cell cycle and carcinogenesis related gene.     

Peripheral and central structures involved in insect gustation

Studies in insect gustation have a long history in general physiology, particularly with work on fly labellar and tarsal sensilla and in the general field of insect-plant interactions, where work on immature Lepidoptera and chrysomelid beetles has been prominent. Much more emphasis has been placed on the physiological characteristics of the sensory cells than on the central cellular mechanisms of taste processing. This is due to the fairly direct access for physiological experimentation presented by many taste sensilla and to the obvious importance of tastants in insect feeding and oviposition behaviour. In some of the insect models used for gustatory studies, advances have been made in understanding the basic morphology of the central neuropils involved in the first stages of taste processing. There is much less known about the physiology of interneurons involved. In this review, we concentrate on four insect models (Manduca sexta, Drosophila melanogaster, Neobellieria bullata (and other large flies), and Apis mellifera) to summarize morphological knowledge of peripheral and central aspects of insect gustation. Our views of current interpretations of available data are discussed and some important areas for future research are highlighted.     

Mammary ductal and alveolar development: lesson learned from genetically manipulated mice

The mammary gland has been an area of great interest to developmental biologists for many years because its formation involves many fundamental processes that are central to the development of other organs. Although mammary development has been well described structurally, the molecules and signaling mechanisms that are involved are still largely undefined. For the last several years, intensive effort has been made to understand the molecular mechanisms involved in mammary development. With the recent advances in transgenic and knockout technologies, the ability to delete and/or alter the expression of certain genes in the mouse genome has allowed us to begin to elucidate the mechanisms underlying mammary gland development. In this review, we discuss several mouse models that have provided insight into the molecules and signaling mechanisms that govern ductal development and lobuoloalveolar differentiation in the mammary gland.     

Mammalian collagen IV

Four decades have passed since the first discovery of collagen IV by Kefalides in 1966. Since then collagen IV has been investigated extensively by a large number of research laboratories around the world. Advances in molecular genetics have resulted in identification of six evolutionary related mammalian genes encoding six different polypeptide chains of collagen IV. The genes are differentially expressed during the embryonic development, providing different tissues with specific collagen IV networks each having unique biochemical properties. Newly translated alpha-chains interact and assemble in the endoplasmic reticulum in a chain-specific fashion and form unique heterotrimers. Unlike most collagens, type IV collagen is an exclusive member of the basement membranes and through a complex inter- and intramolecular interactions form supramolecular networks that influence cell adhesion, migration, and differentiation. Collagen IV is directly involved in a number of genetic and acquired disease such as Alport's and Goodpasture's syndromes. Recent discoveries have also highlighted a new and direct role for collagen IV in the development of rare genetic diseases such as cerebral hemorrhage and porencephaly in infants and hemorrhagic stroke in adults. Years of intensive investigations have resulted in a vast body of information about the structure, function, and biology of collagen IV. In this review article, we will summarize essential findings on the structural and functional relationships of different collagen IV chains and their roles in health and disease.     

Structure and function of the yeast vacuole and its role in autophagy

The vacuole of the yeast Saccharomyces cerevisiae plays an important role in pH- and ion-homeostasis, and is used as a storage compartment for ions. Another important function of the vacuole, especially during nutrient limitation, is the bulk degradation of proteins and even whole organelles. To carry these proteins into the vacuolar lumen, sophisticated transport pathways have evolved. In this review, starvation-induced autophagy and its relationship to the specific cytoplasm to vacuole targeting (cvt-) pathway of proaminopeptidase I is discussed. A further topic is the specific vacuolar uptake and degradation of peroxisomes in Pichia pastoris cells via micro- and macroautophagy.     

Proteomic snapshot analyses of preribosomal ribonucleoprotein complexes formed at various stages of ribosome biogenesis in yeast and mammalian cells

Proteomic technologies powered by advancements in mass spectrometry and bioinformatics and coupled with accumulated genome sequence data allow a comprehensive study of cell function through large-scale and systematic protein identifications of protein constituents of the cell and tissues, as well as of multi-protein complexes that carry out many cellular function in a higher-order network in the cell. One of the most extensively analyzed cellular functions by proteomics is the production of ribosome, the protein-synthesis machinery, in the nucle(ol)us--the main site of ribosome biogenesis. The use of tagged proteins as affinity bait, coupled with mass spectrometric identification, enabled us to isolate synthetic intermediates of ribosomes that might represent snapshots of nascent ribosomes at particular stages of ribosome biogenesis and to identify their constituents--some of which showed dynamic changes for association with the intermediates at various stages of ribosome biogenesis. In this review, in conjunction with the results from yeast cells, our proteomic approach to analyze ribosome biogenesis in mammalian cells is described.     

Evolutionary significance of myosin heavy chain heterogeneity in birds

This article reviews the complexity, expression, genetics, regulation, function, and evolution of the avian myosin heavy chain (MyHC). The majority of pertinent studies thus far published have focussed on domestic chicken and, to a much lesser extent, Japanese quail. Where possible, information available about wild species has also been incorporated into this review. While studies of additional species might modify current interpretations, existing data suggest that some fundamental properties of myosin proteins and genes in birds are unique among higher vertebrates. We compare the characteristics of myosins in birds to those of mammals, and discuss potential molecular mechanisms and evolutionary forces that may explain how avian MyHCs acquired these properties.     

Growth of olfactory epithelial tissue in vitro: lectin staining of axons.

In vitro methods have been used to study several aspects of development of olfactory epithelium. In this paper, the value of growing olfactory tissue in explant cultures is reviewed and some experiments are reported on the identification of lectin receptors on olfactory axons by the use of lectin-gold complexes. Both concanavalin A-gold (con A-gold) and wheat germ agglutinin-gold consistently decorated olfactory axons in explant cultures. Con A-gold also bound to the tips of growth cone filopodia, suggesting the glycoconjugate molecules containing alpha-methyl-pyranoside are important in adherence of growth cones to their substrate. The wide range in density of lectin-gold particles suggested that axons, and the sensory cells from which they arise, are not a uniform population, i.e., they have different molecular fingerprints. This was supported by the observation that soybean agglutinin-gold stained some axons very well, but others remained unstained. Peanut agglutinin did not bind to any axons.