Alterations In Cellular Structure Research Articles

Comprehensive identification of SUMO2/3 targets and their dynamics during mitosis.

During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.

Stimulative Effects of Hominis Placental Pharmacopuncture Solution Combined with Zinc-oxide Nanoparticles on RAW 264.7 Cells: ZnO HPPS more easily stimulates RAW 264.7 cells.

Objectives:The purpose of this study is to examine whether Hominis Placental pharmacopuncture solution (HPPS) combined with zinc-oxide nanoparticles (ZnO NP) activates RAW 264.7 cells.Methods:We soaked ZnO nanoparticles in the Hominis Placenta pharmacopuncture solution, thereby making a combined form (ZnO NP HPPS). The effect of ZnO NP HPPS on the intracellular reactive oxygen species (ROS) production was measured by 2', 7'-dichlorofluorescin diacetate (DCFH-DA) assay. The effect of ZnO NP HPPS on NF-κ B was measured by using a luciferase assay. The effect of ZnO NP HPPS on the cytokine expression was assessed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cellular uptake of ZnO NP HPPS was measured by using a flow cytometric analysis, and cellular structural alterations were analyzed by using transmission electron microscopy (TEM).Results:Neither the HPPS nor the ZnO NPs induced intracellular ROS production in RAW 264.7 cells. Neither of the materials activated NF-κ B or it’s dependent genes, such as TNF-α, IL-1, and MCP-1. However, ZnO NP HPPS, the combined form of ZnO NPs and HPPS, did induce the intracellular ROS production, as well as prominently activating NF-κ B and it’s dependent genes. Also, compared to ZnO NPs, it effectively increased the uptake by RAW 264.7 cells. In addition, cellular structural alterations were observed in groups treated with ZnO NP HPPS.Conclusions:Neither ZnO NP nor HPPS activated RAW 264.7 cells, which is likely due to a low cellular uptake. The ZnO NP HPPS, however, significantly activated NF-κ B and up-regulated its dependent genes such as TNF-α, IL-1, and MCP-1. ZnO NP HPPS was also more easily taken into the RAW 264.7 cells than either ZnO NP or HPPS.

Fractal Dimension as a Measure of Altered Actin Cytoskeleton in MC3T3-E1 Cells Under Simulated Microgravity Using 3-D/2-D Clinostats

Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.

TACC3-TSC2 maintains nuclear envelope structure and controls cell division

Studies of the role of tuberous sclerosis complex (TSC) proteins (TSC1/TSC2) in pathology have focused mainly on their capacity to regulate translation and cell growth, but their relationship with alterations of cellular structures and the cell cycle is not yet fully understood. The transforming acidic coiled-coil (TACC) domain-containing proteins are central players in structures and processes connected to the centrosome. Here, TACC3 interactome mapping identified TSC2 and 15 other physical interactors, including the evolutionary conserved interactions with ch-TOG/CKAP5 and FAM161B. TACC3 and TSC2 co-localize and co-purify with components of the nuclear envelope, and their deficiency causes morphological alterations of this structure. During cell division, TACC3 is necessary for the proper localization of phospho-Ser939 TSC2 at spindle poles and cytokinetic bridges. Accordingly, abscission alterations and increased frequency of binucleated cells were observed in Tacc3- and Tsc2-deficient cells relative to controls. In regulating cell division, TSC2 acts epistatically to TACC3 and, in addition to canonical TSC/mTOR signaling and cytokinetic associations, converges to the early mitotic checkpoint mediated by CHFR, consistently with nuclear envelope associations. Our findings link TACC3 to novel structural and cell division functions of TSC2, which may provide additional explanations for the clinical and pathological manifestations of lymphangioleiomyomatosis (LAM) disease and TSC syndrome, including the greater clinical severity of TSC2 mutations compared to TSC1 mutations.

Halothane-induced alterations in cellular structure and proliferation of A549 cells

Genotoxicity, cytotoxicity or teratogenicity are among the well-known detrimental effects of the volatile anaesthetics. The aim of the present work was to study the structural changes, proliferative activity and the possibility of alveolar A549 cells to recover after in vitro exposure to halothane at 1.5 and 2.1 mM concentrations. Our data indicated significant reduction of viability, suppression of mitotic activity more than 60%, and that these alterations were accompanied by disturbances of nuclear and nucleolar structures. The most prominent negative effect was the destruction of the lamellar bodies, the main storage organelles of pulmonary surfactant, substantial for the lung physiology. In conclusion, halothane applied at clinically relevant concentrations exerts genotoxic and cytotoxic effect on the alveolar cells in vitro, most likely as a consequence of stress-induced apoptosis, thus modulating the respiratory function.

Emerging Roles for Ubiquitin and Protein Degradation in Neuronal Function

Alterations in cellular structure and synapse composition are central to proper nervous system function. Recent work has identified the ubiquitin-proteasome system (UPS) as a key regulator of neuronal biology. The UPS is essential for the growth and development of immature neurons and is a critical mediator of synaptic adaptability in mature neurons. Furthermore, proteinaceous deposits that accumulate in diverse neurodegenerative disorders are enriched in components of the UPS, suggesting that UPS dysfunction may be pivotal for pathogenesis. Here, we summarize existing knowledge about the role of the UPS in brain function, highlighting recent work delineating its importance in neuronal development, plasticity, and degeneration.

Increased resistance of lipofuscin-loaded prematurely senescent fibroblasts to starvation-induced programmed cell death

Alterations of cellular structures often found in ageing cells is mainly the result of production of reactive oxygen species and a consequence of aerobic life. Both oxidative stress and decreased degradative capacity of lysosomal system cause accumulation of intralysosomal age-related pigment called lipofuscin. To investigate the influence of lipofuscin on cell function, we compared survival of lipofuscin-loaded and control human fibroblasts following complete starvation induced by exposure to phosphate-buffered saline (PBS). Starving of control fibroblasts resulted in lysosomal alkalinisation, relocation of cathepsin D to the cytosol, caspase-3 activation and, finally, cell death, which became evident 72 h after the start of exposure to PBS. Increase of lysosomal pH was significantly less prominent in lipofuscin-loaded cells than in controls and was accompanied neither by leakage of cathepsin D nor by caspase-3 activation even 96 h after the initiation of starvation. Suppression of autophagy by 3-methyladenine (3-MA) accelerated cell death, while inhibition of cathepsin D delayed it, implying an important role of autophagy in cell survival during starvation and showing the involvement of lysosomes in starvation-induced cell death. Disturbed apoptotic response found in lipofuscin-loaded cells can be interpreted as an example of hormesis--an adaptation to low doses of otherwise harmful agents, in this case of lipofuscin, which has a protective effect at moderate amounts but becomes toxic at large quantities.

Costameres, focal adhesions, and cardiomyocyte mechanotransduction

Mechanotransduction refers to the cellular mechanisms by which load-bearing cells sense physical forces, transduce the forces into biochemical signals, and generate appropriate responses leading to alterations in cellular structure and function. This process affects the beat-to-beat regulation of cardiac performance but also affects the proliferation, differentiation, growth, and survival of the cellular components that comprise the human myocardium. This review focuses on the experimental evidence indicating that the costamere and its structurally related structure the focal adhesion complex are critical cytoskeletal elements involved in cardiomyocyte mechanotransduction. Biochemical signals originating from the extracellular matrix-integrin-costameric protein complex share many common features with those signals generated by growth factor receptors. The roles of key regulatory kinases and other muscle-specific proteins involved in mechanotransduction and growth factor signaling are discussed, and issues requiring further study in this field are outlined.

MAGNETIC RESONANCE IN SURGICAL ONCOLOGY: I – ON THE ORIGIN OF THE SPECTRUM

The discovery of the phenomenon of nuclear magnetic resonance occurred just 60 years ago. The profusion of subsequent discoveries in this domain has led to the development of magnetic resonance spectroscopy - refined as an analytical tool to discern molecular structure - and magnetic resonance imaging, a cornerstone of modern radiology. Observable alterations in cellular structure and metabolism can be discerned using the non-destructive chemical analysis of magnetic resonance spectroscopy in vitro or in vivo. Differences may thus be discerned between malignant and normal tissues.

Improved Technique for Electron Microscope Visualization of Yeast Membrane Structure

AbstractYeast cells represent a powerful model system in cell biology mainly due to their amenability to genetic manipulations. Increasingly, studies focus on mutant genes resulting in alterations of cellular structures and organelles. To ascertain the phenotypic changes involved, it is often desirable to use the resolving power of electron microscopy. In contrast to higher eukaryotic cells, yeast cells are particularly difficult to preserve mainly due to the presence of a thick cell wall that acts as a barrier against diffusion of fixatives. Although several procedures are targeted to overcome these difficulties, none of them have become established as a standard procedure. As a consequence, electron microscopy is still not used routinely as a tool in yeast cell biology. This prompted us to develop an easy-to-follow protocol for yeast transmission electron microscopy that should be useful in all cases where membrane integrity and organelle morphology is emphasized. One means of making the yeast cytoplasm more attainable to fixation and staining solutions is by enzymatic digestion of the cell wall. Following this approach, we were able to reliably preserve yeast cells and their cellular organelles. Enzymatic treatment with zymolyase 20T to partially remove the yeast cell wall allowed the fixation, preservation, and visualization of the yeast cytoplasm revealing detailed ultrastructure. The advancement of this technique is demonstrated with mitochondria as a model organelle. Our studies on various yeast mutants clearly show the power of the enzymatic digestion technique in visualizing subtle changes of membrane structure and organelle morphology.

Changes in cellular structures and enzymatic activities during browning of Scots pine callus derived from mature buds.

Visible browning is a typical feature of callus cultures derived from shoot tips of mature Scots pine (Pinus sylvestris L.). Because the ability of callus to regenerate is low, we determined the effect of browning on growth and changes in cellular structure during culture. Striking alterations in cellular structure were detected by LM (light microscopy), EM (electron microscopy) and SEM (scanning electron microscopy). Accumulation of phenolic substances was shown by histochemical staining. Staining for beta-glucosidase activity of soluble proteins that had been subjected to polyacrylamide gel electrophoresis indicated lignification of cells. The measured growth rate of callus was low compared with a hypothetical growth curve. Peroxidase activity increased rapidly soon after the start of the culture period, but especially between the second and third weeks of culture. At this time, the degradation of cell membranes and browning began coincident with the loss of chlorophyll. We conclude that browning is associated with cell disorganization and eventual cell death, making tissue culture of mature pine especially difficult.

Fourier Analysis of Textural Variations in Human Normal and Cataractous Lens Nuclear Fiber Cell Cytoplasm

Variations in cytoplasmic texture of human normal and cataractous lenses were investigated with Fourier analysis. Fixed Vibratome sections of six normal transparent human lenses (age range 55–72 years) and six nuclear cataractous lenses (age range 66–89 years) were examined using transmission electron microscopy. Images were taken of the fiber cell cytoplasm and examined using linear optical density scans, Fourier transforms and autocorrelation analysis. The cytoplasm of normal human lenses was found to be smooth and homogeneous. Radially-averaged plots of Fourier transforms revealed a broad band of intensity over the range of 15–50 nm. Four of the six cataracts also had homogeneous cytoplasm without substantial density variations. Fourier transforms and radially-averaged plots were similar to those of the normal, although slightly lower in intensity for components⩽20 nm. Two of the six cataracts had a slightly granular or textured cytoplasm that showed significant density variations. Fourier transforms and radially-averaged plots revealed significant intensity in the range⩾200 nm. Autocorrelation analysis supported the data obtained by Fourier transforms. Radially-averaged plots of the autocorrelation resultant displays detected only small structural units in the normal and non-textured cataractous cytoplasm, whereas larger structural units were detected in the textured cataractous cytoplasm. The appearance of the textured cataractous cytoplasm suggests that redistribution or loss of protein may be the cause of density variations in these types of cataracts. Importantly, these results indicate that some nuclear cataracts do not contain spatial fluctuations in the cytoplasm large enough to cause significant light scattering. The data suggest that nuclear cataractogenesis is a multi-factorial process involving minor alterations in cellular structure.

Calcium-mediated cell injury and cell death.

The effect of intracellular ion deregulation, particularly of [Ca2+], on the events following acute cell injury and the progression of change from initiation (reversible) to maintenance (reversible-irreversible) phases and finally to cell death has been the major thrust of experimentation in our laboratory for over 20 years. Cell death, which plays an important role in both normal and pathological phenomena, has been classified into two principal types, accidental and programmed. Recent exploration of programmed cell death (or apoptosis) has revealed extensive data showing it is an important mechanism for the normal maintenance and also differentiation of a variety of cell types and organs. From the results from our laboratory and those of others, we continue to expand and refine our working hypothesis: deregulation of [Ca2+] results in a number of phenomena from activation of signaling mechanisms and alterations in cellular structure to alterations in gene expression, all of which contribute to or play a critical role in cellular toxicity, including carcinogenesis and cell death. Therefore, although much more experimentation is needed to clarify some of these phenomena, the implications of such data for understanding the mechanisms and processes involved in carcinogenesis and the chemotherapeutic killing of cancer cells are extremely exciting. These relationships between [Ca2+], cell injury, and cell death are briefly reviewed here within the framework of our hypothesis.

Comparison of structural changes in red spruce ( Picea rubens Sarg.) during cold hardening in mature trees and in seedlings used in pollutant exposure studies

One of the concerns in understanding the causes of tree decline has been extrapolating results obtained in controlled experiments on seedlings to mature trees in the field. The goal of this project was to compare results from trees growing under natural conditions at several sites, with varying levels of red spruce ( Picea rubens Sarg.) decline, with results from experiments conducted at the Boyce Thompson Institute on ozone and acid precipitation exposures of seedlings. One of the major hypotheses for decline of red spruce is winter injury. There is accumulating evidence that pollutants such as ozone or acidic deposition may increase susceptibility to freezing injury in early winter. The transition from a summer active state, to a state which can tolerate the conditions in winter, involves concerted changes in biochemistry, physiology, membrane composition and cellular structure. Mature tree foliage differs from seedlings in several respects, but alterations in cellular structure between mid-summer and mid-winter are similar. Changes in chloroplast numbers and orientation occur in red spruce foliage from trees of all ages, and under approximately the same conditions. Differences in cellular health are obvious from the different field sites and treatments, yet a similar pattern of response to low temperatures in the fall was seen. Individual mesophyll cells undergo alterations in the amount of cytoplasm and in chloroplast arrangement. Results indicate that foliage from young seedlings is a valid model for the response of foliage on mature trees to cold hardening in the fall.

Heat-resistant variants of the Chinese hamster ovary cell: alteration of cellular structure and expression of vimentin.

Three heat-resistant mutant cell lines (78-1, 78-2, 78-3) were previously selected from Chinese hamster ovary cells. In this study, we investigated whether the differences in intrinsic thermal sensitivity result from alteration of stress protein levels or cellular structural changes. Although there was no significant difference in the levels of stress proteins, i.e., constitutive HSP70 in wild type and three heat-resistant mutant strains, there were marked differences in the amounts of vimentin among the cell lines. Two-dimensional gel electrophoresis and Western blot showed a 2.3-2.9-fold increase in the level of vimentin in the mutant cells under normal growth conditions. Northern blot also revealed higher amounts of vimentin mRNA in the mutant cells. Electron microscopy and immunofluorescence suggest that increased amounts of the vimentin-containing intermediate filaments are correlated with the heat-resistant phenotypes.

Occurrence and developmental consequences of aberrant cellular organization in meiotically mature human oocytes after exogenous ovarian hyperstimulation

The extent to which fertilization failure in in vitro inseminated human oocytes obtained from hyperstimulated ovaries was associated with alterations in cellular structure and cytoplasmic organization was examined by a combination of DIC, fluorescence, time-lapse video, and transmission electron microscopy. Detailed analysis of meiotically mature preovulatory (uninseminated) oocytes, and of oocytes that failed to fertilize in vitro, indicated that between 10% and 15% of grossly normal-appearing MII-stage oocytes displayed one of the following cellular perturbations: 1) a subtle change in organelle distribution, 2) a small region(s) of intracellular cytolysis, 3) a massive aggregation of SER tubules, 4) an accumulation of vesicles presumed to be of SER origin, 5) a change in the structural organization of the cortical cytoplasm and overlying plasma membrane, 6) a sudden and rapid internalization of perivitelline fluid by means of an apparently aberrant process of endocytosis, and 7) a premature and partial exocytosis of cortical granules. The description of these disorders is discussed with respect to the developmental consequences for the oocyte.

Morphologic alterations in drug sensitive vs. drug resistant cells due to cytostatic application

Chemotherapeutic agents have proven to be effective in the cure ofsome and the palliation of many human cancers. However, in the majority of lesions there develop tumor cell populations resistant to cytostatic drugs. This is considered to be one of the major causes of failure of clinical chemotherapy. Several mechanisms have been described by which the drug-insensitive tumor cells can resist the toxic agents, i.e. altered activity of cytoplasmic enzymes (e.g. 7-hydrofolatereductase, gluthatione-S-transferase, gluthatione-peroxidase, protein kinase C) (1, 3, 29, 32) and reduced intracellular drug toxification (2, 13), as well as stimulated DNA repair by altered activity of topoisomerase II, polymerase a or b and thymidylate synthase (19, 39, 45). Other mechanisms involve the overproduction of binding and transport proteins, e.g. Mr 170.000 P-glycoprotein, Mr 19.000 protein (34, 35, 42) combined with reduced net drug accumulation (16, 20), increased energy dependent efflux (24, 43) and gene amplification (6, 7, 17, 21, 22, 30, 36, 44). In addition, organelle-associated drug binding and extrusion by means of intracellular drug compartmentalization into vesicular structures have been described (4, 11, 14, 33, 40, 41). The development of acquired chemoresistance observed in vivo can be simulated in vitro by the incubation of tumor cells in stepwise enhanced concentration of cytostatic drugs. The associated changes of functional properties have been intensively studied using biochemical and molecular methods. However, morphological alterations of cellular structures in response to drug application have scarcely been investigated. The present paper summarizes some phenotypic and functional alterations due to cytostatic drug application in cell cultures of human carcinoma cell lines. In particular, differences of the morphologic appearance of sensitive vs. resistant cells are compared.

Histopathological observations on the digestive gland ofLymnaea auricularia infected with larval trematodes

Histological observations were made on the digestive gland of infected and uninfected freshwater snails,Lymnaea auricularia, and the varying degree of histopathogenesis of echinostome cercaria,Cercaria itoi, as well as strigeoid metacercaria,Tetracotyle lymnaei, along withCercaria itoi (double infection) in the digestive gland have been observed. Basically 3 types of lesions, L1, L2 and L3 were found and were associated with the types and size of trematode larvae as well as with the degree of parasitemia. In the early stage of larval development or in mild infection, type I lesions (L1) were commonly observed in which degenerative changes were confined to individual digestive gland tubules, and neighbouring tubules mostly remained healthy or unaffected. The type II lesions (L2) were associated with severe infection and were less common than L1 lesions and large areas of parasitised digestive gland tubules were revealed to be necrotic. Type III lesions (L3) were also associated with severe infection in which digestive gland tubules appeared as rudimentary tubules located between trematode rediae. In the present study, in addition to a description of the different types of lesions, the various alterations of cellular structures of the digestive gland ofLymnaea auricularia due to single or double infections (Cercaria itoi + Tetracotyle lymnaei) are also discussed.

Cytoplasmic membrane is the target organelle for transition metal mediated damage induced by paraquat in Escherichia coli.

Bacterial survival indicates that copper or iron is an essential mediator in paraquat toxicity in Escherichia coli [Kohen, R., & Chevion, M. (1985) Free Radical Res. Commun. 1, 79-88; Korbashi, P., Kohen, R., Katzhendler, J., & Chevion, M. (1986) J. Biol. Chem. 261, 12472-12476]. In this study we have identified the cytoplasmic membrane as a target organelle in metal-mediated paraquat toxicity and have demonstrated the complete correlation of the membrane damage with the levels of adventitious copper (or iron). The extent of membrane damage was related by use of four parameters: (a) the level of cellular ATP, (b) the level of cellular potassium, (c) the cellular capacity to accumulate and retain radiolabeled leucine, and (d) the cellular integrity as reflected by transmission electron microscopy (TEM). Exposure of bacterial cells to a combination of paraquat and copper caused a marked decline in parameters a, b, and c. This decline was found to occur in parallel with, or even to precede, the sharp loss of survival of E. coli under the same conditions. Likewise, TEM micrographs clearly indicated alterations in cellular structure that possibly reflect sites of detachment of the cytoplasmic membrane from the bacterial capsule. In contradistinction, copper alone or paraquat alone could not bring about similar changes in cellular structure. These findings are in accord with the suggested site-specific metal-mediated Haber-Weiss mechanism for paraquat toxicity and support our notion that specific chelators of transition metals could reduce or prevent the biological deleterious effects of this herbicide.

Alterations in cellular structure of Mytilus edulis resulting from exposure to environmental contaminants under field and experimental conditions

Alterations in cellular structure of Mytilus edulis resulting from exposure to environmental contaminants under field and experimental conditions