Fragmentation Of Endoplasmic Reticulum Research Articles

Fission and Fusion of the Neuronal Endoplasmic Reticulum

The endoplasmic reticulum (ER) is central for protein synthesis and is the largest intracellular Ca2+ store in neurons. The neuronal ER is classically described to have a continuous lumen spanning all cellular compartments. This allows neuronal ER to integrate spatially separate events in the cell. Recent in vitro as well as in vivo findings, however, demonstrate that the neuronal ER is a structurally dynamic entity, capable of rapid fragmentation, i.e., ER fission. The ER fragments can fuse back together and reinstate ER continuity. This reversible phenomenon can be induced repeatedly within the same cell, is temperature-dependent, and compatible with cell survival. The key trigger for dendritic ER fission is N-methyl D-aspartate (NMDA) receptor stimulation in the presence of extracellular Ca2+. However, the exact molecular machinery responsible for the fission and fusion of neuronal ER remains unknown. Reversible ER fission represents a new cell biological event downstream of NMDA receptor-gated Ca2+ influx and may thus influence many aspects of neuronal function in physiology and disease. Hence, it constitutes a new field for exploration in neuroscience that will benefit greatly from recent advances in light microscopy imaging techniques allowing dynamic characterization of cellular events in vitro and in vivo.

A Ca2+-dependent Mechanism of Neuronal Survival Mediated by the Microtubule-associated Protein p600

In acute and chronic neurodegeneration, Ca(2+) mishandling and disruption of the cytoskeleton compromise neuronal integrity, yet abnormalities in the signaling roles of cytoskeletal proteins remain largely unexplored. We now report that the microtubule-associated protein p600 (also known as UBR4) promotes neuronal survival. Following depletion of p600, glutamate-induced Ca(2+) influx through NMDA receptors, but not AMPA receptors, initiates a degenerative process characterized by endoplasmic reticulum fragmentation and endoplasmic reticulum Ca(2+) release via inositol 1,4,5-trisphosphate receptors. Downstream of NMDA receptors, p600 associates with the calmodulin·calmodulin-dependent protein kinase IIα complex. A direct and atypical p600/calmodulin interaction is required for neuronal survival. Thus, p600 counteracts specific Ca(2+)-induced death pathways through regulation of Ca(2+) homeostasis and signaling.

Intracellular localization of the BCL-2 family member BOK and functional implications.

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

Neuropathies of spinal cord development in rat pups maternally fed with fried potato chips

Objective: Acrylamide is a neurotoxic material and recently elevated levels of acrylamide in varieties of foodstuffs were reported. The present study aimed to illustrate the demyelination of spinal cord of pups maternally fed a diet containing fried potato chips. Methods: Eighty fertile virgin female Wistar rats were made pregnant after mating with healthy male. Zero dates of gestation were determined and dams were arranged into three groups as control, acrylamide-treated (15 mg/kg body weight, p.o.) and 50% fried potato chips containing diet group. Treatments were carried every other day from 6th day of gestation until 3-week post-partum. The cervical spinal cord was separated and subjected for SDS-PAGE analysis and light and transmission electron microscopy. Results: Comparing with acrylamide-treatment, protein expression in spinal cord of pups maternally fed with fried potatoes was altered. Necrosis of motor neuronal cells within grey matter, hyperplasia of ependymal lining cells and fragility of white matter was detected. At ultrastructural level, the sensory and motor neuronal cells showed convoluted nuclear envelope and either chromatolysis or compacted chromatin material. Fragmentation of rough endoplasmic reticulum and damage of mitochondria become well evident in pups maternally fed with potato chips. The neuronal axons possessed vacuolation and demyelination associated with apparent damage of mitochondria. Conclusion: Supplementation of fried potato chips exerted neurotoxicity either directly through their content of acrylamide or via its metabolite glycidamide. Both components were reported to find their way across the placenta during gestation and breast milk during the lactation period, interfering with spinal cord differentiation and adversely affected demyelination.

Ultrastructural and antioxidative changes in lupine embryo axes in response to salt stress

Embryo axes of lupine (<em>Lupinus luteus</em> L. ‘Mister’) were subjected to 0.1 M NaCl salt stress for 24 and 48 h. The ultrastructure modification and adjustment of antioxidant enzymes activities and izoenzymes profiles were observed. In cells of lupine embryo axes grown for 48 hours in medium with 0.1 M NaCl mitochondria took the forked shape and bulges of the outer mitochondrial membranes appeared. Moreover, the inflating and swelling of rough endoplasmic reticulum (RER) lumen and fragmentation of RER were noticed. The level of H<sub>2</sub>O<sub>2</sub> was higher in salt treated embryo axes after 24 hours and increase of thiobarbituric acid reactive substances was observed after both 24 and 48 h of salt treatment. Native gel electrophoresis showed increased intensities of bands for catalase isozymes in response to salt stress, whereas activity of catalase was higher only in embryo axes grown for 48 h in control conditions. Appearance of two new isoforms of ascorbate peroxidase was observed after 48 h only under control condition, however increased activities were stated for both control and salt-stress condition after 48 h. No changes in isozymes pattern for superoxide dismutase were observed, but significant decrease in superoxide dismutase activity was noticed in relation to time and salt stress. Possible role of these enzymes in salt stress tolerance is discussed. The 0.1 M salt stress is regarded as a middle stress for lupine embryo axes and the efficiency of stress prevention mechanisms is proposed.

Yeast Programed Cell Death and Aging

Yeast Programed Cell Death and Aging

New imidazolidine derivatives as anti-Trypanosoma cruzi agents: structure–activity relationships

Imidazolidine derivatives are key components for the development of bioactive compounds for the treatment of many diseases, especially Chagas. In fact, others studies showed that the imidazolidine-2,4-dione has stood out by presenting a wide spectrum of pharmacological activities including anticonvulsants, antiarrhythmic, and antiparasitic. In the present study, we investigated the morphological alterations induced by imidazolidine derivates LPSF/NN-52 and LPSF/NN-100 on trypomastigotes forms of Trypanosoma cruzi through ultrastructural analysis by electron microscopy. Many concentrations were used to measure the antiparasitic propriety promoted by imidazolidine derivatives, and our study indicates that parasites treated with 13 μg mL(-1) of the imidazolidine derivates for 24 h revealed severe damage to the parasite's mitochondrial complex. Beyond that, also observed in treated parasites were the following: myelin bodies, enlargement of cytoplasm vacuole, fragmentation of endoplasmic reticulum, and some treated samples clearly showed signs of necrosis. To confirm the ultrastructural results, some assays were performed for knowledge cellular death induction promoted by imidazolidine derivates against immune spleen cells. The induction of the necrotic process through derivatives LPSF/NN-52 and LPSF/NN-100 showed similar results in relation to nifurtimox and benznidazole. In the last assays, it was demonstrated that NN-100 was efficient against epimastigotes and trypomastigotes forms and these results reinforce the mechanisms of action of both new imidazolidine derivatives against T. cruzi.

The ultrastructure of the articular cartilage in rats with experimental gonarthrosis under effects of low-intensity laser radiation, Diclofenac and Glucosamine

The influence of different combinations of low-intensity infrared laser radiation, Diclofenac and Glucosamine on the ultrastructural organization of an articular cartilage of rats with experimental osteoarthrosis was studied with help of methods of transmission electronic microscopy. It was found out that glucocorticoid-induced osteoarthrosis was accompanied by considerable damages in the ultrastructural organization of the articular cartilage. Chondrocytes revealed heterochromatization of the cell nucleus, areas of destruction in cytoplasm, fragmentation and degranulation of endoplasmic reticulum, and an increased amount of lysosomes in cytoplasm. Conservative treatment of the animals with experimental osteoarthrosis by the laser-Glucosamine-Diclofenac complex promoted activation of chondrocyte metabolism and enhancement of biosynthetic processes in chondrocytes.

Alcohol Disrupts Endoplasmic Reticulum Function and Protein Secretion in Hepatocytes

Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo. HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP). VL-17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced. Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD.

Effect of fipronil on brain and muscle ultrastructure of Nilaparvata lugens (Stål) (Homoptera: Delphacidae)

The ultrastructure of Nilaparvata lugens brain cells was damaged by treatment at different fipronil concentrations. The cell showed swollen mitochondria and vacuolization, but no mitochondrial cristae. Rough endoplasmic reticulum (RER) fragmentation and degranulation were seen. The dilatation of endoplasmic reticulum cisterns was very prominent, and the predominant lamellar RERs were arranged chaotically. The Golgi apparatus demonstrated obvious changes in configuration, as dilated with closed cisternae and atypical vesicles. The mitochondria mainly showed large vacuolization in muscles. Nuclear degeneration and condensation and increased numbers of large hydropic vacuoles and lysosomes were observed. It was concluded that the effect on cellular components was fipronil-specific. Changes in cellular ultrastructure seem to be an appropriate ecotoxicological indicator of the insecticide's efficacy.

Rapid Fragmentation of the Endoplasmic Reticulum in Cortical Neurons of the Mouse Brain in situ Following Cardiac Arrest

Neuronal endoplasmic reticulum (ER), continuous from soma to dendritic spines, undergoes rapid fragmentation in response to N-methyl-D-aspartate (NMDA) receptor stimulation in hippocampal slices and neuronal primary cultures. Here, we show that ER fragments in the mouse brain following cardiac arrest (CA) induced brain ischemia. The ER structure was assessed in vivo in cortical pyramidal neurons in transgenic mice expressing ER-targeted GFP using two-photon laser scanning microscopy with fluorescence recovery after photobleaching (FRAP). Endoplasmic reticulum fragmentation occurred 1 to 2 minutes after CA and once induced, fragmentation was rapid (<15 seconds). We propose that acute ER fragmentation may be a protective response against severe ischemic stress.

‘Functional Histology’ in Brain Ischemia Studies

Classical histology and biochemistry constitute two very different ways to study biology. Classical histology allows direct visualization of tissue, but is limited by the non-specificity of organic dyes and the need to stain fixed tissue in postmortem samples. Classical biochemistry allows direct observation of the activities of tissue constituents, however, at the expense of complete cellular disruption, and is, in a sense, ‘blind' in that kinetic or equilibrium expressions are required intermediaries between data and interpretation. But advances in microscope technology, protein engineering, and transgenic animal technology have blurred this classical distinction. One can today speak of a ‘functional histology' that goes considerably beyond that discussed by Salthouse (1976) many years ago. It is well known that brain ischemia induces morphological alterations in postischemic neurons. Studies using classical histology cataloged end-stage morphologies such as ischemic cell change (Rosenblum, 1997). However, the past decade has seen increasing application of ‘functional histology' methods that literally illustrate ischemia-induced morphological changes at earlier time periods of the injury course. Two notable examples are the Hu et al (2000) discovery of the accumulation in postischemic neurons of ubiquitin–protein clusters (Ge et al, 2007) and our own work on mRNA-containing structures (DeGracia et al, 2008), both of which occur within minutes of reperfusion. In this issue of the Journal of Cerebral Blood Flow and Metabolism, the article by Krzysztof et al (2011) advances the application of ‘functional histology' to a completely new level in the field. The authors utilized fluorescent photobleaching technology and transgenic animals bred to contain a version of green fluorescent protein fused with the calreticulin endoplasmic reticulum-targeting sequence. Using this system, the authors visualized from the cortical surface of intact whole animals the real-time dynamics of the endoplasmic reticulum of cortical neurons during the ischemic period. On the basis of the time of recovery of fluorescent signal after photobleaching, the authors elegantly and convincingly demonstrated that fragmentation of the endoplasmic reticulum occurs within minutes of the onset of global ischemia. The results are illustrated with striking three-dimensional images of fragmented endoplasmic reticulum in cortical neurons. The techniques and analysis are straightforward and unambiguous, leaving little room for controversy. To my knowledge, this study now marks the earliest observation of a clear-cut ‘functional morphological' alteration in postischemic neurons. It is also notable the authors attribute this change to activation of ‘stress responses' in the postischemic neurons. This interpretation reflects the increasing realization that postischemic neuronal death may not be because of only ischemia-induced damage but also because of a competition between ischemia-induced damage and the cell's innate ability to respond to damage (Endres et al, 2008; Lo, 2008; DeGracia, 2010). In sum, the paper by Krzysztof et al (2011) literally illustrates the power of a ‘functional histological' approach by demonstrating the very rapid response of the neuron to the ischemic perturbation. We expect the general approach used here to become increasingly prevalent, and the current paper represents an important milestone on this path.

Ultrastructural changes in the hepatopancreas of Penaeus monodon Fabricius 1798 given aflatoxin B1 diets

Fungal contamination of shrimp feeds affects the shelf life leading to substantial economic losses. Ultrastructural changes in Penaeus monodon sub-adults fed three different doses (50, 1000 and 2000 ppb) of aflatoxin B1 (AFB1) were studied. At the end of 4 and 8 weeks of experiment, the hepatopancreas of shrimps were observed for ultrastructural changes. The prominent ultrastructural changes in hepatopancreas of the shrimps fed 1000 and 2000 ppb AFB1 were rupture of cell membrane and microvillus border, damage and swelling of mitochondria, fragmentation of endoplasmic reticulum, nuclear vacuolation, chromatin condensation and autophagy. This study helps to decipher the ultrastructural changes and relate the effects on biochemical, pathological, immunological and histological architecture of the shrimps fed AFB1-incorporated feed. The observed ultrastructural changes could serve as indicators in shrimps exposed to toxicants.

Morphogenetic status of somatic embryos of Citrus sinensis from mature polyembryonic seeds and those produced in vitro

In tissue culture of sweet orange (Citrus sinensis (L.) Osbeck, cv. Tarocco), we obtained mass regeneration of somatic embryos with two morphologically distinct cotyledons about 3 mm in length, their numbers amounting to 110–150 embryos per petri dish and 60 to 80% of the population. The morphogenetic state of somatic embryos was compared using the embryos with the cotyledons of different size (from 3 to 10 mm) from mature polyembryonic seeds as a test system and the cell number, size, and ultrastructural organization, and the number of protein bodies in the cotyledon cells as morphological and biochemical criteria. Cell number in the cotyledons of different size was related to the content of protein bodies therein. Typical protein bodies where 33 kD polypeptide of storage proteins was identified were detected only in the cotyledons, which size was identical to that of embryonic cotyledons from monoembryonic seeds of citrus plants. In the cells of smaller cotyledons, we detected protein-accumulating vacuoles with electron-dense inclusions that irrespective of their size, shape and structure accumulated the gold label. The number of the cells with protein depositions in vacuoles decreased when the cotyledons became smaller. Irrespective of the origin of embryos (in vivo or in vitro), lipids were the major storage metabolites in the cells of 3-mm cotyledons. As the cotyledon-forming cells became smaller and less numerous, their metabolic activity tended to decrease in line with the fragmentation of endoplasmic reticulum, the absence of polysomic complexes, and indistinct inner organization of mitochondria and plastids. It was concluded that somatic embryos developing in vivo and in vitro were physiological dwarfs, that is, the structures with diminutive storage organ with characteristically incomplete cell differentiation. Apparently such forms emerged due to the suppression of cotyledon growth at the initial stages of their organogenesis; as a result, the cell population could not properly realize both organo- and histogenesis.

An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship

Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best-known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca(2+) signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.

Protein Metabolic Profiles and Ultrastructural Studies in Rat Liver During Cypermethrin Toxicosis

Nagarjuna, A. and Doss, P.J. 2010. Protein metabolic profiles and ultrastructural studies in rat liver during cypermethrin toxicosis. J. Appl. Anim. Res., 37: 171–176. To evaluate the toxic effect of cypermethrin on rat liver its sublethal doses (41 mg/kg bw) were administered orally as single, double and multiple doses at 48 h interval. The protein metabolic profiles and histological changes in different groups and ultrastructural changes in multiple dose treated groups were studied. Total proteins decreased, whereas, free amino acids and the activity of protease, AST, ALT and GDH as well as ammonia and urea increased (P<0.05) in cypermethrin—exposed rats. The ultrastructural changes were dense chromatin condensation (clumping) at the centre of nucleus, fragmentation of rough endoplasmic reticulum, ruptured mitochondria and peroxysomes and disappearance of secretory vesicles. These changes indicated cypermethrin's toxic effects on cellular functioning. Such changes can lead to apoptotic and necrotic cell death due to various cellular organelles dysfunction.

Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity

Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity

The Endoplasmic Reticulum of Dorsal Root Ganglion Neurons Contains Functional TRPV1 Channels

Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca(2+) channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1(ER)). We have studied the properties of TRPV1(ER) in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1(ER) with capsaicin or other vanilloids produced an increase of cytosolic Ca(2+) due to Ca(2+) release from the ER. The decrease of [Ca(2+)](ER) was directly revealed by an ER-targeted aequorin Ca(2+) probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1(ER) to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1(ER) was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca(2+) because the sensitivity to capsaicin was increased by loading the cells with the Ca(2+) chelator BAPTA. Decreasing [Ca(2+)](ER) did not affect the sensitivity of TRPV1(ER) to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Delta35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1(ER) for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatidylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1(ER) to agonists may be critical for neuron health, because otherwise Ca(2+) depletion of ER could lead to ER stress, unfolding protein response, and cell death.

Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin

Establishment and maintenance of proper architecture is essential for endoplasmic reticulum (ER) function. Homotypic membrane fusion is required for ER biogenesis and maintenance, and has been shown to depend on GTP hydrolysis. Here we demonstrate that Drosophila Atlastin--the fly homologue of the mammalian GTPase atlastin 1 involved in hereditary spastic paraplegia--localizes on ER membranes and that its loss causes ER fragmentation. Drosophila Atlastin embedded in distinct membranes has the ability to form trans-oligomeric complexes and its overexpression induces enlargement of ER profiles, consistent with excessive fusion of ER membranes. In vitro experiments confirm that Atlastin autonomously drives membrane fusion in a GTP-dependent fashion. In contrast, GTPase-deficient Atlastin is inactive, unable to form trans-oligomeric complexes owing to failure to self-associate, and incapable of promoting fusion in vitro. These results demonstrate that Atlastin mediates membrane tethering and fusion and strongly suggest that it is the GTPase activity that is required for ER homotypic fusion.

Histology and ultrastructure of Aedes albopictus larval midgut infected with Bacillus thuringiensis var. israelensis

Histological and ultrastrucutural alterations in the midgut of Aedes albopictus larvae infected with Bacillus thuringiensis var. israelensis (Bti) were observed by light and transmission electron microscopy (TEM). Two formulations of Bti were used: granulated and powder, with 0.2% active ingredient in 90 larvae of Ae. albopictus distributed in three containers containing 30 larvae each (one control group and two test groups). The midgut epithelium of the control group presented flattened and elongated cells with mace-shape with a narrow base. Midgut epithelium cells' surface was convex and had a large circular nucleus located in the median-apical portion of the cell. These cells also presented a basal lamina with a small accumulation of extracellular fibrous matrix, thus characterizing a basal membrane, with a muscle layer and a peritoneal membrane more externally. After Bti ingestion, the larvae stopped/slowed their natural movements down in 5 min. After 30 min approximately, the swimming movements stopped completely. Internally, the intestinal cells showed a disorganization of the basal processes, dilatation and fragmentation of the rough endoplasmic reticulum, with intense cytoplasmic vacuolization. There were concentric dense laminas accumulated in the cytoplasm, and these residual membranous bodies were seen greatly increased in size after 60 min. Mitochondria, fragments of rough endoplasmic reticulum and other remainder organelles were surrounded and segregated from the cytoplasm by exocytosis. This article reports the histopathological alterations in the midgut of Ae. albopictus after infection with Bti and contributes to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against mosquito larvae.