Density Of Endoplasmic Reticulum Research Articles

Dynamic changes in the association between maternal mRNAs and endoplasmic reticulum during ascidian early embryogenesis

Axis formation is one of the most important events occurring at the beginning of animal development. In the ascidian egg, the antero-posterior axis is established at this time owing to a dynamic cytoplasmic movement called cytoplasmic and cortical reorganisation. During this movement, mitochondria, endoplasmic reticulum (ER), and maternal mRNAs (postplasmic/PEM RNAs) are translocated to the future posterior side. Although accumulating evidence indicates the crucial roles played by the asymmetrical localisation of these organelles and the translational regulation of postplasmic/PEM RNAs, the organisation of ER has not been described in sufficient detail to date owing to technical difficulties. In this study, we developed three different multiple staining protocols for visualising the ER in combination with mitochondria, microtubules, or mRNAs in whole-mount specimens. We defined the internally expanded “dense ER” using these protocols and described cisterna-like structures of the dense ER using focused ion beam-scanning electron microscopy. Most importantly, we described the dynamic changes in the colocalisation of postplasmic/PEM mRNAs and dense ER; for example, macho-1 mRNA was detached and excluded from the dense ER during the second phase of ooplasmic movements. These detailed descriptions of the association between maternal mRNA and ER can provide clues for understanding the translational regulation mechanisms underlying axis determination during ascidian early embryogenesis.

A novel automated image analysis pipeline for quantifying morphological changes to the endoplasmic reticulum in cultured human cells

BackgroundIn mammalian cells the endoplasmic reticulum (ER) comprises a highly complex reticular morphology that is spread throughout the cytoplasm. This organelle is of particular interest to biologists, as its dysfunction is associated with numerous diseases, which often manifest themselves as changes to the structure and organisation of the reticular network. Due to its complex morphology, image analysis methods to quantitatively describe this organelle, and importantly any changes to it, are lacking.ResultsIn this work we detail a methodological approach that utilises automated high-content screening microscopy to capture images of cells fluorescently-labelled for various ER markers, followed by their quantitative analysis. We propose that two key metrics, namely the area of dense ER and the area of polygonal regions in between the reticular elements, together provide a basis for measuring the quantities of rough and smooth ER, respectively. We demonstrate that a number of different pharmacological perturbations to the ER can be quantitatively measured and compared in our automated image analysis pipeline. Furthermore, we show that this method can be implemented in both commercial and open-access image analysis software with comparable results.ConclusionsWe propose that this method has the potential to be applied in the context of large-scale genetic and chemical perturbations to assess the organisation of the ER in adherent cell cultures.

Ultrastructural analysis of the dehydrated tardigrade Hypsibius exemplaris unveils an anhydrobiotic-specific architecture

Tardigrades can cope with adverse environmental conditions by turning into anhydrobiotes with a characteristic tun shape. Tun formation is an essential morphological adaptation for tardigrade entry into the anhydrobiotic state. The tun cell structure and ultrastructure have rarely been explored in tardigrades in general and never in Hypsibius exemplaris. We used transmission electron microscopy to compare cellular organization and ultrastructures between hydrated and anhydrobiotic H. exemplaris. Despite a globally similar cell organelle structure and a number of cells not significantly different between hydrated and desiccated tardigrades, reductions in the sizes of both cells and mitochondria were detected in dehydrated animals. Moreover, in anhydrobiotes, secretory active cells with a dense endoplasmic reticulum network were observed. Interestingly, these anhydrobiote-specific cells are in a close relationship with a specific extracellular structure surrounding each cell. It is possible that this rampart-like extracellular structure resulted from the accumulation of anhydrobiotic-specific material to protect the cells. Interestingly, after five hours of rehydration, the number of secretory cells decreased, and the specific extracellular structure began to disappear. Twenty-four hours after the beginning of rehydration, the cellular structure and ultrastructure were comparable to those observed in hydrated tardigrades.

Increased mitochondrial distribution in early-cleaving embryos indicate successful pre-implantation development

The timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early have higher developmental viability compared to their late counterparts. During embryonic development, cleavage is affected by cellular metabolic processes performed by mitochondria and its synergistic interaction with endoplasmic reticulum (ER). However, in depth study on differences of mitochondria and ER ultrastructures in early- cleaving (EC) versus late- cleaving (LC) embryos is limited. This study compares mitochondria and ER ultrastructures of EC versus LC embryos using Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM). Embryos were obtained from female ICR superovulated mice, 28-30 hours post hCG. Two-cell embryos were categorized as early-cleaving (EC), while zygotes with the second polar body and two pronuclei present were categorized as late-cleaving (LC). The LC embryos were cultured in vitro until the 2- cell stage. In EC embryos, mitochondria were mostly found at the perinuclear region and closely associated with dense ER. Meanwhile, mitochondria of LC embryos were distributed uniformly within the cytoplasm. Mitochondrial fluorescence intensity was significantly higher in EC versus LC [(18.7 ± 0.4) versus (14.6 ± 0.4)] x 105 pixel, (p<0.01). Development to the blastocyst stage was also significantly higher in EC compared to LC embryos (96.7% versus 60.9%) (p<0.01). Higher viability of EC embryos is attributed to the close association of their mitochondria to ER. This contributed to better mitochondrial fission, resulting in enhanced energy generating processes and preimplantation development.

Histología y ultraestructura del sistema reproductor masculino de la langosta espinosa de la India Panulirus homarus (Decapoda: Palinuridae)

The spiny lobster Panulirus homarus, distributed along the Southeast and Southwest coasts of India, is an important commercial species having mariculture potential. Despite its importance, the structural and ultrastructure features of male gonads from this species have received scarce attention. Hence this study was aimed to describe the male reproductive tract of the species, using standard histological and electron microscopy techniques. Gonads from 94 specimens of P. homarus ranging in carapace length 37mm-92mm from Vizhinjam (Southwest coast of India.) were obtained and processed for the study (Histology-70 numbers & ultrastructure-24 numbers). The male reproductive system consists of paired testis and vas deferens located in the cephalo-thoracic region. Macroscopically, the reproductive tract was observed in lobsters > 35mm carapace length. In immature testis, spermatogonia were seen which measured 6.9-13.8 microm in diameter and in the mature testis primary (5.4-5.9 microm) and secondary spermatocytes (2.8-3 microm) and spermatids (2.2-2.4 microm) were present. Each vas deferens consists of proximal and distal portions. The spermatophoric mass begins formation in the proximal vas deferens. In the distal vas deferens the spermatophoric mass containing the spermatozoa are arranged in packets towards the periphery by the gelatinous matrix produced by the typhlosole. Ultrastructurally, the spermatogonia have lamina, nucleus and mitochondria like bodies, the primary spermatocytes have nucleus, dense chromatin and vacuolated cytoplasm and the spermatids have mitochondria, endoplasmic reticulum and centrioles. The endoplasmic reticulum and the nuclear envelope in the spermatids form the acrosome. The radial arms with microtubules are formed in association with the dense endoplasmic reticulum, near the nucleus. The sperm has a spherical structure with the nucleus, lamellar region, spikes and acrosome. This is the first comprehensive report of the structure of the male gametes and spermatogenesis in P. homarus from Indian waters.

Paxillin Associates with Poly(A)-binding Protein 1 at the Dense Endoplasmic Reticulum and the Leading Edge of Migrating Cells

Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.

Elevation of Intracellular Glucosylceramide Levels Results in an Increase in Endoplasmic Reticulum Density and in Functional Calcium Stores in Cultured Neurons

Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca(2+)](i) response to glutamate, and a large increase in [Ca(2+)](i) release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co-incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B(1), completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca(2+)](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C(8)-glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre-incubation with ryanodine, suggesting that [Ca(2+)](i) release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease.

Chapter 5: Targeting GFP to Organelles

This chapter shows that, through suitable chimeras, green fluorescent protein (GFP) can be targeted to various intracellular locations (the cytosol, the nucleus, the mitochondria, the endoplasmic reticulum, the Golgi, the subplasmalemmal space), thus providing powerful in vivo markers of these compartments. The GFP chimeras described in this chapter are currently utilized in lab for monitoring the dynamic changes of organelle structure that occur in living cells. Two types of instruments are currently used for detecting GFP fluorescence: (a) a wide-field fluorescence imaging system, based on a Zeiss Axiovert inverted microscope and a Princeton Instruments back-illuminated camera, and (b) a Nikon RCM 8000 confocal microscope. In both cases, the microscope is equipped with motorized control of the Z stage, thus allowing the acquisition of three-dimensional images. It is obvious that by confocal imaging, the dense endoplasmic reticulum structure of HeLa cells can be finely resolved, and the continuous rearrangement of organelle structure can be clearly appreciated.

Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase.

Galactosylceramide (GalCer) is the major glycolipid in brain. In order to characterize the activity of brain UDPgalactose: ceramide galactosyltransferase (CGalT), it has been stably expressed in CGalT-negative Chinese hamster ovary (CHO) cells. After fractionation of transfected cells, CHO-CGT, on sucrose gradients, the activity resides at the density of endoplasmic reticulum and not of Golgi. A lipid chromatogram from CHO-CGT cells revealed two new iodine-staining spots identified as GalCer, since they comigrate with GalCer standards, can be metabolically labelled with [3H]galactose, are recognized by anti-GalCer antibodies, and are resistant to alkaline hydrolysis. A third [3H]galactose lipid was identified as galactosyldiglyceride. In the homogenate CGalT displays a 25-fold preference for hydroxy fatty acid-containing ceramides. Remarkably, endogenous GalCer of transfected cells contains exclusively non-hydroxy fatty acids: fast atom bombardment and collision-induced dissociation mass spectrometric analysis revealed mainly C16:0 in the lower GalCer band on TLC and mainly C22:0 and C24:0 in the upper band. Our results suggest that CGalT galactosylates both hydroxy- and non-hydroxy fatty acid-containing ceramides and diglycerides, depending on their local availability. Thus, CGalT alone may be responsible for the synthesis of hydroxy- and non-hydroxy-GalCer, and galactosyldiglyceride in myelin.

The ultrastructure of Heleopera petricola an agglutinate soil amoeba; with comments on feeding and silica deposition

The ultrastructure of H. petricola is described from cultured specimens. Siliceous shell plates are bound by a continuous layer of organic cement which is thicker in the posterior half of the body. The cytoplasm is divided into two distinct portions: anteriorly it is vacuolar and composed of numerous pseudopodial strands; posteriorly it is a compact mass containing the organelles, two or three contractile vacuoles, a typical 'ovular' nucleus with several nucleoli that occupies a central position in a region of dense endoplasmic reticulum, and numerous dictyosomes. Prey is captured by a loose arrangement of pseudopodial strands and digestion occurs within specific areas of compact cytoplasmic strands. It is suggested that the cyst wall of naked amoebae is ruptured by specific enzymatic activity and the contents digested after penetration by cytoplasmic strands. Predated siliceous body plates, enclosed in membrane bound vacuoles are modified by the addition of silica to their surfaces. Some organic particles, like bacteria, may also be coated with biogenic silica. Comparisons are made both with the predation of fungal spores by giant naked amoebae and silica biomineralization in another testate amoeba, Netzelia tuberculata.

Ultrastructure of the vegetative organisation and initial stages of silica plate deposition in the soil testate amoebaCorythion dubium

The siliceous body plates ofCorythion dubium are bound by a band of organic cement which is thickest at the lateral margins. The anterior vacuolar cytoplasm is separated by a pigment zone, which forms a dark band in the mid-body region, from the compact posterior region containing a typical vesicular nucleus surrounded by a region of dense endoplasmic reticulum. A pellicular basket of microtubules surrounds the posterior cytoplasm. The large Golgi complex lies between the nucleus and the fundus. Numerous coated and uncoated vesicles from the Golgi cisternae are seen in the peripheral cytoplasm alongside developing plates. These small siliceous plates are enclosed in silicon deposition vesicles lying in surface ruffles of the plasmalemma, often in association with a pair of microtubules. Observations are made on the formation of these vesicles and the early stages of silica deposition. A comparison is drawn between silica deposition inC. dubium and choanoflagellates where there is a similar association between silicon deposition vesicles and microtubules.

The biology and ultrastructure of the testate amoeba, Difflugia lucida penard (Protozoa, Rhizopoda)

Two strains and one population of Difflugia lucida have been studied, from Germany and Japan. The ultrastructure is described from selectively cultured specimens with thin shells; such agglutinate shells have a slightly different morphology from those provided with ample mineral grains. The cytoplasm is described and of particular interest are details of the 'ovular' nucleus, which has an additional lamina to the nuclear membrane, several nucleoli and changes its position within the region of dense endoplasmic reticulum. Two types of organic cement vesicles are secreted by the dictyosomes. The building blocks are spherical in the cytoplasm but when discharged often become elongate strips. Smaller cement vesicles discharge fibrous material which is used either to line the inner shell wall or possibly form the enclosing membrane of cysts. Some cytoplasmic differences associated with the first stage of division and precystic organisation are included. Comparisons are made with recent light microscope observations.

Morphology of identified interneurons in the CA1 regions of guinea pig hippocampus.

Identified interneurons in the CA1 region of guinea pig hippocampus were examined using light and electron microscopic (EM) techniques. The HRP was intracellularly injected into cells, recorded in the in vitro slice preparation, which met physiological criteria for interneurons. These neurons were characterized at the light and electron microscopic level, and used as standards for identifying interneurons which had not been HRP-labeled. Pyramidal basket cell somata were found in stratum oriens and stratum pyramidale; they were easily identifiable by their convoluted nucleus, dense endoplasmic reticulum, and numerous organelles. Aspinous dendrites reached into stratum oriens, where they received profuse synaptic input (primarily asymmetric synapses). Many of the asymmetric synapses degenerated following commissural lesions, suggesting that much of the input to interneurons was from extrinsic afferents. Dendrites were characterized by their spindled appearance, especially at distal sites. They showed postsynaptic degenerative changes following commissural lesions. Interneuron axons were extremely fine, with regular enlargements or "beads" which made apparent synaptic contacts primarily on pyramidal cell somata. The axon of a single, HRP-injected interneuron made many apparent contacts on large numbers of pyramidal cells; axons arborized over distances of several hundred micra within stratum pyramidale. This study provides direct evidence that neurons, with an identified inhibitory interneuron function in hippocampus, can mediate feed-forward as well as feed-back (recurrent) inhibition. Interneuron output showed extreme divergence, with influence over large distances. The high density of intracellular organelles in these cells suggested high metabolic activity and demand, perhaps making these interneurons exceptionally vulnerable to trauma-induced damage.

ATP and GTP Stimulate Membrane-bound but not Digitonin-solubilized -Glucan Synthases from Saprolegnia monoica

Summary: Glucan synthases of Saprolegnia assayed in the presence of MgCl2 [(1→4)-β-glucan production] were stimulated by ATP or GTP, while the non-phosphorylating nucleotide analogues adenylyl (β,γ-methylene)diphosphonate and guanylyl (β,γ-methylene)diphosphonate did not stimulate glucan synthesis. The analogues adenosine 5′-O-(3-thiotriphosphate) and guanosine 5′-O-(3-thiotriphosphate) stimulated the enzyme activity but to a lesser extent than the nucleotides. The activators stimulated membrane-bound enzymes, particularly those equilibrating at the endoplasmic reticulum density in sucrose gradients. Glucan synthases solubilized by digitonin were not stimulated by nucleotides. Nucleotides, whose action seems to be related to the integrity of the phosphate groups, may produce their stimulative effect by increasing the translocation of the substrate through membranes to the enzymes.

Variations cytoplasmiques dans l'épithelium pigmenté en fonction du cycle lumineux; observations préliminaires*

Cytochemical and stereological studies on frog (rana ridibunda) RPE during a 12 hrs dark/12 hrs light cycle. Rapid changes in surface density of endoplasmic reticulum and in volume density of myeloid bodies shortly after the onset of light. Numerical and volumetric density of microperoxisomes increased after the fourth hour of light, with a concomitant decrease in the volume of phagosomes.

Gilbert's Syndrome: Analytical Subcellular Fractionation of Liver Biopsy Specimens. Enzyme Activities, Organelle Pathology and Evidence for Subpopulations of the Syndrome

1. The hepatic organelle pathology of 12 patients with Gilbert's syndrome was studied by analytical subcellular fractionation in combination with enzymic microanalysis of liver biopsy material. 2. All patients showed increased activities of the endoplasmic reticulum marker enzyme, neutral α-glucosidase. Seven patients showed a decrease in the modal density of the endoplasmic reticulum, from 1·20 to 1·15 g/ml. These patients also showed striking hypertrophy of the hepatocyte smooth endoplasmic reticulum on electron microscopy. The remaining five patients showed normal endoplasmic reticulum density distribution with a peak at 1·20 g/ml, and had normal appearance of the endoplasmic reticulum on electron microscopy. 3. All patients showed increased activity of three lysosomal marker enzymes: N-acetyl-β-glucosaminidase, acid phosphatase and β-glucuronidase. The distribution of these enzymes in the sucrose gradients showed less enzyme in the high-density region of the gradients, indicating a reduced equilibrium density of the lysosomes. Assays of latent and sedimentable N-acetyl-β-glucosaminidase, a measure of lysosomal integrity, were normal. 4. Marker enzyme activities and density gradient distribution of other organelles, including plasma membrane (5′-nucleotidase), mitochondria (malate dehydrogenase), biliary canaliculi (γ-glutamyl transferase) and cytosol (lactate dehydrogenase) were normal. Increased catalase activities were noted.

Elektronenmikroskopische Untersuchung an intrazellulär lebenden Einzellern in Foraminiferen / Electron Microscopic Study on Intracellular Microorganisms in Foraminifera

In the cytoplasmic vacuoles and lacunas of the monothalamous foraminifera Myxotheca arenilega and Allogromia laticollaris unicellular organisms of about 2,5-6 µm appear, which persist intracellularly, showing no disintegration, even after a long starvation period of the host. The mono- or multinucleated cells show a well developed dense endoplasmic reticulum, a large number of ribosomes, elongated mitochondria of the tubulus type, a few Golgi - complexes and paired centrioles or basal bodies. The cells are surrounded by a sheath of about 0.1 μm, consisting of numerous lamellae in a loose arrangement together with osmiophilic substances. It is presumed that this protects the cells against digestion by the host. Since foraminifera which contain these organisms show no differences in growth and size at the end of their developing period compared to those cells which are free from these organisms, it is doubted that these organisms are parasites. They are also presumably not symbiontic organisms, since they possess no thylacoids. It is likely that they are harmless commensals. Their systematic position is unknown.

The ultrastructure and histochemistry of a nematode-induced giant cell.

The development of giant cells induced by the nematode Meloidogyne in tomato roots has been followed under controlled growth conditions and the ultrastructure and histochemistry of these structures have been examined. Entry of the nematode larvae into the roots took place within 24 hours; giant cell formation started on the 4th day and involved breakdown of the cell walls accompanied by thickening of a surrounding giant cell wall and an increase in density and area of the cytoplasm. The nuclei increased in number by simultaneous mitosis throughout a single giant cell. The peak of cytoplasmic density was reached after moulting and during egg production. The rate of protein synthesis in the giant cell is correlated with the rate of growth of the nematode. The giant cell wall is a thick, irregularly surfaced structure which contains all the normal polysaccharide components of a cell wall. The cytoplasm is rich in protein and RNA and contains mitochondria, proplastids, Golgi bodies, and a dense endoplasmic reticulum. The nuclei are large and irregular in shape and contain large nucleoli and a number of Feulgen-positive bodies scattered irregularly along the nuclear envelope. The nucleolus contains RNA and fat as well as Feulgen-positive granules which are revealed after treatment with ribonuclease. It consists of a dense outer cortex surrounding a much lighter central core and is connected at times with the Feulgen-positive bodies in the nucleus. Speculation is provided on the role of these bodies in cytoplasmic protein synthesis.