Changes In Cellular Organelles Research Articles

Subcellular responses of fish cells to sewage effluents: Cell line-based and whole-animal based approaches

Acute toxicity determination is essential in the ecological risk assessment. Traditionally, acute toxicity testing requires substantial numbers of animals and uses death as an apical end point which requires large number of experimental animals and takes days to obtain the results. Application of fish cell lines can provide a possible alternative to traditional acute toxicity test. However, cell-based assay may show several orders of magnitude less sensitive than the animal-based results. Some changes in cellular organelles could have the sensitivity in responding to pollutants. For this reason, a cell-based fluorescent assay was developed using rabbitfish fin cells as model and fluorescent probes to visualize the subcellular responses. The subcellular responses under sewage effluents exposure were captured by confocal microscopy. These cellular responses were quantified and several subcellular indexes represented the toxicity. The optimized assay was then used to determine the toxicity of sewage effluents displaying toxicity to aquatic animals. Through visualization of cellular responses, we further screened several cellular indexes including lysosomal number and mitochondrial size which had a good linear relationship with sewage effluents content. Besides, these cellular indexes had a good agreement between in vivo and in vitro results, demonstrating the accuracy of cellular parameters in representing the acute toxicity of sewage effluents. The developed cell-based testing assay presented here has the characteristics of a faster and cheaper method, which does not require complex facilities and large amount of testing samples. The developed assay may be further applied in predicting the acute toxicity to sewage effluents.

Long-term in-situ real-time fluorescence imaging of lipid droplets during cell ferroptosis process enabled by an epindolidione-based fluorescent probe

Ferroptosis, as a new form regulation of cell death discovered recently, is triggered by an iron-dependent accumulation of lipid reactive oxygen species and thus closely associated with cellular lipid droplets (LDs). However, directly limited by the un-sufficient photostability and low LDs specificity of common fluorescent probes, visualization of LDs during ferroptosis process particular in a long-term, in-situ, and real-time manner is highly preferred but extremely challenge. To this end, here a new LDs fluorescent probe Lipi-EP featuring with outstanding photostability and high LDs specificity have been rationally developed based on the four-ring-fused epindolidione. Experimentally, the photostability and LDs specificity of this new probe are much higher than the representative LDs probe BODIPY and Nile Red. Consequently, time-lapse multicolor imaging and time-lapse three-dimensional imaging employed this new probe have been successfully conducted to in-situ real-time monitor the LDs change during ferroptosis process up to 6 h. The morphology changes of cellular organelles (LDs, mitochondria and nucleus) and the variations of space distribution of LDs have been impressively visualized. Moreover, the sizes and numbers of LDs have been also determined based on the imaging. This work providing abundant information of LDs would significantly promote the in-depth study of LDs during ferroptosis.

Improved efficacy of foliar application of zinc oxide nanoparticles on zinc biofortification, primary productivity and secondary metabolite production in dragonhead

Improving medicinal plants' performance in semi-arid regions facing climate change could support sustainable food and agriculture production. Nano-fertilization is considered an emerging strategy for increasing plant production, while avoiding agroecosystem contamination. The objective of this study was to evaluate potential beneficial effects of foliar applications of two sources of zinc (Zn oxide nanoparticles [ZnONPs] as a novel nano-fertilizer and Zn suspension [ZnS] as a conventional fertilizer) on Zn biofortification, primary productivity (biomass) and secondary metabolite production in a medicinally important dragonhead (Dracocephalum moldavica L.) under semi-arid conditions. After 60- and 74-day post-planting, the desired fertilizers were sprayed on to plant foliage. The results showed that leaf Zn bioaccumulation, total chlorophyll, and total flavonoids increased in a dose-dependent manner for both Zn compounds. Sub-toxic levels of Zn-based NPs uptake (up to on average 152.6 mg Zn/kg upon foliar treatment with 160 mg ZnONPs treatment) led to stimulatory effects on total chlorophyll (0.93 g/kg FW), shoot biomass (2168 kg/ha), and essential oil content (3.4 g/kg) and yield (7.4 kg/ha) compared to ZnS treatment or control. The levels of secondary metabolites including neral, geraniol and geranial increased significantly with 160 mg/L ZnONPs compared to equivalent concentration of ZnS or control. The TEM analyses of leaf ultrastructure revealed changes in cellular organelles at 400 mg/L ZnONPs, likely a result of higher oxidative stress (malondialdehyde and H2O2). Higher efficacy of ZnONPs will support greater economic return for dragonhead growers and curtail environmental pollution. Foliar application of 160 mg/L ZnONPs is recommended for optimal micronutrient Zn biofortification, biomass production and essential oil yield in dragonhead, and may serve as a sustainable strategy to promote agricultural yield in (semi-) arid regions.

Using soft X-ray tomography for rapid whole-cell quantitative imaging of SARS-CoV-2-infected cells

SummaryHigh-resolution and rapid imaging of host cell ultrastructure can generate insights toward viral disease mechanism, for example for a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Here, we employ full-rotation soft X-ray tomography (SXT) to examine organelle remodeling induced by SARS-CoV-2 at the whole-cell level with high spatial resolution and throughput. Most of the current SXT systems suffer from a restricted field of view due to use of flat sample supports and artifacts due to missing data. In this approach using cylindrical sample holders, a full-rotation tomogram of human lung epithelial cells is performed in less than 10 min. We demonstrate the potential of SXT imaging by visualizing aggregates of SARS-CoV-2 virions and virus-induced intracellular alterations. This rapid whole-cell imaging approach allows us to visualize the spatiotemporal changes of cellular organelles upon viral infection in a quantitative manner.

Iron Accumulation and Changes in Cellular Organelles in WDR45 Mutant Fibroblasts.

Iron overload in the brain, defined as excess stores of iron, is known to be associated with neurological disorders. In neurodegeneration accompanied by brain iron accumulation, we reported a specific point mutation, c.974-1G>A in WD Repeat Domain 45 (WDR45), showing iron accumulation in the brain, and autophagy defects in the fibroblasts. In this study, we investigated whether fibroblasts with mutated WDR45 accumulated iron, and other effects on cellular organelles. We first identified the main location of iron accumulation in the mutant fibroblasts and then investigated the effects of this accumulation on cellular organelles, including lipid droplets, mitochondria and lysosomes. Ultrastructure analysis using transmission electron microscopy (TEM) and confocal microscopy showed structural changes in the organelles. Increased numbers of lipid droplets, fragmented mitochondria and increased numbers of lysosomal vesicles with functional disorder due to WDR45 deficiency were observed. Based on correlative light and electron microscopy (CLEM) findings, most of the iron accumulation was noted in the lysosomal vesicles. These changes were associated with defects in autophagy and defective protein and organelle turnover. Gene expression profiling analysis also showed remarkable changes in lipid metabolism, mitochondrial function, and autophagy-related genes. These data suggested that functional and structural changes resulted in impaired lipid metabolism, mitochondrial disorder, and unbalanced autophagy fluxes, caused by iron overload.

Toward Raman Subcellular Imaging of Endothelial Dysfunction.

Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.

Gene Expression-Related Changes in Morphologies of Organelles and Cellular Component Organization in Mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases characterized by accumulation of incompletely degraded glycosaminoglycans (GAGs) in lysosomes. Although primary causes of these diseases are mutations in genes coding for enzymes involved in lysosomal GAG degradation, it was demonstrated that storage of these complex carbohydrates provokes a cascade of secondary and tertiary changes affecting cellular functions. Potentially, this might lead to appearance of cellular disorders which could not be corrected even if the primary cause of the disease is removed. In this work, we studied changes in cellular organelles in MPS fibroblasts relative to control cells. All 11 types and subtypes of MPS were included into this study to obtain a complex picture of changes in organelles in this group of diseases. Two experimental approaches were employed, transcriptomic analyses and electron microscopic assessment of morphology of organelles. We analyzed levels of transcripts of genes grouped into two terms included into the QuickGO database, ‘Cellular component organization’ (GO:0016043) and ‘Cellular anatomical entity’ (GO:0110165), to find that number of transcripts with significantly changed levels in MPS fibroblasts vs. controls ranged from 109 to 322 (depending on MPS type) in GO:0016043, and from 70 to 208 in GO:0110165. This dysregulation of expression of genes crucial for proper structures and functions of various organelles was accompanied by severe changes in morphologies of lysosomes, nuclei, mitochondria, Golgi apparatus, and endoplasmic reticulum. Interestingly, some observed changes occurred in all/most MPS types while others were specific to particular disease types/subtypes. We suggest that severe changes in organelles in MPS cells might arise from dysregulation of expression of a battery of genes involved in organelles’ structures and functions. Intriguingly, normalization of GAG levels by using recombinant human enzymes specific to different MPS types corrected morphologies of some, but not all, organelles, while it failed to improve regulation of expression of selected genes. These results might suggest reasons for inability of enzyme replacement therapy to correct all MPS symptoms, particularly if initiated at advanced stages of the disease.

Toxicity of copper oxide nanoparticles on spring barley (Hordeum sativum distichum)

The rapid growth of copper oxide nanoparticles (CuO NPs) production and its abundant uses in many industries, and increasing release into an environment from both intentional and unintentional sources, create risks to spring barley (Hordeum sativum distichum), one of the most important staple food crop. Thereby, the aim of this study was to investigate the phytotoxicity of CuO NPs on H. sativum growth in hydroponic system. The CuO NPs inhibited H. sativum growth by affecting the germination rate, root and shoot lengths, maximal quantum yield of photosystem II, and transpiration rate. Structural and ultrastructural examination of H. sativum tissues using light, transmission and scanning electron microscopy showed effects on stomatal aperture and root morphology, metaxylem size and changes in cellular organelles (plastids, mitochondria), as well as in plastoglobules, starch granules, protoplasm, and membranes. The formation of electron-dense materials was noted in the intercellular space of cells of CuO NPs-treated plants. In addition, relative root length was one-third (35%) that of the control, and relative shoot length (10%) was also reduced. Further, the Cu content of roots and leaves of CuO NPs-treated plants was 5.7 and 6.4-folds higher than the control (without CuO NPs), respectively. Presented data were significant at p ≤ 0.05 compared to control. Conclusively, the results provide insights into our understanding of CuO NPs toxicity on H. sativum, and findings could be used for developing strategies for safe disposal of NPs.

Single-prolonged stress induce different change in the cell organelle of the hippocampal cells: A study of ultrastructure

MRI studies have revealed structural and functional changes in the hippocampus of post-traumatic stress disorder (PTSD) patients. Previous studies conducted by us in a PTSD animal model found that single prolonged stress (SPS) induced abnormal morphological changes in hippocampal cells. The effects of SPS on cellular organelles of the hippocampal neurons remain unknown; however, these changes have been involved in SPS-induced abnormal hippocampal function. The aim of the present study is to examine ultrastructural changes in cellular organelles, including the lysosomes, mitochondria (Mit), Golgi apparatus, and endoplasmic reticulum (ER), following SPS exposure using transmission electron microscopy, enzyme histochemistry, and enzyme cytochemistry. First, morphological changes of the hippocampal cells and ultrastructural changes in cellular organelles, including lysosomes, ER, and Mit-induced by SPS were observed. Results from histo- and cytochemistry demonstrated that the Mit marker enzyme, cytochrome c oxidase (COX), and the lysosomal enzyme acid phosphatase, (ACP), increased following exposure to SPS. SPS induced COX release from Mit and led to a wider distribution of ACP in round lysosomes, NLY, and the Golgi. In addition, we found that SPS increased the presence of autophagosomes and induced changes in the autophagy-related protein, Beclin. These results indicated the differential effects of SPS on cellular organelles, that is, a positive effect on lysosomes as well as a negative effect on the Mit and ER. Increased lysosomal function may serve as protection against SPS-induced cell damage. Structural changes in the Mit and ER may be involved in SPS-induced disorders of energy metabolism and protein synthesis and export.

Ultrastructural and X-ray microanalysis of U-937 cells in hypertonia-induced apoptosis

The results of this study of the ultrastructural changes in U-937 cells in the apoptotic state are largely consistent with analogous data available in the literature. However, we also obtained original data on the ultrastructural changes in cellular organelles and immunocytochemical localization and distribution of proteasomes. It was shown that, when apoptosis was induced by incubation of cells in a hypertonic sucrose solution (200–400 mM), the Golgi apparatus (GA) was localized close to the plasma membrane. This fact is indirect evidence that cytoskeletal elements (particularly microtubules, which hold GA in the center of the cell) are depolymerized already at the early stages of apoptosis. At the late stages of apoptosis, the distance between individual GA cisterns significantly increases, which is apparently due to the cleavage of their binding proteins, golgins. Mitochondria in apoptotic cells do not change significantly. They have regularly spaced cristae and a fairly dense matrix containing single vacuoles. At all stages of apoptosis, the nucleus and cytoplasm of U-937 cells contained proteasomes, which were represented by small rod-shaped osmiophilic particles approximately 12 × 30 nm in size. These particles formed aggregates of varying density and size, not covered with a membrane. We assumed that they are similar to the “processing bodies,” or aggresomes, which were described in the literature. The latter are detected in cells under conditions of suppressed transcription in the nucleus and, according to some researchers, are involved in the storage and degradation of various mRNAs, RNPs, and proteins. X-ray spectral analysis revealed changes in the intracellular content of Na+ and K+ at the level of single cells during apoptosis induced by osmotic shock. An increased ratio of intracellular Na+/K+ compared to the control for the majority of cells in apoptosis was shown.

Germination and ultrastructural studies of seeds produced by a fast-growing, drought-resistant tree: implications for its domestication and seed storage.

Seed ageing during storage is one of the main causes of reduction in seed quality and this results in loss of vigour and failure to thrive. Finding appropriate storage conditions to ameliorate deterioration due to ageing is, therefore, essential. Ultrastructural changes in cellular organelles during storage and seed germination rates are valuable indices of damage that occurs during seed ageing. There is increasing interest in Moringa oleifera Lam. because of its multiple uses as an agroforestry crop. Seeds of this species lose their viability within 6-12 months of harvest but no scientific information is available on the longevity of seed stored in the fruit (capsules). In most undeveloped countries, seeds are still stored inside the fruit by traditional methods in special handmade structures. In this experiment we tried to simulate these traditional storage conditions. Capsules of Moringa were stored at ambient room temperature for 12, 24 and 36 months. The ultrastructure, solute leakage and viability of seed were investigated. The ultrastructure of 1-year-old seed showed no sign of deterioration. It was evident, however, that some cells of the 3-year-old seed had deteriorated. The remnants of the outer and inner two integuments that remain tightly attached to the cotyledons probably play a role in seed dormancy. No significant difference was found between germination percentage of fresh and 1-year-old seed. The germination percentage decreased significantly from 2 years of storage onward. The decrease in seed viability during storage was associated with a loss in membrane integrity which was evidenced by an increase in electrolyte leakage. Our findings indicate that the longevity of M. oleifera seeds can be maintained if they are stored within their capsules.

Monthly variation in the morphological characteristics of Trichodina sp. (Ciliophora: Peritrichida) found on whiting Merlangius merlangus euxinus

To determine the existence and extent of seasonal variation in morphological characteristics of Trichodina sp., sixty whiting Merlangius merlangus euxinus were sampled monthly for one year, from the Eastern Black Sea coast of Turkey, using a line and hook. Strong seasonal morphological variations were detected; the denticle span, blade length and ray length showed significant seasonal variation, especially in June. The timing of primary production in the Black Sea and the changes in cellular organelles suggested a negative relationship between the body diameter of trichodinids and food availability in the surrounding environment.

Protein quality control: the who's who, the where's and therapeutic escapes.

In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.

A mechanism behind the antitumour effect of 6-diazo-5-oxo- l-norleucine (DON): disruption of mitochondria

6-diazo-5-oxo- l-norleucine (DON) exerts a growth inhibitory effect selectively on the neuroendocrine tumour cell line BON and is proposed as an antitumour drug. The mechanism behind this has not yet been clarified. In the present study, transmission electron microscopy was used for the assessment of changes in cellular organelles. Furthermore, the methylthiazolyldiphenyl tetrazolium (MTT) assay for mitochondrial enzymatic activity, a fluorescent marker (rhodamine 123) for mitochondrial integrity and [2- 11C]-acetyl-carnitine which is a substrate of the tricarboxylic acid cycle of mitochondria were employed. The studies were performed in parallel in BON and in a neuroblastoma cell line LAN, with the cells grown as monolayers or as multicellular aggregates. Severe morphological changes of intracellular organelles were observed in BON aggregates treated with low-doses of DON. Especially striking was the disruption of mitochondrial internal membrane structures. Other features included the swelling of endoplasmic reticulum, autophagocytosis of secretory granules and nuclear condensation (apoptosis). In LAN cells, no ultrastructural changes were seen after DON treatment. The MTT assay indicated inhibition of mitochondrial enzymatic activity in BON cells but not in LAN cells after 5 h treatment with DON. The mitochondrial damage was also demonstrated as a reduced metabolism of [2- 11C]-acetyl-carnitine. The observations revealed mitochondrial damage by DON treatment and suggest that the mitochondria might be a primary target for the antitumour effect in neuroendocrine cells.

Ultrastructural changes of the myocardium in the embryonic rat heart

Ultrastructural changes of the embryonic heart have been described, and quantitative studies have reported the changes of cellular organelles in late fetal and postnatal development. However, no specific data are available on the quantitative morphology of the individual segments and intersegmental junctions of the early embryonic heart, although these components must have different functions. We measured the absolute volumes of glycogen, Golgi complex, myofibrils, mitochondria, and the surface areas of the rough endoplasmic reticulum and mitochondrial cristae in the different regions of the embryonic rat heart by using stereological tools. During embryonic development, the cardiac segments and intersegmental junctions increase their glycogen volume. The sinoatrial junction and primary fold show a more rapid increase than all the other cardiac regions, whereas the atrioventricular canal shows a high level of glycogen content throughout the period studied. The Golgi complex and rough endoplasmic reticulum show a conspicuous decrease from day 15 onward. The cellular content of myofibrils and mitochondria and the surface area of the mitochondrial cristae show a gradual increase from day 11 to day 17 of development, but full maturation apparently takes place in late fetal and early postnatal stages. At day 15 of development, the cellular volumes of myofibrils and mitochondria show a temporary decrease. The glycogen content cannot be explained on the basis of metabolism alone. The storage of glycogen is hypothesized to serve mechanical cell stability and may also be related to a target mechanism for ingrowing nerves. Myofibrillar and mitochondrial contents of the myocytes indicate a relatively late differentiation of the venous pole of the heart. Uninterrupted maturation is only started at the time of septation.

Gossypolone suppresses progesterone synthesis in bovine luteal cells

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 μM) induced morphological changes in cellular organelles.

Relationship between sarcolemmal damage and appearance of amorphous matrix densities in mitochondria following occlusion of coronary artery in rats

The relationship between the progression of sarcolemmal damage and the appearance of amorphous matrix densities (AMDs) within myocardial mitochondria in the early phase of ischemia was studied in coronary-ligated rats by means of electron microscopy. The animals were divided into six groups according to the duration of ischemia (from 10 to 120 min). The severity of sarcolemmal damage was graded into four classes according to the ultrastructural ischemic changes, as follows: grade 0, normal cells; grade 1, slight ischemic changes in cellular organelles but intact sarcolemma; grade 2, formation of subsarcolemmal blebs, but overall sarcolemmal integrity; and grade 3, sarcolemmal disruption. In the experiment, grade 1 cells decreased and those of grade 3 increased with a longer period of ischemia. The appearance of grade 3 cells was distinct at 30 min of ischemia. This cellular damage proceeded from the subendocardial layer to the subepicardial layer indicating the “wavefront phenomenon.” The number of AMDs within the mitochondria increased as the sarcolemmal damage became more severe. It was noteworthy that AMDs could be found in cells with an intact sarcolemma after subjection to 10 min of ischemia. Myoglobin staining of the myocardium obtained after 10 min of ischemia followed by 6 hr of reperfusion revealed no loss of myoglobin, indicating no irreversible cell damage. This finding suggests that the formation of AMDs may occur prior to the disruption of the sarcolemma or irreversible cell damage.

Changes in Storage Protein Components of Developing Soybean Seeds (Glycine max var. Enrei).

In a study of the relationship between the dynamic changes in cellular organelles and biosynthesis of soybean storage proteins, we found that as the protein components accumulated, the α and α′-subunits of 7S globulin appeared first, followed successively by the acidic and basic subunits of 11S globulin and finally by the β-subunit of 7S globulin. Labeling experiments using tissue slices showed that the radioactivities of (3H)leucine and (14C)N-acetyl-d-glycosamine were incorporated into the trichloroacetic acid insoluble fraction and that (14C)N-acetyl-d-glycosamine was specifically incorporated into 7S.

Changes in storage protein components of developing soybean seeds (Glycine max var. Enrei).

In a study of the relationship between the dynamic changes in cellular organelles and biosynthesis of soybean storage proteins, we found that as the protein components accumulated, the α and α′-subunits of 7S globulin appeared first, followed successively by the acidic and basic subunits of 11S globulin and finally by the β-subunit of 7S globulin. Labeling experiments using tissue slices showed that the radioactivities of (3H)leucine and (14C)N-acetyl-d-glycosamine were incorporated into the trichloroacetic acid insoluble fraction and that (14C)N-acetyl-d-glycosamine was specifically incorporated into 7S.

Fine structure of the lutein cell in the house musk shrew, Suncus murinus.

The ultrastructure of lutein cells during pregnancy and the post partum period was examined by transmission electron microscopy in the house musk shrew, Suncus murinus. Lutein cells on day 13 of pregnancy contained an extensive system of anastomosing tubules or cisternae of smooth ER and many enlarged mitochondria with numerous tubulovesicular cristae. From day 13 on, the number of small granules, possibly lysosomes, increased gradually. Between day 20 and 25, the loss of smooth ER began and elongated, or flattened mitochondria increased. Regressing lutein cells observed after parturition were characterized by abundant large dense bodies, bizarre mitochondria and a decrease in the amount of smooth ER. Unusual forms of mitochondria were always observed after day 5 of pregnancy. Two types could be distinguished; one, found frequently in the second third of pregnancy, was ring-, disc-, cup- or dumb-bell-shaped with tubulovesicular cristae, and the other, found exclusively in the last third of pregnancy and after parturition, was elongated, flattened and sometimes twisted. The paucity of lipid droplets was a characteristic feature of the lutein cells of this species. The significance of these ultrastructural changes of cellular organelles is discussed in relation to the ovarian and plasma levels of progesterone.