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Biology of the Cell Jun 2021Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used...
BACKGROUND INFORMATION
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil).
RESULTS
Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space.
CONCLUSIONS AND SIGNIFICANCE
This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.
Topics: Animals; COVID-19; Chlorocebus aethiops; Electron Microscope Tomography; Endoplasmic Reticulum; Humans; Intracellular Membranes; Microscopy, Electron, Transmission; Nuclear Envelope; SARS-CoV-2; Vero Cells; Virus Assembly; Virus Replication
PubMed: 33600624
DOI: 10.1111/boc.202000146 -
Journal of Cell Communication and... Jun 2021Glutamine (gln) metabolism has emerged as a cancer therapeutic target in past few years, however, the effect of gln-deprivation of bCSCs remains elusive in breast...
Glutamine (gln) metabolism has emerged as a cancer therapeutic target in past few years, however, the effect of gln-deprivation of bCSCs remains elusive in breast cancer. In this study, effect of glutamine on stemness and differentiation potential of bCSCs isolated from MCF-7 and MDAMB-231 were studied. We have shown that bCSCs differentiate into CD24 epithelial population under gln-deprivation and demonstrated increased expression of epithelial markers such as e-cadherin, claudin-1 and decreased expression of mesenchymal protein n-cadherin. MCF-7-bCSCs showed a decrease in EpCAM population whereas MDAMB-231-bCSCs increased CD44 population in response to gln-deprivation. The expression of intracellular stem cell markers such sox-2, oct-4 and nanog showed a drastic decrease in gene expression under gln-deprived MDAMB-231-bCSCs. Finally, localization of β-catenin in MCF-7 and MDAMB-231 cells showed its accumulation in cytosol or perinuclear space reducing its efficiency to transcribe downstream genes. Conclusively, our study demonstrated that gln-deprivation induces differentiation of bCSCs into epithelial subtypes and also reduces stemness of bCSCs mediated by reduced nuclear localization of β-catenin. It also suggests that basal and luminal bCSCs respond differentially towards changes in extracellular and intracellular gln. This study could significantly affect the gln targeting regimen of breast cancer therapeutics.
PubMed: 33511560
DOI: 10.1007/s12079-020-00603-1 -
Redox Biology Jan 2021Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular...
Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca and reactive oxygen species (ROS, i.e. hydrogen peroxide, HO). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular HO levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded HO-responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular HO map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral HO levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of HO in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral HO or PRX2 oxidation but rather lead to increased nuclear HO and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1 MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular HO levels and local cellular responses dependent on mitochondrial ROS.
Topics: Hydrogen Peroxide; Mitochondria; Mitochondrial Proteins; Oxidation-Reduction; Reactive Oxygen Species
PubMed: 33341544
DOI: 10.1016/j.redox.2020.101818 -
ELife Dec 2020The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis,...
The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology. These defects are dependent on a hitherto unknown chromatin-binding region of LAP1 that we have delineated. LAP1-induced NE abnormalities are efficiently suppressed by expression of wild-type but not ATPase-deficient Torsins. Furthermore, a dominant-negative Torsin induces chromosome segregation defects in a LAP1-dependent manner. These results indicate that association of LAP1 with chromatin in the nucleus can be modulated by Torsins in the perinuclear space, shedding new light on the LAP1-Torsin interplay.
Topics: Adenosine Triphosphatases; Carrier Proteins; Cell Line, Tumor; Chromatin; Chromosome Segregation; Gene Knockout Techniques; HCT116 Cells; HSC70 Heat-Shock Proteins; HeLa Cells; Hep G2 Cells; Humans; Mitosis; Molecular Chaperones; Nuclear Envelope
PubMed: 33320087
DOI: 10.7554/eLife.63614 -
Journal of Microscopy and Ultrastructure 2020The damage of the adrenal gland by snake venoms needs to be clarified. Lethality (LD) of () venom was established by intraperitoneally mice injections. Preparation of...
The damage of the adrenal gland by snake venoms needs to be clarified. Lethality (LD) of () venom was established by intraperitoneally mice injections. Preparation of specimens for transmission electron microscopy samples from cortex adrenal gland biopsies at 3, 6, and 24 h was processed. The quantitative description by the principal component analysis (PCA) of the adrenal gland was as follows: thickening of the capillary endothelium, area of the capillary lumen, cell nucleus area, enlargement of the perinuclear space, number of mitochondria, area of the mitochondria, number of mitochondrial cristae, number of cristae per mitochondrial unit, and tubular diameter of the smooth endoplasmic reticulum (SER). Sections of the adrenal cortex, after 3 h postinjection with venom showed in the cortical cells: mitochondria with tubular cristae and slightly swollen SER cisternae, nucleus with variable heterochromatin content, irregular edges, and swollen nuclear envelope. After 6 h, cells with swollen nucleus envelope, electron dense lipids and mitochondria with loss of their cristae were observed. Myelin figures, close to the microvilli of the cortical cell, multivesicular bodies, swollen profiles of the SER, and electron dense lipid drops were noticed. After 24 h, thickening of the endothelial wall, fenestrae and projections into the capillary lumen, loss of the mitochondrial cristae, destruction of the capillary and the plasma membrane of the cortical cell, multivesicular body, SER loss, and an enlargement of the perinuclear space were detected. In the quantitative PCA, there were significant changes after the venom treatments.
PubMed: 33282685
DOI: 10.4103/JMAU.JMAU_49_19 -
BMC Ophthalmology Oct 2020Hurler syndrome-associated keratopathy is an exceedingly rare corneal disorder that requires corneal transplantation in advanced stages. Precise assessment of the...
BACKGROUND
Hurler syndrome-associated keratopathy is an exceedingly rare corneal disorder that requires corneal transplantation in advanced stages. Precise assessment of the corneal condition is necessary for deciding which type of keratoplasty (i.e., deep anterior lamellar or penetrating) should be proposed. We aimed to confront the results of multimodal imaging with those of histology in a case of Hurler syndrome-associated keratopathy.
CASE PRESENTATION
A 16-year-old patient with Hurler's syndrome treated with hematopoietic stem cell transplantation was referred for decreased vision related to advanced keratopathy. The patient was treated with deep anterior lamellar keratoplasty (DALK) in both eyes with uncomplicated outcome. Visual acuity improved from 0.1 (20/200) preoperatively to 0.32 (20/63) and 0.63 (20/32) after transplantation. The corneal endothelial cell density was 2400 cells/mm in both eyes 3 years after transplantation. In vivo confocal microscopy (IVCM) and spectral domain optical coherence tomography (SD-OCT) were performed preoperatively. The corneal buttons retrieved during keratoplasty were processed for histology. In SD-OCT scans, corneal opacities appeared as diffuse stromal hyperreflectivity associated with increased corneal thickness. IVCM showed diffuse cytoplasmic granular hyperreflectivity and rounded/ellipsoid aspects of keratocytes, presence of small intracellular vacuoles, and hyperreflective epithelial intercellular spaces. Bowman's layer was thin and irregular. The corneal endothelium was poorly visualized but no endothelial damage was observed. Histology showed irregular orientation and organization of stromal lamellae, with the presence of macrophages whose cytoplasm appeared clear and granular. A perinuclear clear halo was visible within the epithelial basal cells. Bowman's layer featured breaks and irregularities.
CONCLUSIONS
The observed corneal multimodal imaging features in mucopolysaccharidosis-related keratopathy were concordant with histology. Compared with standard histology, multimodal imaging allowed additional keratocyte features to be observed. It revealed both morphological and structural changes of all corneal layers but the endothelium. This information is essential for therapeutic management which should include DALK as the first-choice treatment in case of impaired visual acuity.
Topics: Adolescent; Corneal Diseases; Corneal Transplantation; Humans; Keratoplasty, Penetrating; Mucopolysaccharidosis I; Multimodal Imaging
PubMed: 33129306
DOI: 10.1186/s12886-020-01689-2 -
Memorias Do Instituto Oswaldo Cruz 2020Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown.
BACKGROUND
Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown.
OBJECTIVE
To analyse the cytotoxic effects of KPF on Giardia duodenalis trophozoites and to identify a likely parasite target of this compound.
METHODS
We used inhibitory concentrations of KPF (IC25, IC50 and IC100) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron microscopy (TEM) whilst apoptosis/necrosis, production of reactive oxygen species (ROS) and cell cycle progression were assessed by flow cytometry (FCM) and confocal laser microscopy (CLM). Ligand-protein docking analyses were carried out using KPF structure from a drug library and crystal structure of a G. duodenalis aldose reductase (GdAldRed) homolog.
RESULTS
KPF provoked appearance of perinuclear and periplasmic spaces devoid of cytosolic content and multilamellar structures. KPF induced proapoptotic death associated with partial arrest in the S phase without ROS production. Bioinformatics approaches predicted that GdAldRed is a viable KPF target (ΔG = -7.09 kCal/mol), exhibiting 92% structural identity and a similar coupling pattern as its human homolog.
CONCLUSIONS
KPF exerted a proapoptotic effect on G. duodenalis trophozoites involving partial interruption of DNA synthesis without oxidative stress or structure damage to chromatin and cytoskeletal structures. GdAldRed is a likely target underlying its antigiardial activity.
Topics: Animals; Computational Biology; Giardia lamblia; Giardiasis; Humans; Kaempferols; Trophozoites
PubMed: 33111756
DOI: 10.1590/0074-02760200127 -
Nanomaterials (Basel, Switzerland) Oct 2020Barium ferrite nanoparticles (BaFeNPs) were investigated as vehicles for Ra radionuclide in targeted α-therapy. BaFe nanoparticles were labeled using a hydrothermal Ba...
Barium ferrite nanoparticles (BaFeNPs) were investigated as vehicles for Ra radionuclide in targeted α-therapy. BaFe nanoparticles were labeled using a hydrothermal Ba cations replacement by Ra with yield reaching 61.3 ± 1.8%. Radiolabeled nanoparticles were functionalized with 3-phosphonopropionic acid (CEPA) linker followed by covalent conjugation to trastuzumab (Herceptin). Thermogravimetric analysis and radiometric method with the use of [I]-labeled trastuzumab revealed that on average 19-21 molecules of trastuzumab are attached to the surface of one BaFe-CEPA nanoparticle. The hydrodynamic diameter of BaFe-CEPA-trastuzumab conjugate is 99.9 ± 3.0 nm in water and increases to 218.3 ± 3.7 nm in PBS buffer, and the zeta potential varies from +27.2 ± 0.7 mV in water to -8.8 ± 0.7 in PBS buffer. The [Ra]BaFe-CEPA-trastuzumab radiobioconjugate almost quantitatively retained Ra (>98%) and about 96% of Bi and 94% of Pb over 30 days. The obtained radiobioconjugate exhibited high affinity, cell internalization and cytotoxicity towards the human ovarian adenocarcinoma SKOV-3 cells overexpressing HER2 receptor. Confocal studies indicated that [Ra]BaFe-CEPA-trastuzumab was located in peri-nuclear space. High cytotoxicity of the [Ra]BaFe-CEPA-trastuzumab bioconjugate was confirmed by radiotoxicity studies on SKOV-3 cell monolayers and 3D-spheroids. In addition, the magnetic properties of the radiobioconjugate should allow for its use in guide drug delivery driven by magnetic field gradient.
PubMed: 33092037
DOI: 10.3390/nano10102067 -
Cells Sep 2020Cell migration requires reposition and reshaping of the cell nucleus. The nuclear lamina is highly important for migration of both primary and cancer cells. B-type...
Cell migration requires reposition and reshaping of the cell nucleus. The nuclear lamina is highly important for migration of both primary and cancer cells. B-type lamins are important for proper migration of epicardial cells and neurons and increased lamin B to lamin A ratio accelerates cancer cell migration through confined spaces. Moreover, a positive association between lamin B1 levels and tumor formation and progression is found in various cancer types. Still, the molecular mechanism by which B-type lamins promote cell migration is not fully understood. To better understand this mechanism, we tested the effects of lamin B1 on perinuclear actin organization. Here we show that induction of melanoma cell migration leads to the formation of a cytosolic Linker of Nucleoskeleton and Cytoskeleton (LINC) complex-independent perinuclear actin rim, which has not been detected in migrating cells, yet. Significantly, increasing the levels of lamin B1 but not the levels of lamin A prevented perinuclear actin rim formation while accelerated the cellular migration rate. To interfere with the perinuclear actin rim, we generated a chimeric protein that is localized to the outer nuclear membrane and cleaves perinuclear actin filaments in a specific manner without disrupting other cytosolic actin filaments. Using this tool, we found that disruption of the perinuclear actin rim accelerated the cellular migration rate in a similar manner to lamin B1 over-expression. Taken together, our results suggest that increased lamin B1 levels can accelerate cell migration by inhibiting the association of the nuclear envelope with actin filaments that may reduce nuclear movement and deformability.
Topics: Actins; Cell Line, Tumor; Cell Movement; Cell Nucleus; Gelsolin; Humans; Lamin Type B; Melanoma
PubMed: 32987785
DOI: 10.3390/cells9102161 -
JACC. Basic To Translational Science Aug 2020Excessive autophagy induces a defined form of cell death called autosis, which is characterized by unique morphological features, including ballooning of perinuclear... (Review)
Review
Excessive autophagy induces a defined form of cell death called autosis, which is characterized by unique morphological features, including ballooning of perinuclear space and biochemical features, including sensitivity to cardiac glycosides. Autosis is observed during the late phase of reperfusion after a period of ischemia and contributes to myocardial injury. This review discusses unique features of autosis, the involvement of autosis in myocardial injury, and the molecular mechanism of autosis. Because autosis promotes myocardial injury under some conditions, a better understanding of autosis may lead to development of novel interventions to protect the heart against myocardial stress.
PubMed: 32875173
DOI: 10.1016/j.jacbts.2020.04.014