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1.
Physiol Int ; 106(3): 225-235, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31560236

RESUMO

OBJECTIVES: Impaired intestinal barrier function has been demonstrated in the pathophysiology of diarrhea-predominant irritable bowel syndrome (IBS-D). This study aimed to describe the intestinal ultrastructural findings in the intestinal mucosal layer of IBS-D patients. METHODS: In total, 10 healthy controls and 10 IBS-D patients were analyzed in this study. The mucosa of each patient's rectosigmoid colon was first assessed by confocal laser endomicroscopy (CLE); next, biopsied specimens of these sites were obtained. Intestinal tissues of IBS-D patients and healthy volunteers were examined to observe cellular changes by transmission electron microscopy (TEM). RESULTS: CLE showed no visible epithelial damage or inflammatory changes in the colonic mucosa of IBS-D compared with healthy volunteers. On transmission electron microscopic examination, patients with IBS-D displayed a larger apical intercellular distance with a higher proportion of dilated (>20 nm) intercellular junctional complexes, which was indicative of impaired mucosal integrity. In addition, microvillus exfoliation, extracellular vesicle as well as increased presence of multivesicular bodies were visible in IBS-D patients. Single epithelial cells appeared necrotic, as characterized by cytoplasmic vacuolization, cytoplasmic swelling, and presence of autolysosome. A significant association between bowel habit, frequency of abdominal pain, and enlarged intercellular distance was found. CONCLUSION: This study showed ultrastructural alterations in the architecture of intestinal epithelial cells and intercellular junctional complexes in IBS-D patients, potentially representing a pathophysiological mechanism in IBS-D.


Assuntos
Diarreia/patologia , Mucosa Intestinal/ultraestrutura , Síndrome do Intestino Irritável/patologia , Dor Abdominal/patologia , Colo Sigmoide/ultraestrutura , Citoplasma/patologia , Citoplasma/ultraestrutura , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Feminino , Humanos , Junções Intercelulares/ultraestrutura , Masculino , Pessoa de Meia-Idade , Reto/patologia , Reto/ultraestrutura
2.
Nucleic Acids Res ; 47(18): e109, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31340014

RESUMO

Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.


Assuntos
Citoplasma/ultraestrutura , DNA/ultraestrutura , Hibridização in Situ Fluorescente/métodos , RNA/química , Núcleo Celular/ultraestrutura , DNA/isolamento & purificação , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico
3.
Methods Mol Biol ; 1992: 231-238, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148042

RESUMO

Optical tweezers allow for noninvasive manipulation of subcellular compartments to study their physical interactions and attachments. By measuring (delay of) displacements, (semi)quantitative force measurements within a living cell can be performed. In this chapter, we provide practical tips for setting up such experiments paying special attention to the technical considerations for integrating optical tweezers into a confocal microscope. Next, we describe experimental approaches we have taken to trap intracellular structures in plant cells.


Assuntos
Microscopia Confocal/instrumentação , Pinças Ópticas , Células Vegetais/ultraestrutura , Plantas/ultraestrutura , Citoplasma/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Desenho de Equipamento , Microscopia Confocal/métodos
4.
PLoS Genet ; 15(6): e1008061, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170156

RESUMO

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/genética , Proteínas de Schizosaccharomyces pombe/genética , Transporte Ativo do Núcleo Celular/genética , Ciclo Celular/genética , Divisão Celular/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Citoplasma/genética , Citoplasma/ultraestrutura , Humanos , Meiose/genética , Microscopia de Fluorescência , Membrana Nuclear/genética , Poro Nuclear/ultraestrutura , Ligação Proteica/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
5.
Nanoscale ; 11(21): 10320-10328, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31106790

RESUMO

Precise localization and biophysical characterization of cellular structures is a key to the understanding of biological processes happening both inside the cell and at the cell surface. Atomic force microscopy is a powerful tool to study the cell surface - topography, elasticity, viscosity, interactions - and also the viscoelastic behavior of the underlying cytoplasm, cytoskeleton or the nucleus. Here, we demonstrate the ability of atomic force microscopy to also map and characterize organelles and microorganisms inside cells, at the nanoscale, by combining stiffness tomography with super-resolution fluorescence and electron microscopy. By using this correlative approach, we could both identify and characterize intracellular compartments. The validation of this approach was performed by monitoring the stiffening effect according to the metabolic status of the mitochondria in living cells in real-time.


Assuntos
Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Microscopia de Força Atômica , Microtúbulos/ultraestrutura , Elasticidade , Células HeLa , Humanos , Viscosidade
6.
Methods Cell Biol ; 151: 419-432, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948022

RESUMO

The cellular cortex-consisting of the plasma membrane and the adjacent outer few microns of the cytoplasm-is a critically important, dynamic and complex region in the sea urchin egg and embryo. Some 40 years ago it was discovered that isolated cortices could be obtained from eggs adhered to glass coverslips and since that time this preparation has been used in a wide range of studies, including seminal research on fertilization, exocytosis, the cytoskeleton, and cytokinesis. In this chapter, we discuss methods for isolating cortices from eggs and embryos, including those undergoing cell division. We also provide protocols for analyzing cortical architecture and dynamics using specific localization methods combined with super-resolution Structured Illumination and Stimulated Emission Depletion light microscopy and platinum replica transmission electron microscopy.


Assuntos
Citoplasma/ultraestrutura , Imagem Molecular/métodos , Óvulo/ultraestrutura , Ouriços-do-Mar/ultraestrutura , Animais , Membrana Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Embrião não Mamífero , Exocitose/genética , Fertilização/genética , Ouriços-do-Mar/crescimento & desenvolvimento
7.
Acta Histochem ; 121(4): 484-490, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31003695

RESUMO

Phospholipase C (PLC)ß has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCß isoforms in mouse salivary glands in situ. A single major band was detected for PLCß3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCß1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCß3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCß3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.


Assuntos
Fosfolipase C beta/metabolismo , Glândulas Salivares/metabolismo , Células Acinares/metabolismo , Células Acinares/ultraestrutura , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Immunoblotting , Masculino , Camundongos , Microscopia Imunoeletrônica , Glândula Parótida/metabolismo , Glândula Parótida/ultraestrutura , Glândulas Salivares/ultraestrutura , Glândula Sublingual/metabolismo , Glândula Sublingual/ultraestrutura , Glândula Submandibular/metabolismo , Glândula Submandibular/ultraestrutura
9.
Ultrastruct Pathol ; 43(1): 56-65, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30758240

RESUMO

Electron-microscopic analysis of the ultrastructure of the Krebs-2 carcinoma ascites cells in the first 90 min immediately after their exposure to fragmented double-stranded DNA has been performed. Morphological attributes of the treated cancer cells indicate the induction in these cells of destructive processes of presumably apoptotic type. The predominance of dystrophic-destructive changes in cells after the addition of DNA is supposed to be a consequence of the disturbance in metabolic processes caused by the experimental action.


Assuntos
Carcinoma Krebs 2/ultraestrutura , Membrana Celular/ultraestrutura , Citoplasma/ultraestrutura , DNA/ultraestrutura , Animais , Apoptose/fisiologia , Ascite , DNA/metabolismo , Camundongos , Microscopia Eletrônica/métodos
10.
PLoS Genet ; 15(2): e1007968, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30811383

RESUMO

Activation-induced deaminase (AID) converts C to U and 5-methyl-C to T. These mutagenic activities are critical to immunoglobulin (Ig) gene diversification and epigenetic reprogramming, but they must be tightly controlled to prevent compromising cell fitness. AID acts in the nucleus but localizes predominately to the cytoplasm. To address this apparent paradox, we have carried out time-lapse imaging of AID in single living B cells and fibroblasts. We demonstrate that AID enters the nucleus in brief (30 min) pulses, evident in about 10% of cells in the course of a single cell cycle (24 hr imaging). Pulses do not depend on AID catalytic activity, but they are coordinated with nuclear accumulation of P53. Pulsing may protect cells from pathologic consequences of excess exposure to AID, or enable AID to synchronize its activity with transcription of genes that are AID targets or with nuclear entry of factors that act at sites of AID-catalyzed DNA deamination to promote Ig gene diversification or epigenetic reprogramming.


Assuntos
Núcleo Celular/ultraestrutura , Citidina Desaminase/metabolismo , Citoplasma/ultraestrutura , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos , Proteína Supressora de Tumor p53/metabolismo , Linfócitos B/citologia , Linfócitos B/enzimologia , Linfócitos B/ultraestrutura , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibroblastos/ultraestrutura , Variação Genética , Humanos , Imunoglobulinas/genética , Microscopia de Fluorescência , Transporte Proteico
11.
Nat Commun ; 10(1): 497, 2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700705

RESUMO

Determining the path of single ribonucleoprotein (RNP) particles through the 100 nm-wide nuclear pore complex (NPC) by fluorescence microscopy remains challenging due to resolution limitation and RNP labeling constraints. By using high-pressure freezing and electron tomography, here we captured snapshots of the translocation of native RNP particles through NPCs in yeast and analyzed their trajectory at nanometer-scale resolution. Morphological and functional analyses indicate that these particles mostly correspond to pre-ribosomes. They are detected in 5-6% of the NPCs, with no apparent bias for NPCs adjacent to the nucleolus. Their path closely follows the central axis of the NPC through the nuclear and inner rings, but diverges at the cytoplasmic ring, suggesting interactions with the cytoplasmic nucleoporins. By applying a probabilistic queueing model to our data, we estimated that the dwell time of pre-ribosomes in the yeast NPC is ~90 ms. These data reveal distinct steps of pre-ribosome translocation through the NPC.


Assuntos
Tomografia com Microscopia Eletrônica , Poro Nuclear/metabolismo , Ribossomos/ultraestrutura , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Microscopia de Fluorescência , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura
12.
Dev Comp Immunol ; 96: 9-17, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30790604

RESUMO

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram - human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Proteínas de Peixes/farmacologia , Psychrobacter/efeitos dos fármacos , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Mutação , Psychrobacter/fisiologia , Coelhos , Testes de Toxicidade
13.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30659121

RESUMO

The linker of nucleoskeleton to cytoskeleton (LINC) complex is an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots, which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. By using bioinformatic, biochemical and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with Zea mays (Zm)SUN2 in vivo FRAP experiments using domain deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Associadas aos Microtúbulos/genética , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas de Plantas/genética , Zea mays/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Divisão Celular , Cromatina/metabolismo , Cromatina/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Ontologia Genética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Anotação de Sequência Molecular , Família Multigênica , Membrana Nuclear/ultraestrutura , Matriz Nuclear/ultraestrutura , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Zea mays/metabolismo
14.
Soft Matter ; 15(2): 190-199, 2019 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-30488938

RESUMO

During physiological processes, cells can undergo morphological changes that can result in a significant redistribution of the cytoskeleton causing anisotropic behavior. Evidence of anisotropy in cells under mechanical stimuli exists; however, the role of cytoskeletal restructuring resulting from changes in cell shape in mechanical anisotropy and its effects remain unclear. In the present study, we examine the role of cell morphology in inducing anisotropy in both intracellular mechanics and dynamics. We change the aspect ratio of cells by confining the cell width and measuring the mechanical properties of the cytoplasm using optical tweezers in both the longitudinal and transverse directions to quantify the degree of mechanical anisotropy. These active microrheology measurements are then combined with intracellular movement to calculate the intracellular force spectrum using force spectrum microscopy (FSM), from which the degree of anisotropy in dynamics and force can be quantified. We find that unrestricted cells with aspect ratio (AR) ∼1 are isotropic; however, when cells break symmetry, they exhibit significant anisotropy in cytoplasmic mechanics and dynamics.


Assuntos
Citoplasma/ultraestrutura , Corrente Citoplasmática , Citoesqueleto/ultraestrutura , Fibroblastos/citologia , Animais , Anisotropia , Fenômenos Biomecânicos , Linhagem Celular , Forma Celular , Tamanho Celular , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Camundongos , Movimento (Física) , Pinças Ópticas , Reologia
15.
Toxicol Pathol ; 47(1): 35-43, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30407147

RESUMO

We characterized pancreatic islet lesions induced by several quinolones using functional and morphological examinations of the pancreatic islets in male rats orally administered gatifloxacin, lomefloxacin, or levofloxacin at 300 mg/kg for 14 consecutive days. Consequently, in contrast to lomefloxacin or levofloxacin, gatifloxacin increased serum glucose and glycosylated albumin on day 14 and elevated serum glucose tended to decrease insulin in the intravenous glucose tolerance test. Microscopically, only gatifloxacin induced cytoplasmic vacuoles containing eosinophilic homogenous contents in islet cells. Immunohistochemical examination revealed that vacuolated islet cells were positively stained for insulin, demonstrating they were pancreatic ß cells. Electron microscopy showed that the cytoplasmic vacuoles represented dilated cisterna of the rough endoplasmic reticulum filled with electron-lucent materials in pancreatic ß cells. Moreover, insulin secretory granules were drastically decreased in vacuolated islet cells, suggesting impaired insulin synthesis and/or transport. This gatifloxacin-induced pancreatic toxicity in rats was considered to be associated with high pancreatic drug distribution. These results demonstrated that gatifloxacin provoked functional and morphological pancreatic ß cell alteration associated with impaired insulin synthesis and/or transport, leading to hyperglycemia.


Assuntos
Antibacterianos/toxicidade , Gatifloxacina/toxicidade , Ilhotas Pancreáticas/efeitos dos fármacos , Administração Oral , Animais , Antibacterianos/sangue , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Gatifloxacina/sangue , Glucose/metabolismo , Teste de Tolerância a Glucose , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Masculino , Ratos Sprague-Dawley , Distribuição Tecidual
16.
Methods ; 157: 106-114, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30419335

RESUMO

The sequestration of DNA within the membrane-bound nucleus is a defining characteristic of eukaryotic cells. Replication and transcription are therefore restricted to the nucleus, however, the regulation of these events relies on cytoplasmic processes including protein synthesis and signal transduction pathways. Because a variety of cellular activities depend on nuclear transport, researchers from diverse fields have found it useful to examine the nuclear localization of proteins of interest. Here we present some important technical considerations for studying nuclear and cytoplasmic localization, and provide guidance for quantifying protein levels using fluorescence microscopy and ImageJ software. We include discussion of the use of regions of interest and image segmentation for quantification of protein localization. Nucleocytoplasmic transport is fundamentally important for controlling protein levels and activity in the nucleus or cytoplasm, and quantitative analysis can provide insight into how biological output is achieved.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Citoplasma/genética , Microscopia de Fluorescência/métodos , Núcleo Celular/genética , Citoplasma/ultraestrutura , Fluorescência , Humanos , Transporte Proteico/genética , Transdução de Sinais/genética
17.
Protein Pept Lett ; 26(3): 221-226, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30543160

RESUMO

BACKGROUND: Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published. OBJECTIVE: Here, the protealysin cellular localization was studied for the first time using immunochemical methods. METHODS AND RESULTS: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm. CONCLUSION: The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Citoplasma/enzimologia , Peptídeo Hidrolases/metabolismo , Serratia/enzimologia , Animais , Anticorpos Antibacterianos/química , Citoplasma/ultraestrutura , Coelhos , Serratia/ultraestrutura
18.
ACS Nano ; 13(1): 187-202, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30566836

RESUMO

The intracellular delivery of nucleic acids and proteins remains a key challenge in the development of biological therapeutics. In gene therapy, the inefficient delivery of small interfering RNA (siRNA) to the cytosol by lipoplexes or polyplexes is often ascribed to the entrapment and degradation of siRNA payload in the endosomal compartments. A possible mechanism by which polyplexes rupture the endosomal membrane and release their nucleic acid cargo is commonly defined as the "proton sponge effect". This is an osmosis-driven process triggered by the proton buffering capacity of polyplexes. Herein, we investigate the molecular basis of the "proton sponge effect" through direct visualization of the siRNA trafficking process, including analysis of individual polyplexes and endosomes, using stochastic optical reconstruction microscopy. We probe the sequential siRNA trafficking steps through single molecule super-resolution analysis of subcellular structures, polyplexes, and silencing RNA molecules. Specifically, individual intact polyplexes released in the cytosol upon rupture of the endosomes, the damaged endosomal vesicles, and the disassembly of the polyplexes in the cytosol are examined. We find that the architecture of the polyplex and the rigidity of the cationic polymer chains are crucial parameters that control the mechanism of endosomal escape driven by the proton sponge effect. We provide evidence that in highly branched and rigid cationic polymers, such as glycogen or polyethylenimine, immobilized on silica nanoparticles, the proton sponge effect is effective in inducing osmotic swelling and rupture of endosomes.


Assuntos
Citoplasma/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Prótons , RNA Interferente Pequeno/metabolismo , Transporte Biológico , Citoplasma/ultraestrutura , Endossomos/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Nanopartículas/química , Células PC-3 , RNA Interferente Pequeno/ultraestrutura , Imagem Individual de Molécula/métodos
19.
Parasitol Res ; 118(2): 493-504, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30588541

RESUMO

Vitellogenesis and vitellocytes of Cainocreadium labracis were studied by transmission electron microscopy (TEM) and TEM cytochemistry. Four developmental stages were distinguished during vitellogenesis: (I) stem cell of high nucleo-cytoplasmic ratio; (II) early differentiation with chief activity focused on the beginning of protein synthesis and shell globule formation; (III) advanced differentiation with rapid intensification of protein synthesis, progressive fusion of single shell globules into large globule clusters, and formation of unsaturated lipid droplets surrounded by ß-glycogen particles; and (IV) mature vitellocyte. Early vitellogenesis with vitellocyte maturation consists of: (1) increase in cell volume; (2) increased development of large, parallel cisternae of GER with production of proteinaceous granules; (3) development of small Golgi complexes that package granules; and (4) within vacuoles, progressive enlargement of proteinaceous granules into shell globule clusters formed during vitellogenesis. Three types of inclusions accumulate in large amounts in mature vitelline cells: (1) shell globule clusters, important component in the formation of egg shell; (2) numerous unsaturated lipid droplets. Though fewer, there are also diphasic droplets consisting of saturated and unsaturated lipids in the same droplet, and (3) a relatively small amount of ß-glycogen particles, usually surround a few groups of lipid droplets. The ß-glycogen and lipid droplets are nutritive reserves for embryogenesis. General pattern and functional ultrastructure of vitellogenesis greatly resemble those observed in some lower cestodes, such as bothriocephalideans and diphyllobothrideans. Variations and differences in the amount of lipids and of glycogen during vitellogenesis in lower cestodes and other trematodes are compared and discussed.


Assuntos
Bass/parasitologia , Trematódeos/crescimento & desenvolvimento , Trematódeos/ultraestrutura , Vitelogênese , Animais , Tamanho Celular , Citoplasma/ultraestrutura , Glicogênio/metabolismo , Complexo de Golgi , Histocitoquímica , Microscopia Eletrônica de Transmissão , Trematódeos/química , Trematódeos/citologia
20.
Elife ; 72018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30516133

RESUMO

Eukaryotes thought to have evolved clonally for millions of years are referred to as ancient asexuals. The oldest group among these are the arbuscular mycorrhizal fungi (AMF), which are plant symbionts harboring hundreds of nuclei within one continuous cytoplasm. Some AMF strains (dikaryons) harbor two co-existing nucleotypes but there is no direct evidence that such nuclei recombine in this life-stage, as is expected for sexual fungi. Here, we show that AMF nuclei with distinct genotypes can undergo recombination. Inter-nuclear genetic exchange varies in frequency among strains, and despite recombination all nuclear genomes have an average similarity of at least 99.8%. The present study demonstrates that AMF can generate genetic diversity via meiotic-like processes in the absence of observable mating. The AMF dikaryotic life-stage is a primary source of nuclear variability in these organisms, highlighting its potential for strain enhancement of these symbionts.


Assuntos
Núcleo Celular/genética , DNA Fúngico/genética , Genoma Fúngico , Micorrizas/genética , Recombinação Genética , Núcleo Celular/ultraestrutura , Citoplasma/genética , Citoplasma/ultraestrutura , Variação Genética , Genótipo , Plantas/microbiologia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Simbiose
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