Tauopathies are characterised by intracellular deposits of fibrillar tau tangles. However, the interneuronal spread of pathological tau species precedes the development of major tau burdens. Two amyloid motifs, VQIINK in repeat 2 and VQIVYK in repeat 3, of tau repeat domain, assemble into β-sheet-rich fibrils on their own but alone do not form seed-competent fibrils. In contrast, the entire R3 region self-aggregates and forms seed-competent fibrils. Our study aimed to identify the minimal regions in the tau repeat domain that define seeding and its impact on intracellular tau phosphorylation and aggregation. Using peptides of individual repeats, we show that R2, like R3, forms seed-competent fibrils when assembled in the presence of heparin. However, R3, but not R2, forms seed-competent fibrils when assembled without heparin, even though both R2 and R3 have identical N-terminal hexapeptide and cysteine residue sequences. Moreover, cysteine to alanine substitution in R3 abrogates its self-aggregation and seeding potency. Tau RD P301S biosensor cells and Tau P301L (0N4R)-expressing HEK293 cells seeded with R2 and R3 fibrils show the induction of pathological phosphorylation of tau at Ser262/Ser396/Ser404 positions and oligomerisation of native tau. Protein fractions of biosensor cells seeded with R2 and R3 fibrils reseed endogenous tau aggregation when introduced into a fresh set of biosensor cells. Our findings suggest that R3 may be the minimal region for pathological seed generation under physiological conditions, whereas R2 might need polyanionic cofactors to generate pathogenic seeds. Lastly, R2 and R3 fibrils induce template-induced misfolding and pathological hyperphosphorylation of intracellular tau, making intracellular tau seed-competent.
- MeSH
- Alzheimer Disease * metabolism MeSH
- Cysteine MeSH
- HEK293 Cells MeSH
- Heparin MeSH
- Humans MeSH
- tau Proteins genetics metabolism MeSH
- Tauopathies * metabolism pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Publication type
- Meeting Abstract MeSH
Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.
- MeSH
- Antigens, Surface analysis MeSH
- Cell Membrane radiation effects ultrastructure MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Microscopy, Atomic Force MeSH
- Nanostructures * MeSH
- Surface Properties * MeSH
- Hot Temperature * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Immunohistochemistry and immunocytochemistry (ICC) play an irreplaceable role in research and diagnostics. It is well known that antigen retrieval (AR) can, as a technique, have beneficial outcomes on immunohistochemistry results when using formalin-fixed, paraffin-embedded tissue samples. The main purpose of AR is to break protein crosslinks which are formed during formalin fixation. Although AR was originally designed for formalin-fixed, paraffin-embedded samples, the usefulness of AR in ICC has been described in previous studies. Cytologic samples are often fixed in alcohol-based fixatives which does not lead to the formation of crosslinks. Therefore, alcohol-fixed samples can be successfully immunostained without AR. We investigated the effect of heat-induced antigen retrieval (HIAR) on alcohol-fixed HEK293 cell line samples and patient cytologic samples from thyroid gland obtained by fine needle aspiration technique. We compared indirect 2-step ICC staining results performed according to the protocol with or without HIAR in citrate buffer pH 6 for several antibodies. Utilizing HIAR against intracellular antigens has beneficial effects. Therefore, more diluted antibodies can be used for satisfactory results. However, surface antigens were probably damaged by HIAR treatment. We demonstrated evident changes in cell surface topography after HIAR treatment by atomic force microscopy. Staining specificity of patient samples improves and background staining is reduced, allowing higher dilutions of primary antibody. Improving staining specificity is necessary for accurate diagnostics. Although we have shown the beneficial effect of HIAR for immunostaining intracellular antigens, proper staining protocol should be tested on appropriate controls for individual antibodies.
- MeSH
- Antigens chemistry MeSH
- Adult MeSH
- Ethanol chemistry MeSH
- Tissue Fixation * MeSH
- HEK293 Cells MeSH
- Immunohistochemistry * MeSH
- Middle Aged MeSH
- Humans MeSH
- Retrospective Studies MeSH
- Aged MeSH
- Thyroid Gland chemistry MeSH
- Hot Temperature * MeSH
- Paraffin Embedding * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
INTRODUCTION: Cytochrome (CYP) epoxygenases (CYP2C and CYP2J) and soluble epoxide hydrolase (sEH) participate in the metabolism of arachidonic acid and may also have a potential role in enterocyte differentiation. The first critical step in the study of intestinal cell differentiation is the determination of a suitable in vitro model, which must be as similar as possible to the conditions of a living organism. It is known that HT-29 and Caco2 cell lines derived from human colorectal carcinomas can differentiate into enterocyte-like cells in appropriate culture conditions. MATERIAL AND METHODS: We tested 4 different approaches of enterocyte-like differentiation and determined the most appropriate culture conditions for each model. Subsequently, the changes in the expression of CYP epoxygenases and sEH in undifferentiated and differentiated cells were measured by In-Cell ELISA. These results were compared with immunohistochemical profiles of expression of CYP epoxygenases and sEH in samples of human embryonic and fetal intestines as well as adult duodenum and colon. RESULTS: Our results show that sodium butyrate (NaBt)-differentiated HT-29 cells and spontaneously differentiated Caco2 cells resemble CYP epoxygenases and sEH profiles, corresponding with different types of intestines. CONCLUSION: Our study revealed that the most suitable models for the study of the role of CYP epoxygenases and sEH expression in differentiation of intestinal epithelium are NaBt-differentiated HT-29 cells and spontaneously differentiated Caco2 cells.
- MeSH
- Cell Differentiation * MeSH
- HT29 Cells MeSH
- Caco-2 Cells MeSH
- Enterocytes enzymology MeSH
- Epoxide Hydrolases metabolism MeSH
- Arachidonic Acid metabolism MeSH
- Humans MeSH
- Intestines cytology embryology MeSH
- Intestinal Mucosa * embryology metabolism MeSH
- Cytochrome P-450 Enzyme System metabolism MeSH
- In Vitro Techniques MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Photodynamic therapy is a very promising and constantly evolving diagnostic and therapeutic method that is used mainly for malignant and non-malignant tumors treatment. This study deals with the utilization of zinc photosensitizer (λmax ~ 660 nm) from the group of phthalocyanines in photodynamic therapy. The aim of this study is to evaluate in vitro effect of the 5 Jcm-2 zinc phthalocyanine photosensitizer-mediated photodynamic therapy in EC50 concentration (30 nM) on cytoskeletal apparatus of the tumor cell line-HeLa (cervical cancer cells). For the measurement, the tandem mass spectrometry, atomic force and fluorescent confocal microscopy techniques were used. The results showed, that compared to the control cells zinc-derivative mediated photodynamic therapy caused in HeLa cells significant change of the cell height and extensive cytoskeletal actin rearrangement although the levels of beta actin, gamma actin and F-actin did not change significantly. This is probably caused by decreased level of the ARPC2 actin-related protein which is responsible for actin polymerization. Its level decreased 24 hours after therapy by 56%. The cytoskeletal apparatus is one of the basic cellular structures that provides cell shape, cell division and the intracellular transport. After in vitro 5 Jcm(-2) zinc derivative-mediated photodynamic therapy, the cervical carcinoma cells showed a significant damage of the cytoskeletal structure followed by changes of cell shape leading to cell death. Considering these results and low effective concentration (EC50 = 30 nM), the therapy used is potentially very promising antitumor treatment.
Silver nanoparticles (AgNPs) have been widely studied for their beneficial antimicrobial effect and have been considered by some to be a safe ingredient, as penetration of metal nanoparticles through the skin in vivo has not been proven. However, AgNPs are becoming a commonly applied nanomaterial for surface modifications of medical products which come into contact with damaged skin. In our experiments, we tested two commercially available AgNPs samples manufactured by electrolysis. AFM was used to characterize tested AgNPs morphology and their mean particle size which was assessed as 30.6nm and 20.4nm. An important mechanism of AgNPs cytotoxicity is generation of reactive oxygen species (ROS), chemically reactive species containing oxygen. Although ROS occur in cell metabolism naturally, their overproduction can induce oxidative stress - imbalance between production and antioxidant defenses. This can be associated with cytotoxicity and DNA damage. Conventional in vitro tests were used to evaluate the cytotoxic potential and DNA damage in BJ human fibroblasts cell lines. We found that both tested AgNPs samples induced ROS generation and caused the DNA damage in fibroblasts. One of the key concerns about the association with cytotoxic or genotoxic responses of nanoparticles is the capability of these materials to penetrate through cellular membrane. Cellular uptake studies were performed using Raman imaging as a label-free microscopic technique. In combination with a univariate image analysis, results demonstrate cellular uptake and distribution of the AgNPs which were taken up by BJ cells within 24h of incubation in a growth medium. The study demonstrates the potential of Raman imaging to unambiguously identify and localize AgNPs in fixed cells.
- MeSH
- Cell Line MeSH
- Fibroblasts metabolism MeSH
- Metal Nanoparticles * MeSH
- Humans MeSH
- Spectrum Analysis, Raman MeSH
- Reactive Oxygen Species metabolism MeSH
- Silver metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.
- MeSH
- Cell Membrane metabolism MeSH
- Cell Nucleus metabolism MeSH
- Exocytosis physiology MeSH
- Fibroblasts cytology metabolism MeSH
- HeLa Cells MeSH
- Cells, Cultured MeSH
- Skin cytology metabolism MeSH
- Humans MeSH
- Actin Cytoskeleton metabolism MeSH
- Myosin Type I metabolism MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Cell Movement MeSH
- Cell Shape MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Silver nanoparticles (AgNPs) are the most frequently applied nanomaterials. In our experiments, we tested AgNPs (size 27 nm) manufactured by the Tollens process. Physico-chemical methods (TEM, DLS, AFM and spectrophotometry) were used for characterization and imaging of AgNPs. The effects of AgNPs and Ag(+) were studied in two experimental models (plant and mammalian cells). Human keratinocytes (SVK14) and mouse fibroblasts (NIH3T3) cell lines were selected to evaluate the cytotoxicity and genotoxicity effect on mammalian cells. Higher sensitivity to AgNPs and Ag(+) was observed in NIH3T3 than in SVK14 cells. AgNPs accumulated in the nucleus of NIH3T3 cells, caused DNA damage and increased the number of apoptotic and necrotic cells. Three genotypes of Solanum spp. (S. lycopersicum cv. Amateur, S. chmielewskii, S. habrochaites) were selected to test the toxicity of AgNPs and Ag(+) on the plant cells. The highest values of peroxidase activity and lipid peroxidation were recorded after the treatment of S. habrochaites genotype with AgNPs. Increased ROS levels were likely the reason for observed damaged membranes in S. habrochaites. We found that the cytotoxic and genotoxic effects of AgNPs depend not only on the characteristics of nanoparticles, but also on the type of cells that are treated with AgNPs.
- MeSH
- Apoptosis drug effects MeSH
- Comet Assay MeSH
- Metal Nanoparticles administration & dosage chemistry MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mice MeSH
- Oxidation-Reduction MeSH
- Lipid Peroxidation drug effects MeSH
- DNA Damage drug effects MeSH
- Cell Proliferation drug effects MeSH
- Spectrum Analysis, Raman MeSH
- Reactive Oxygen Species metabolism MeSH
- Solanum lycopersicum cytology drug effects MeSH
- Silver chemistry pharmacology MeSH
- In Vitro Techniques MeSH
- Blotting, Western MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Metalloporphyrins are an important group of sensitizers with a porphyrin skeleton. Their photophysical properties are significantly affected by the nature of the central ion. In this work, we focus on the mechanical properties of a cervix carcinoma cell line which underwent photodynamic treatment (PDT) with MgTPPS4 photosensitzer. Atomic force microscopy alongside confocal microscopy was used to quantify and qualify the structural characteristics before and after PDT. Cells before PDT showed a fine actin network and higher elasticity with the median of Young modulus 12.2 kPa. After PDT, the median of Young modulus was 13.4 kPa and a large redistribution in the actin network was observed.
- MeSH
- Cytoskeleton drug effects physiology radiation effects MeSH
- Photosensitizing Agents chemistry pharmacology MeSH
- HeLa Cells MeSH
- Microscopy, Confocal MeSH
- Humans MeSH
- Metalloporphyrins chemistry pharmacology MeSH
- Microscopy, Atomic Force MeSH
- Elastic Modulus MeSH
- Light MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
