Intracytoplasmic filaments were demonstrated in the chondrocytes of articular cartilage of cattle at 82 days post partum and at 3 and 4 years of age using transmission electron microscopy and immunocytochemistry. Samples of cartilage for electron microscopy were processed in a standard way. Immunocytochemical procedures were performed with semithin sections made from the material prepared for electron microscopy after embedding medium and osmium compounds had been eliminated. Vimentin filaments were demonstrated by means of anti-vimentin monoclonal antibody visualized by peroxidase-labelled immunoglobulin. Both techniques showed that intracytoplasmic filaments were situated around the nucleus and only occasionally at the cell periphery, e.g. in cell projections.

• MeSH
• cytoskelet chemie   ultrastruktura   MeSH
• kloubní chrupavka chemie   ultrastruktura   MeSH
• skot anatomie a histologie   MeSH
• stárnutí patologie   MeSH
• vimentin analýza   MeSH
• zvířata MeSH
• Check Tag
• skot anatomie a histologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• vimentin MeSH

It is now possible to generate large volumes of high-quality images of biomolecules at near-atomic resolution and in near-native states using cryogenic electron microscopy/electron tomography (Cryo-EM/ET). However, the precise annotation of structures like filaments and membranes remains a major barrier towards applying these methods in high-throughput. To address this, we present TARDIS (Transformer-based Rapid Dimensionless Instance Segmentation), a machine-learning framework for fast and accurate annotation of micrographs and tomograms. TARDIS combines deep learning for semantic segmentation with a novel geometric model for precise instance segmentation of various macromolecules. We develop pre-trained models within TARDIS for segmenting microtubules and membranes, demonstrating high accuracy across multiple modalities and resolutions, enabling segmentation of over 13,000 tomograms from the CZI Cryo-Electron Tomography data portal. As a modular framework, TARDIS can be extended to new structures and imaging modalities with minimal modification. TARDIS is open-source and freely available at https://github.com/SMLC-NYSBC/TARDIS, and accelerates analysis of high-resolution biomolecular structural imaging data.

• Klíčová slova
• CNN, Cryo-EM/ET, DIST, Filaments, Instance Segmentation, Membranes, Microtubules, Point Cloud, Segmentation, Semantic Segmentation, TARDIS, TEM EM/ET,
• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

• MeSH
• aorta ultrastruktura   MeSH
• arteria pulmonalis ultrastruktura   MeSH
• cytoskelet ultrastruktura   MeSH
• králíci MeSH
• svaly hladké cévní ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The expression of cytokeratins 7, 8, 14, 18, 19 and vimentin was examined in 100 cases of ductal invasive breast carcinomas. While the predominantly diffuse immunohistological positivity of simple epithelia cytokeratins 7 (in 93), 8 (in 100), 18 (in 100) and 19 (in 97) cases represents a constant feature of these tumors, cytokeratin 14 was detected in only 36 cases which were mostly of low grade and in a focal pattern. Vimentin positivity was found in 53 intermediate and high grade tumors and, again the pattern was also rarely diffuse. The ductal carcinomas can be grouped into four classes according to vimentin and cytokeratin 14 immunoreactivity. This grouping correlates well with tumor grade and with simple histological classification of ductal breast carcinoma, consisting of the low, intermediate and high malignancy categories, as proposed here. The types ofductal carcinomas can be sorted into prognostically different subgroups, according to ICD-O morphologic terminology and commonly adopted results of morphologic and prognostic studies.

• MeSH
• duktální karcinom prsu patologie   MeSH
• intermediární filamenta ultrastruktura   MeSH
• keratiny metabolismus   MeSH
• lidé MeSH
• nádory prsu patologie   MeSH
• prognóza MeSH
• vimentin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• keratiny MeSH
• vimentin MeSH

A protein of M(r) 210,000 was identified in 3T3 cells by immunoblotting and by immunoprecipitation with a monoclonal antibody MA-01. The protein was thermolabile and was located on 3T3 microtubules prepared by taxol-driven polymerization in vitro. On fixed cells the MA-01 antigen was located on interphase and mitotic microtubular structures, vinblastine paracrystals, taxol bundles and colcemid-resistant microtubules. Microinjection experiments with purified MA-01 antibody followed by double immunofluorescence have shown that the injection of antibody led to disruption of vimentin filaments, whereas the distribution of cytoplasmic microtubules was unchanged. The collapse of vimentin filaments started 30 minutes after injecting the antibody at immunoglobulin concentrations 2 mg ml-1 or higher and reached its maximum 3-6 hours after the injection. Within 20 hours after the injection vimentin filaments became reconstituted. Microinjection of the antibody into cells pre-treated with vinblastine resulted in localization of the MA-01 antigen on vinblastine paracrystals as well as on coiled vimentin filaments. The data presented suggest that the MA-01 antigen is a new microtubule-interacting protein that mediates, directly or indirectly, an interaction between microtubules and vimentin intermediate filaments.

• MeSH
• antigeny imunologie   metabolismus   MeSH
• buňky 3T3 MeSH
• demekolcin farmakologie   MeSH
• fluorescenční protilátková technika MeSH
• kompartmentace buňky účinky léků   MeSH
• mikrofilamenta metabolismus   MeSH
• mikroinjekce MeSH
• mikrotubuly metabolismus   MeSH
• monoklonální protilátky MeSH
• myši MeSH
• paclitaxel farmakologie   MeSH
• proteiny asociované s mikrotubuly imunologie   izolace a purifikace   metabolismus   MeSH
• vimentin metabolismus   MeSH
• vinblastin farmakologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny MeSH
• demekolcin MeSH
• monoklonální protilátky MeSH
• paclitaxel MeSH
• proteiny asociované s mikrotubuly MeSH
• vimentin MeSH
• vinblastin MeSH

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.

• Klíčová slova
• Filament self-assembly, GCP-free γ-tubulin, Mitotic spindle, Nucleus, γ-Tubulin,
• MeSH
• Arabidopsis chemie   genetika   MeSH
• cytoskelet chemie   genetika   MeSH
• mikrofilamenta chemie   genetika   ultrastruktura   MeSH
• mikrotubuly chemie   genetika   MeSH
• mitóza genetika   MeSH
• polymerizace MeSH
• proteinové agregáty genetika   MeSH
• proteiny asociované s mikrotubuly chemie   genetika   MeSH
• tubulin chemie   genetika   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteinové agregáty MeSH
• proteiny asociované s mikrotubuly MeSH
• tubulin MeSH

Intermediate filaments constitute the third component of the cellular skeleton. Unlike actin and microtubule cytoskeletons, the intermediate filaments are composed of a wide variety of structurally related proteins showing distinct expression patterns in tissues and cell types. Changes in the expression patterns of intermediate filaments are often associated with cancer progression; in particular with phenotypes leading to increased cellular migration and invasion. In this review we will describe the role of vimentin intermediate filaments in cancer cell migration, cell adhesion structures, and metastasis formation. The potential for targeting vimentin in cancer treatment and the development of drugs targeting vimentin will be reviewed.

• Klíčová slova
• EMT, amoeboid, cancer drugs, cancer treatment, cell adhesion, invasion, mechanotransduction, mesenchymal, vimentin,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The relationship between cytoskeleton and morphology of cumulus granulosa cells in expanding bovine oocyte-cumulus complexes (OCCs) cultured in vitro has been investigated by the means of indirect immunofluorescence and transmission electron microscopy. The round-shaped cells in unstimulated control OCCs displayed a homogeneous distribution of cytoskeletal networks and cytoplasmic organelles. Luteinizing hormone (LH) stimulation caused the redistribution of microfilaments (MFs), accelerated the development of Golgi apparatus, and led to the generation of lipid droplets in cumulus cells. These changes culminated in the elongation and polarization of cumulus cells and in the extension of the cytoplasmic networks of microtubules (MTs) and intermediate filaments (IFs) into the newly formed cytoplasmic projections. The culture of OCCs in the presence of microfilament disruptor cytochalasin B prevented cumulus expansion, formation of cellular projections and cell elongation and suppressed the development of the Golgi apparatus. On the contrary, cytochalasin had no effect on the abundance and distribution of lipid droplets and on the integrity of IFs and MTs. The present data support the hypothesis that the response of cumulus granulosa cells to LH is partially mediated by F-actin.

• MeSH
• aktiny chemie   MeSH
• cytochalasin B farmakologie   MeSH
• elektronová mikroskopie MeSH
• fluorescenční protilátková technika MeSH
• folikulární buňky ultrastruktura   MeSH
• intermediární filamenta fyziologie   MeSH
• luteinizační hormon farmakologie   MeSH
• mikrofilamenta fyziologie   MeSH
• mikrotubuly fyziologie   MeSH
• oocyty účinky léků   fyziologie   ultrastruktura   MeSH
• skot anatomie a histologie   MeSH
• zvířata MeSH
• Check Tag
• skot anatomie a histologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• cytochalasin B MeSH
• luteinizační hormon MeSH

Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth.

• MeSH
• aktiny metabolismus   ultrastruktura   MeSH
• mikrofilamenta metabolismus   ultrastruktura   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   ultrastruktura   MeSH
• Saccharomyces cerevisiae cytologie   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Alzheimer's disease (AD) involves the propagation of filaments of tau protein throughout the cerebral cortex. Imaging tau filaments and oligomers in human brain at high resolution would help contribute insight into the mechanism and progression of tauopathic diseases. STED microscopy is a nano-scale imaging technique and we aimed to test the abilities of this method for resolving tau structures within human brain. Using autopsied 50μm AD brain sections, we demonstrate that STED microscopy can resolve immunolabelled tau filaments at 77nm resolution. Ribbon-like tau filaments imaged by STED appeared smooth along their axis with limited axial undulations. STED also resolved 70-80nm wide tau puncta. Of the fluorophores tested, STAR635p was optimal for STED imaging in this tissue. This was in part due to brain tissue autofluorescence within the lower wavelength ranges (488-590nm). Further, the stability and minimal photobleaching of STAR635p allowed STED z-stacks of neurons packed with tau filaments (neurofibrillary tangles) to be collated. There was no loss of x-y image resolution of individual tau filaments through the 20μm z-stack. This demonstrates that STED can contribute to nano-scale analysis and characterisation of pathologies within banked human autopsied brain tissue. Resolving tau structures at this level of resolution provides promising avenues for understanding mechanisms of pathology propagation in the different tauopathies as well as illuminating what contributes to disease heterogeneity.

• Klíčová slova
• AD, Alzheimer’s disease, Human tissue, PHF, STED, Super resolution, Tau filament, Tau protein,
• MeSH
• Alzheimerova nemoc diagnostické zobrazování   patologie   MeSH
• barvení a značení MeSH
• lidé MeSH
• neurofibrilární klubka patologie   ultrastruktura   MeSH
• optické zobrazování MeSH
• proteiny tau chemie   ultrastruktura   MeSH
• šedá hmota diagnostické zobrazování   patologie   MeSH
• zobrazování trojrozměrné MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MAPT protein, human MeSH Prohlížeč
• proteiny tau MeSH