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1.
Nat Commun ; 12(1): 5773, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599159

RESUMO

Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community.


Assuntos
Inflamação/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Lipopolissacarídeos/farmacologia , Proteômica , Algoritmos , Anti-Infecciosos/metabolismo , Anti-Inflamatórios/metabolismo , Apresentação do Antígeno , Autofagossomos/metabolismo , Teorema de Bayes , Pontos de Checagem do Ciclo Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Forma Celular , Humanos , Imunidade , Inflamação/patologia , Leucemia/imunologia , Ativação Linfocitária/imunologia , Lisossomos/metabolismo , Proteínas de Neoplasias/metabolismo , Transporte Proteico , Proteoma/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Células THP-1 , Fatores de Tempo , Vesículas Transportadoras/metabolismo , Regulação para Cima , Proteínas rho de Ligação ao GTP/metabolismo
2.
Nat Commun ; 12(1): 5528, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545085

RESUMO

Inferring cellular trajectories using a variety of omic data is a critical task in single-cell data science. However, accurate prediction of cell fates, and thereby biologically meaningful discovery, is challenged by the sheer size of single-cell data, the diversity of omic data types, and the complexity of their topologies. We present VIA, a scalable trajectory inference algorithm that overcomes these limitations by using lazy-teleporting random walks to accurately reconstruct complex cellular trajectories beyond tree-like pathways (e.g., cyclic or disconnected structures). We show that VIA robustly and efficiently unravels the fine-grained sub-trajectories in a 1.3-million-cell transcriptomic mouse atlas without losing the global connectivity at such a high cell count. We further apply VIA to discovering elusive lineages and less populous cell fates missed by other methods across a variety of data types, including single-cell proteomic, epigenomic, multi-omics datasets, and a new in-house single-cell morphological dataset.


Assuntos
Algoritmos , Genômica , Análise de Célula Única , Animais , Ciclo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Forma Celular , Hematopoese , Humanos , Ilhotas Pancreáticas/citologia , Proteínas com Homeodomínio LIM/metabolismo , Mesoderma/citologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Organogênese , Fatores de Transcrição/metabolismo
3.
Phys Rev Lett ; 127(11): 118002, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34558936

RESUMO

Predicting the densest random disc packing fraction is an unsolved paradigm problem relevant to a number of disciplines and technologies. One difficulty is that it is ill defined without setting a criterion for the disorder. Another is that the density depends on the packing protocol and the multitude of possible protocol parameters has so far hindered a general solution. A new approach is proposed here. After formulating a well-posed form of the general protocol-independent problem for planar packings of discs, a systematic criterion is proposed to avoid crystalline hexagonal order as well as further topological order. The highest possible random packing fraction is then derived exactly: ϕ_{RCP}=0.852 525…. The solution is based on the cell order distribution that is shown to (i) yield directly the packing fraction; (ii) parametrize all possible packing protocols; (iii) make it possible to define and limit all topological disorder. The method is further useful for predicting the highest packing fraction in specific protocols, which is illustrated for a family of simply sheared packings that generate maximum-entropy cell order distributions.


Assuntos
Modelos Teóricos , Forma Celular , Entropia
4.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576127

RESUMO

Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.


Assuntos
Diferenciação Celular , Radicais Livres/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Proteínas/metabolismo , Acetofenonas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Células HL-60 , Humanos , Radical Hidroxila , Monócitos/efeitos dos fármacos , NADP/metabolismo , Coloração e Rotulagem , Acetato de Tetradecanoilforbol/farmacologia , Células U937
5.
Radiol Oncol ; 55(3): 292-304, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34384011

RESUMO

BACKGROUND: Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Different isolation tech-niques based on their biological or physical features were established. Currently, the most widely used methods for visualization after their separation are based on immunofluorescent staining, which does not provide the information on the morphology. MATERIALS AND METHODS: The aim of this study was to evaluate how two different separation techniques affect cell morphology and to analyse cell morphology with techniques used in routine cytopathological laboratory. A direct side-by-side comparison of physical (Parsortix®) and biological (MACS®) separation technique was performed. RESULTS: In the preclinical setting, both isolation techniques retained the viability and antigenic characteristics of MCF7 breast cancer cells. Some signs of degeneration such as cell swelling, cytoplasmic blebs, villous projections and vacuolization were observed. In metastatic breast cancer patient cohort, morphological features of isolated CTCs were dependent on the separation technique. After physical separation, CTCs with preserved cell morphology were detected. After biological separation the majority of the isolated CTCs were so degenerated that their identity was difficult to confirm. CONCLUSIONS: Taken together, physical separation is a suitable technique for detection of CTCs with preserved cell morphology for the use in a routine cytopathological laboratory.


Assuntos
Neoplasias da Mama/patologia , Separação Celular/métodos , Forma Celular , Células Neoplásicas Circulantes/patologia , Corantes Azur , Neoplasias da Mama/sangue , Sobrevivência Celular , Corantes , Feminino , Humanos , Células MCF-7/patologia
6.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443413

RESUMO

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography-tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid-ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11-2.25, 0.012-0.227, and 0.02-0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Glutamina/metabolismo , Ácido Láctico/metabolismo , Tamoxifeno/farmacologia , Espectrometria de Massas em Tandem , Calibragem , Contagem de Células , Forma Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células MCF-7 , Ácido Pirúvico/metabolismo , Reprodutibilidade dos Testes
7.
Biomed Res Int ; 2021: 8463161, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337053

RESUMO

Meso-Xanthin (Meso-Xanthin F199™) is a highly active antiaging injection drug of the latest generation. The main acting compound is fucoxanthin, supplemented with several growth factors, vitamins, and hyaluronic acid. Previous examination of fucoxanthin on melanocytes showed its ability to inhibit skin pigmentation through different signaling pathways focused on suppression of melanogenic-stimulating receptors. In turn, the anticancer property of fucoxanthin is realized through MAPK and PI3K pathways. We aimed to evaluate the effect of fucoxanthin and supplemented growth factors on melanocyte growth and transformation at a proteomic level. The effect of fucoxanthin on melanocytes cultivated in three-dimensional (3D) condition was examined using high-throughput proteomic and system biology approaches to disclose key molecular events of the targeted action. Our results demonstrated significant inhibition of cell differentiation and ubiquitination processes. We found that the negative regulation of PSME1 and PTGIS largely determines the inhibition of NF-κB and MAPK2. Besides, fucoxanthin selectively inhibits cell differentiation via negative regulation of Raf signaling and the upstream activation of IL-1 signaling. It is assumed that inhibition of Raf influences the Notch-4 signaling and switches off the MAPK/MAPK2 cascade. Blockage of MAPK/MAPK2 is feasible due to suppression of Ras and NF-κB by the addressed action of IKKB, IKK2, and TRAF6. Suggestively, Meso-Xanthin F199™ can manage processes of proliferative activity and inhibition of apoptosis due to composition of fucoxanthin and growth-stimulating factors, which may increase the risk of skin cancer development under certain condition.


Assuntos
Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células , Sistema de Sinalização das MAP Quinases , Melanócitos/citologia , Melanócitos/metabolismo , Receptores Notch/metabolismo , Xantina/farmacologia , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteoma/metabolismo
8.
Biosens Bioelectron ; 193: 113521, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34380102

RESUMO

In this work, we investigated the ability of impedance flow cytometry to measure the shape of single cells/particles. We found that the impedance pulses triggered by micro-objects that are asymmetric in morphology show a tilting trend, and there is no such a tilting trend for symmetric ones. Therefore, we proposed a new metric, tilt index, to quantify the tilt level of the impedance pulses. Through simulation, we found that the value of tilt index tends to be zero for perfectly symmetrical objects, while the value is greater than zero for asymmetrical ones. Also, this metric was found to be independent on the trajectories (i.e., lateral, and z-direction shift) of the target micro-object. In experiments, we adopted a home-made lock-in amplifier and performed experiments on 10 µm polystyrene beads and Euglena gracilis (E. gracilis) cells with varying shapes. The experimental results coincided with the simulation results and demonstrated that the new metric (tilt index) enables the impedance cytometry to characterize the shape single cells/particles without microscopy or other optical setups.


Assuntos
Técnicas Biossensoriais , Forma Celular , Impedância Elétrica , Citometria de Fluxo , Poliestirenos
9.
Molecules ; 26(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34443489

RESUMO

Hydrogel formulations (masks or patches, without tissue support) represent the new frontier for customizable skin beauty and health. The employment of these materials is becoming popular in wound dressing, to speed up the healing process while protecting the affected area, as well as to provide a moisturizing reservoir, control the inflammatory process and the onset of bacterial development. Most of these hydrogels are acrylic-based at present, not biodegradable and potentially toxic, due to acrylic monomers residues. In this work, we selected a new class of cellulose-derived and biodegradable hydrogel films to incorporate and convey an active compound for dermatological issues. Films were obtained from a combination of different polysaccharides and clays, and berberine hydrochloride, a polyphenolic molecule showing anti-inflammatory, immunomodulatory, antibacterial and antioxidant properties, was chosen and then embedded in the hydrogel films. These innovative hydrogel-based systems were characterized in terms of water uptake profile, in vitro cytocompatibility and skin permeation kinetics by Franz diffusion cell. Berberine permeation fitted well to Korsmeyer-Peppas kinetic model and achieved a release higher than 100 µg/cm2 within 24 h. The latter study, exploiting a reliable skin model membrane, together with the biological assessment, gained insights into the most promising formulation for future investigations.


Assuntos
Berberina/administração & dosagem , Sistemas de Liberação de Medicamentos , Metilgalactosídeos/química , Pele/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células HaCaT , Humanos , Cinética , Permeabilidade , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Difração de Raios X
10.
Biomed Res Int ; 2021: 1340281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34336999

RESUMO

The purpose of this study was to develop an efficient vitrification system for cryopreservation of dog skin tissues as a source of stable autologous stem cells. In this study, we performed vitrification using four different cryoprotectants, namely, ethylene glycol (EG), dimethyl-sulfoxide (Me2SO), EG plus Me2SO, and EG plus Me2SO plus sucrose, and analyzed the behaviors of cells established from warmed tissues. Tissues vitrified with 15% EG, 15% Me2SO, and 0.5 M sucrose had a normal histological appearance and the highest cell viability after cell isolation, and thus, this cocktail of cryoprotectants was used in subsequent experiments. We evaluated proliferation and apoptosis of cells derived from fresh and vitrified tissues. These cells had a normal spindle-like morphology after homogenization through subculture. Dog dermal skin stem cells (dDSSCs) derived from fresh and vitrified tissues had similar proliferation capacities, and similar percentages of these cells were positive for mesenchymal stem cell markers at passage 3. The percentage of apoptotic cell did not differ between dDSSCs derived from fresh and vitrified tissues. Real-time PCR analysis revealed that dDSSCs at passage 3 derived from fresh and vitrified tissues had similar expression levels of pluripotency (OCT4, SOX2, and NANOG), proapoptotic (BAX), and antiapoptotic (BCL2 and BIRC5) genes. Both types of dDSSCs successfully differentiated into the mesenchymal lineage (adipocytes and osteocytes) under specific conditions, and their differentiation potentials did not significantly differ. Furthermore, the mitochondrial membrane potential of dDSSCs derived from vitrified tissues was comparable with that of dDSSCs derived from fresh tissues. We conclude that vitrification of dog skin tissues using cocktail solution in combination of 15% EG, 15% Me2SO, and 0.5 M sucrose allows efficient banking of these tissues for regenerative stem cell therapy and conservation of genetic resources.


Assuntos
Células-Tronco Mesenquimais/citologia , Pele/citologia , Vitrificação , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Derme/citologia , Cães , Feminino , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo
11.
Elife ; 102021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282727

RESUMO

Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the conserved protein kinase Pak1 with cell shape through the RNA-binding protein Sts5. Pak1 (also called Shk1 and Orb2) prevents Sts5 association with P bodies by directly phosphorylating its intrinsically disordered region (IDR). Pak1 and the cell polarity kinase Orb6 both phosphorylate the Sts5 IDR but at distinct residues. Mutations preventing phosphorylation in the Sts5 IDR cause increased P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to stress granules with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to stress granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through ribonucleoprotein granules during normal and stressed growth conditions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Forma Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Quinases Ativadas por p21/metabolismo , Polaridade Celular/fisiologia , Citoesqueleto/metabolismo , Fosforilação , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Transdução de Sinais
12.
Nutrients ; 13(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209455

RESUMO

Glucose-based solutions remain the most used osmotic agents in peritoneal dialysis (PD), but unavoidably they contribute to the loss of peritoneal filtration capacity. Here, we evaluated at a molecular level the effects of XyloCore, a new PD solution with a low glucose content, in mesothelial and endothelial cells. Cell viability, integrity of mesothelial and endothelial cell membrane, activation of mesothelial and endothelial to mesenchymal transition programs, inflammation, and angiogenesis were evaluated by several techniques. Results showed that XyloCore preserves mesothelial and endothelial cell viability and membrane integrity. Moreover XyloCore, unlike glucose-based solutions, does not exert pro-fibrotic, -inflammatory, and -angiogenic effects. Overall, the in vitro evidence suggests that XyloCore could represent a potential biocompatible solution promising better outcomes in clinical practice.


Assuntos
Soluções para Diálise/farmacologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Glucose/farmacologia , Inflamação/patologia , Mesoderma/metabolismo , Neovascularização Fisiológica , Diálise Peritoneal , Biomarcadores/metabolismo , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Mesoderma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203758

RESUMO

Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-ß1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-ß1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis.


Assuntos
Condrogênese/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Interferon gama/farmacologia , Células-Tronco Mesenquimais/imunologia , Líquido Sinovial/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Cavalos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Óxido Nítrico/metabolismo
14.
PLoS Comput Biol ; 17(7): e1009193, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34297718

RESUMO

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Aprendizado de Máquina , Modelos Biológicos , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/fisiopatologia , Adaptação Fisiológica , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Fenômenos Biofísicos , Caderinas/metabolismo , Adesão Celular/fisiologia , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Forma Celular/fisiologia , Biologia Computacional , Matriz Extracelular/patologia , Matriz Extracelular/fisiologia , Feminino , Humanos , Metástase Neoplásica/patologia , Metástase Neoplásica/fisiopatologia , Microambiente Tumoral/fisiologia , Vimentina/metabolismo
15.
Biomolecules ; 11(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066168

RESUMO

BACKGROUND: Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease with deformed red blood cells (RBCs), so-called acanthocytes, as a typical marker of the disease. Erythrocyte sedimentation rate (ESR) was recently proposed as a diagnostic biomarker. To date, there is no treatment option for affected patients, but promising therapy candidates, such as dasatinib, a Lyn-kinase inhibitor, have been identified. METHODS: RBCs of two ChAc patients during and after dasatinib treatment were characterized by the ESR, clinical hematology parameters and the 3D shape classification in stasis based on an artificial neural network. Furthermore, mathematical modeling was performed to understand the contribution of cell morphology and cell rigidity to the ESR. Microfluidic measurements were used to compare the RBC rigidity between ChAc patients and healthy controls. RESULTS: The mechano-morphological characterization of RBCs from two ChAc patients in an off-label treatment with dasatinib revealed differences in the ESR and the acanthocyte count during and after the treatment period, which could not directly be related to each other. Clinical hematology parameters were in the normal range. Mathematical modeling indicated that RBC rigidity is more important for delayed ESR than cell shape. Microfluidic experiments confirmed a higher rigidity in the normocytes of ChAc patients compared to healthy controls. CONCLUSIONS: The results increase our understanding of the role of acanthocytes and their associated properties in the ESR, but the data are too sparse to answer the question of whether the ESR is a suitable biomarker for treatment success, whereas a correlation between hematological and neuronal phenotype is still subject to verification.


Assuntos
Acantócitos/efeitos dos fármacos , Biomarcadores/sangue , Sedimentação Sanguínea/efeitos dos fármacos , Dasatinibe/uso terapêutico , Eritrócitos/efeitos dos fármacos , Neuroacantocitose/tratamento farmacológico , Acantócitos/patologia , Adulto , Forma Celular/efeitos dos fármacos , Humanos , Masculino , Neuroacantocitose/sangue , Neuroacantocitose/patologia , Uso Off-Label , Inibidores de Proteínas Quinases/uso terapêutico
16.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34124762

RESUMO

During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur simultaneously in different regions of a tissue. Here, using quantitative imaging in Drosophila melanogaster, we investigate how patterned cell shape changes promote tissue bending during early embryogenesis. We find that the transcription factors Twist and Snail combinatorially regulate a multicellular pattern of lateral F-actin density that differs from the previously described Myosin-2 gradient. This F-actin pattern correlates with whether cells apically constrict, stretch or maintain their shape. We show that the Myosin-2 gradient and F-actin depletion do not depend on force transmission, suggesting that transcriptional activity is required to create these patterns. The Myosin-2 gradient width results from a gradient in RhoA activation that is refined through the balance between RhoGEF2 and the RhoGAP C-GAP. Our experimental results and simulations of a 3D elastic shell model show that tuning gradient width regulates tissue curvature.


Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina , Animais , Proteínas de Ciclo Celular , Forma Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Ativadoras de GTPase/metabolismo , Morfogênese/fisiologia , Miosina Tipo II/metabolismo , Proteínas rho de Ligação ao GTP/genética
17.
Cells ; 10(5)2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068012

RESUMO

The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination of modeling and experiment are further informed by the elimination of length-dependent changes to troponin C calcium binding in the presence of blebbistatin. Here, the model suggests that it is the X-ROS signaling-dependent Ca2+ release increase that serves to maintain free myoplasmic calcium concentrations during a change in myocyte length. Together, our experimental and modeling approaches have further defined the relative contributions of X-ROS signaling and the length-dependent calcium buffering by troponin in shaping the myoplasmic calcium transient.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Forma Celular , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Troponina C/metabolismo , Animais , Sítios de Ligação , Acoplamento Excitação-Contração , Ativação do Canal Iônico , Masculino , Modelos Cardiovasculares , Contração Miocárdica , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
18.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34132746

RESUMO

Epithelial cells undergo striking morphological changes during division to ensure proper segregation of genetic and cytoplasmic materials. These morphological changes occur despite dividing cells being mechanically restricted by neighboring cells, indicating the need for extracellular force generation. Beyond driving cell division itself, forces associated with division have been implicated in tissue-scale processes, including development, tissue growth, migration, and epidermal stratification. While forces generated by mitotic rounding are well understood, forces generated after rounding remain unknown. Here, we identify two distinct stages of division force generation that follow rounding: (1) Protrusive forces along the division axis that drive division elongation, and (2) outward forces that facilitate postdivision spreading. Cytokinetic ring contraction of the dividing cell, but not activity of neighboring cells, generates extracellular forces that propel division elongation and contribute to chromosome segregation. Forces from division elongation are observed in epithelia across many model organisms. Thus, division elongation forces represent a universal mechanism that powers cell division in confining epithelia.


Assuntos
Divisão Celular , Forma Celular , Células Epiteliais/fisiologia , Mecanotransdução Celular , Animais , Animais Geneticamente Modificados , Comunicação Celular , Segregação de Cromossomos , Simulação por Computador , Cães , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Células Madin Darby de Rim Canino , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Estresse Mecânico , Fatores de Tempo , Imagem com Lapso de Tempo
19.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34096975

RESUMO

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.


Assuntos
Membrana Celular/metabolismo , Forma Celular , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/ultraestrutura , Movimento Celular , Células HEK293 , Células HL-60 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/ultraestrutura , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Pseudópodes/genética , Pseudópodes/ultraestrutura , Transdução de Sinais , Fatores de Tempo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética
20.
PLoS Biol ; 19(6): e3001277, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138841

RESUMO

Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.


Assuntos
Proteínas Arqueais/metabolismo , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Haloferax volcanii/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Bioensaio , Forma Celular/efeitos dos fármacos , Bases de Dados de Proteínas , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação/efeitos dos fármacos , Haloferax volcanii/efeitos dos fármacos , Mutação/genética , Fenótipo , Filogenia , Proteômica , Cloreto de Sódio/farmacologia
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