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1.
Nat Commun ; 11(1): 5062, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033254

RESUMO

Septins are GTP-binding proteins involved in diverse cellular processes including division and membrane remodeling. Septins form linear, palindromic heteromeric complexes that can assemble in filaments and higher-order structures. Structural studies revealed various septin architectures, but questions concerning assembly-dynamics and -pathways persist. Here we used high-speed atomic force microscopy (HS-AFM) and kinetic modeling which allowed us to determine that septin filament assembly was a diffusion-driven process, while formation of higher-order structures was complex and involved self-templating. Slightly acidic pH and increased monovalent ion concentrations favor filament-assembly, -alignment and -pairing. Filament-alignment and -pairing further favored diffusion-driven assembly. Pairing is mediated by the septin N-termini face, and may occur symmetrically or staggered, likely important for the formation of higher-order structures of different shapes. Multilayered structures are templated by the morphology of the underlying layers. The septin C-termini face, namely the C-terminal extension of Cdc12, may be involved in membrane binding.


Assuntos
Microscopia de Força Atômica , Septinas/metabolismo , Simulação por Computador , Difusão , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Lipídeos/química , Domínios Proteicos , Septinas/ultraestrutura , Eletricidade Estática
3.
Vestn Oftalmol ; 136(4. Vyp. 2): 251-257, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32880147

RESUMO

The review addresses the current state of atomic force microscopy (AFM) usage in ophthalmology. Briefly presented here are the history of the development of AFM, principles and modes of operation, its advantages, disadvantages, as well as a comparison with other types of microscopy. The review describes the capabilities of AFM in visualization of various structures of the eye. A significant part of the review is devoted to the study of the retina, which arouses great interest among researchers. In particular, the possibilities of AFM for visualization at the submicron level of various structures in the retina, such as the internal limiting membrane, membrane cells, Müller cells, retinal pigment epithelium in their normal state and in the presence of a pathology (age-related macular degeneration, diabetes mellitus) were reviewed. In addition, several study papers were analyzed, providing a base for the judgement of the mechanical properties of said structures. An AFM study of the visual pigment rhodopsin helped identify its dimeric structure. The stability of the rhodopsin molecule was proved to be determined by the degree of strength of its individual segments connected to each other. The AFM method is a highly accurate method that helps solve many fundamental and practical problems, particularly in ophthalmology.


Assuntos
Oftalmologia , Retina , Microscopia de Força Atômica , Rodopsina
4.
Phys Rev Lett ; 125(6): 068102, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32845671

RESUMO

We quantified the equilibrium (un)folding free energy ΔG_{0} of an eight-amino-acid region starting from the fully folded state of the model membrane-protein bacteriorhodopsin using single-molecule force spectroscopy. Analysis of equilibrium and nonequilibrium data yielded consistent, high-precision determinations of ΔG_{0} via multiple techniques (force-dependent kinetics, Crooks fluctuation theorem, and inverse Boltzmann analysis). We also deduced the full 1D projection of the free-energy landscape in this region. Importantly, ΔG_{0} was determined in bacteriorhodopsin's native bilayer, an advance over traditional results obtained by chemical denaturation in nonphysiological detergent micelles.


Assuntos
Bacteriorodopsinas/química , Modelos Químicos , Microscopia de Força Atômica , Dobramento de Proteína , Termodinâmica
5.
Phys Rev Lett ; 125(6): 068101, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32845697

RESUMO

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.


Assuntos
Actinas/química , Fibroblastos/química , Fibroblastos/citologia , Modelos Biológicos , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Força Compressiva , Cães , Elasticidade , Microscopia de Força Atômica , Miosinas/química , Miosinas/fisiologia , Reologia/métodos , Viscosidade
6.
Chemosphere ; 260: 127591, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32758773

RESUMO

This study examines the organization and morphology of Bacillus globigii (BG) spores, a common surrogate for Bacillus anthracis, which were seeded and then recovered at various times from several points within a conventional, pilot-scale activated sludge system. Recovered BG spores were enumerated, microscopically examined, and tested for resistance to chemical (i.e. 5% H2O2 for 8 min), thermal (80 °C for 30 min), and ultraviolet light (8 W, 254 nm UV for 1 min) inactivation. Spores exposed to activated sludge germinated, sporulated, and exhibited unique multilayer clustering patterns and statistically significant changes (p < 0.005) in dimensional morphology. Spores collected in the later experimental stages (i.e., during weeks 6 and 7) were significantly more resistant (p ≤ 0.05) to inactivation than those collected on the first day of testing. These results have direct consequences for sludge treatment requirements at wastewater treatment plants that receive spore-containing waste streams.


Assuntos
Bacillus/fisiologia , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Esgotos/microbiologia , Esporos Bacterianos/isolamento & purificação , Raios Ultravioleta , Purificação da Água/métodos , Microscopia de Força Atômica , Microscopia de Contraste de Fase , Projetos Piloto , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/efeitos da radiação , Esporos Bacterianos/ultraestrutura
7.
J Chromatogr A ; 1626: 461358, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797837

RESUMO

In this study, the in-situ conversion of the synthesized Co-Al layered double hydroxide (Co-Al LDH) nanosheets to three dimensional hierarchical zeolitic imidazolate framework-67 (3D HZIF-67) was presented as a cost-effective, highly efficient, flexible and robust sorbent to carry out the microextraction process. In the first stage, the anodized aluminum foil was prepared electrochemically. Then, the Co-Al LDH precursor was constructed on the surface of the previously-prepared anodized Al foil applying in-situ formation approach. The procedure is followed by the conversion of the prepared Co-Al LDH film to 3D HZIF-67 film via a facile solvothermal method without adding cobalt salt. The in-situ prepared 3D HZIF-67-anodized Al was used for the thin film microextraction (TFME) of caffeine. The effective factors in TFME procedure were investigated and optimized through applying Central Composite Design (CCD). In the obtained optimal condition, the calibration curves for TFME-HPLC-UV of caffeine were linear in the range of 1-200 µg L-1 with the coefficient of determination (r2) higher than 0.9915. The limits of detection were 0.33 and 0.38 µg L-1, in water and urine matrices, respectively. Moreover, the enrichment factors (EFs) and absolute recoveries (%AR) were also calculated as 173-198 and 57.1%-65.3%, respectively. The inter-day relative standard deviations (RSDs) were evaluated as the method precision for 20 and 200 µg L-1 of spiked sample and were between 4.9-6.1%. The repeatability of the preparation step was investigated as batch-to-batch reproducibility and it was found to be 4.9%; as a result, the reproducibility of the presented film was approved. Finally, the proposed method was utilized to determine caffeine (as the model analyte) from different types of real samples including urine, coffee, beverage (Pepsi) and shampoo. The obtained recoveries (higher than 88%) confirmed the capability of the method for real sample analysis.


Assuntos
Alumínio/química , Cafeína/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Cobalto/química , Hidróxidos/química , Imidazóis/química , Raios Ultravioleta , Zeolitas/química , Adsorção , Eletrodos , Limite de Detecção , Microscopia de Força Atômica , Porosidade , Reprodutibilidade dos Testes , Solventes/química , Propriedades de Superfície
8.
Int J Nanomedicine ; 15: 4333-4350, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606681

RESUMO

Background: Different diseases affect both mechanical and chemical features of the involved tissue, enhancing the symptoms. Methods: In this study, using atomic force microscopy, we mechanically characterized human ovarian tissues with four distinct pathological conditions: mucinous, serous, and mature teratoma tumors, and non-tumorous endometriosis. Mechanical elasticity profiles were quantified and the resultant data were categorized using K-means clustering method, as well as fuzzy C-means, to evaluate elastic moduli of cellular and non-cellular parts of diseased tissues and compare them among four disease conditions. Samples were stained by hematoxylin-eosin staining to further study the content of different locations of tissues. Results: Pathological state vastly influenced the mechanical properties of the ovarian tissues. Significant alterations among elastic moduli of both cellular and non-cellular parts were observed. Mature teratoma tumors commonly composed of multiple cell types and heterogeneous ECM structure showed the widest range of elasticity profile and the stiffest average elastic modulus of 14 kPa. Samples of serous tumors were the softest tissues with elastic modulus of only 400 Pa for the cellular part and 5 kPa for the ECM. Tissues of other two diseases were closer in mechanical properties as mucinous tumors were insignificantly stiffer than endometriosis in cellular part, 1300 Pa compared to 1000 Pa, with the ECM average elastic modulus of 8 kPa for both. Conclusion: The higher incidence of carcinoma out of teratoma and serous tumors may be related to the intense alteration of mechanical features of the cellular and the ECM, serving as a potential risk factor which necessitates further investigation.


Assuntos
Microscopia de Força Atômica , Nanopartículas/química , Ovário/patologia , Ovário/ultraestrutura , Adulto , Fenômenos Biomecânicos , Módulo de Elasticidade , Matriz Extracelular/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade
9.
Water Res ; 183: 116068, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32619803

RESUMO

Saturated column experiments were conducted to systematically examine the influence of hydration on the detachment of nano- and micro-sized latex colloids (35 nm and 1 µm, respectively) from sand. The colloids were attached on the sand in primary minima (PM) using high ionic strength (IS) NaCl solutions. The PM were predicted to be shallower and located farther from sand surfaces with increasing IS due to the hydration force. Consequently, a greater amount of colloid detachment occurred in deionized water when the colloids were initially deposited at a higher IS. Atomic force microscopy (AFM) examinations showed that both nanoscale protruding asperities and large wedge-like valleys existed on the sand surface. The influence of these surface features on the interaction energies/forces was modeled by approximating the roughness as cosinoidal waves and two intersecting half planes, respectively. The PM were deep and attachment was irreversible at concave regions for all ISs, even if the hydration force was included. Conversely, colloids were weakly attached at protruding asperities due to a reduced PM depth, and thus were responsible for the detachment upon IS reduction. The AFM examinations confirmed that the adhesive forces were enhanced and reduced (or even completely eliminated) at concave and convex locations of sand surfaces, respectively. These results have important implications for surface cleaning and prediction of the transport and fate of hazardous colloids and colloid-associated contaminants in subsurface environments.


Assuntos
Coloides , Dióxido de Silício , Microscopia de Força Atômica , Concentração Osmolar , Porosidade , Propriedades de Superfície
10.
Anal Chem ; 92(16): 11297-11304, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32683857

RESUMO

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.


Assuntos
Microscopia de Força Atômica/métodos , Nanoestruturas/química , Análise Espectral Raman/métodos , Vírion/química , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Capsídeo/química , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Análise Discriminante , Herpesvirus Humano 1/fisiologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Análise dos Mínimos Quadrados , Levivirus/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Células Vero
11.
Expert Rev Proteomics ; 17(6): 425-432, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32654533

RESUMO

INTRODUCTION: Rapid transmission of the severe acute respiratory syndrome coronavirus 2 has affected the whole world and forced it to a halt (lockdown). A fast and label-free detection method for the novel coronavirus needs to be developed along with the existing enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR)-based methods. AREAS COVERED: In this report, biophysical aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein are outlined based on its recent reported electron microscopy structure. Protein binding sites are analyzed theoretically, which consisted of hydrophobic and positive charged amino acid residues. Different strategies to form mixed self-assembled monolayers (SAMs) of hydrophobic (CH3) and negatively charged (COOH) groups are discussed to be used for the specific and strong interactions with spike protein. Bio-interfacial interactions between the spike protein and device (sensor) surface and its implications toward designing suitable engineered surfaces are summarized. EXPERT OPINION: Implementation of the engineered surfaces in quartz crystal microbalance (QCM)-based detection techniques for the diagnosis of the novel coronavirus from oral swab samples is highlighted. The proposed strategy can be explored for the label-free and real-time detection with sensitivity up to ng level. These engineered surfaces can be reused after desorption.


Assuntos
Betacoronavirus/química , Técnicas de Laboratório Clínico/métodos , Técnicas de Microbalança de Cristal de Quartzo/instrumentação , Glicoproteína da Espícula de Coronavírus/química , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Sítios de Ligação , Técnicas de Laboratório Clínico/instrumentação , Infecções por Coronavirus/diagnóstico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Microscopia de Força Atômica , Conformação Proteica , Técnicas de Microbalança de Cristal de Quartzo/métodos , Espectroscopia de Infravermelho com Transformada de Fourier , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Propriedades de Superfície
12.
Oral Health Prev Dent ; 18(1): 311-318, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32618454

RESUMO

PURPOSE: The aim of this study was to investigate the in vitro effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and fluoride-containing varnish on prevention enamel erosive tooth wear. MATERIALS AND METHODS: A total of 28 enamel samples were prepared from human molars, divided into four groups: CPP-ACPF varnish, TCP-F varnish, NaF varnish, and deionised water. For the remineralisation process stimulated human pooled saliva was used. After treatment, all enamel samples were exposed to 10 ml of Coca Cola. Ca++ release was determined by atomic absorption spectroscopy (AAS). The surface topography was evaluated by atomic force microscopy (AFM). Surface microhardness of enamel was analysed and SMHR % (surface microhardness reduction ) was calculated. Data were analysed with repeated measures analysis of variance (ANOVA). RESULTS: Deionised water demonstrated a statistically significantly higher Ca+2 release compared to those of groups NaF > fTCP > CPP-ACPF, respectively (p <0.01). All groups measured for root-mean-square-roughness (Rrms) showed a statistically significantly difference of 6 × 6 µm2 and 12 × 12 µm2 enamel area (p <0.05) compared with a negative control group. CPP-ACPF varnish showed rougher surfaces than all remineralisation groups. SMHR % of enamels were as follows: CPP-ACPF < fTCP < NaF < deionised water (p <0.01). CONCLUSION: According to the findings of this study; CPP-ACP containing agents have a statistically statistically significant effect on preventing dental erosion. Among these, CPP-ACPF-containing remineralisation agents have the most effect on the remineralisation process.


Assuntos
Esmalte Dentário , Desgaste dos Dentes , Caseínas , Humanos , Microscopia de Força Atômica , Pintura , Pirazinas , Remineralização Dentária
13.
PLoS One ; 15(7): e0227395, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628681

RESUMO

The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters (setpoint, z-speed, z-length, pause time, and relative underpressure) to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells (here the gram negative seawater bacterium Paracoccus seriniphilus and the gram positive bacterium Lactococcus lactis), we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell on a tipless cantilever. Even for the bacterium Lactococcus lactis, which is difficult to immobilze chemically (like seen in an earlier study), immobilization and the measurement of force-distance curves are possible by using the FluidFM technology.


Assuntos
Aderência Bacteriana , Lactococcus lactis/fisiologia , Microscopia de Força Atômica/métodos , Paracoccus/fisiologia , Células Imobilizadas/fisiologia , Vidro/química , Indóis/química , Polímeros/química , Água do Mar/microbiologia , Análise de Célula Única , Propriedades de Superfície , Titânio/química
14.
Nucleic Acids Res ; 48(12): 6458-6470, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32496520

RESUMO

The dynamic topological structure of telomeric DNA is closely related to its biological function; however, no such structural information on full-length telomeric DNA has been reported due to difficulties synthesizing long double-stranded telomeric DNA. Herein, we developed an EM-PCR and TA cloning-based approach to synthesize long-chain double-stranded tandem repeats of telomeric DNA. Using mechanical manipulation assays based on single-molecule atomic force microscopy, we found that mechanical force can trigger the melting of double-stranded telomeric DNA and the formation of higher-order structures (G-quadruplexes or i-motifs). Our results show that only when both the G-strand and C-strand of double-stranded telomeric DNA form higher-order structures (G-quadruplexes or i-motifs) at the same time (e.g. in the presence of 100 mM KCl under pH 4.7), that the higher-order structure(s) can remain after the external force is removed. The presence of monovalent K+, single-wall carbon nanotubes (SWCNTs), acidic conditions, or short G-rich fragments (∼30 nt) can shift the transition from dsDNA to higher-order structures. Our results provide a new way to regulate the topology of telomeric DNA.


Assuntos
DNA/química , Simulação de Dinâmica Molecular , Telômero/química , Quadruplex G , Humanos , Microscopia de Força Atômica , Desnaturação de Ácido Nucleico , Imagem Individual de Molécula
15.
Food Chem ; 328: 126967, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32505057

RESUMO

Different particle sizes in cloudy apple juice were obtained following filtration with different mesh sizes (100, 200, 300, and 400-mesh). The effects of cloud particle size on the stability, nutrient content, and volatile flavor of cloudy apple juice were evaluated. With increasing mesh number, particle size decreased (p < 0.05) and particle shape changed. Particle size had an effect on volatile flavor compounds, especially nitrogen oxides, alcohols, and aromatic compounds. The content of pectin and total phenol decreased with decreasing particle size, while the content of soluble protein was not affected. The reduction of cloud particle size increased absolute value of ζ-potential, cloud stability, and apparent viscosity and decreased turbidity and cloud values. Pearson correlation analysis showed that there was a strong correlation between particle size and quality indicators, except for soluble protein.


Assuntos
Aromatizantes/química , Sucos de Frutas e Vegetais/análise , Malus/química , Álcoois/análise , Malus/metabolismo , Microscopia de Força Atômica , Óxidos de Nitrogênio/análise , Tamanho da Partícula , Fenóis/análise , Viscosidade
16.
Nat Commun ; 11(1): 3114, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561744

RESUMO

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.


Assuntos
Afinidade de Anticorpos , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Sondas Moleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Complexo Antígeno-Anticorpo/ultraestrutura , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Epitopos/imunologia , Epitopos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Sondas Moleculares/imunologia , Sondas Moleculares/metabolismo , Nanotecnologia , Relação Estrutura-Atividade
17.
PLoS Comput Biol ; 16(6): e1007693, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520928

RESUMO

Understanding cellular remodeling in response to mechanical stimuli is a critical step in elucidating mechanical activation of biochemical signaling pathways. Experimental evidence indicates that external stress-induced subcellular adaptation is accomplished through dynamic cytoskeletal reorganization. To study the interactions between subcellular structures involved in transducing mechanical signals, we combined experimental data and computational simulations to evaluate real-time mechanical adaptation of the actin cytoskeletal network. Actin cytoskeleton was imaged at the same time as an external tensile force was applied to live vascular smooth muscle cells using a fibronectin-functionalized atomic force microscope probe. Moreover, we performed computational simulations of active cytoskeletal networks under an external tensile force. The experimental data and simulation results suggest that mechanical structural adaptation occurs before chemical adaptation during filament bundle formation: actin filaments first align in the direction of the external force by initializing anisotropic filament orientations, then the chemical evolution of the network follows the anisotropic structures to further develop the bundle-like geometry. Our findings present an alternative two-step explanation for the formation of actin bundles due to mechanical stimulation and provide new insights into the mechanism of mechanotransduction.


Assuntos
Citoesqueleto de Actina/fisiologia , Resistência à Tração , Actinas/fisiologia , Animais , Anisotropia , Fenômenos Biomecânicos , Células Cultivadas , Simulação por Computador , Fibronectinas/fisiologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Mecanotransdução Celular , Microscopia de Força Atômica , Miócitos de Músculo Liso/metabolismo , Miosinas/fisiologia , Ratos , Estresse Mecânico
18.
Proc Natl Acad Sci U S A ; 117(26): 14936-14947, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541055

RESUMO

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.


Assuntos
Proteínas Arqueais/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Sulfolobus acidocaldarius/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Proteína Homóloga a MRE11/química , Proteína Homóloga a MRE11/genética , Microscopia de Força Atômica , Ligação Proteica , Sulfolobus acidocaldarius/química , Sulfolobus acidocaldarius/enzimologia , Sulfolobus acidocaldarius/metabolismo
19.
Nature ; 582(7811): 294-297, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32523118

RESUMO

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics1,2. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor1,3. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength4-6. Here we applied atomic force microscopy7-12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface13,14, providing information complementary to traditional structural biology approaches.


Assuntos
Bacillus subtilis/citologia , Bacillus subtilis/ultraestrutura , Parede Celular/química , Parede Celular/ultraestrutura , Microscopia de Força Atômica , Staphylococcus aureus/citologia , Staphylococcus aureus/ultraestrutura , Bacillus subtilis/química , Viabilidade Microbiana , Peptidoglicano/química , Peptidoglicano/isolamento & purificação , Peptidoglicano/ultraestrutura , Staphylococcus aureus/química
20.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510478

RESUMO

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.


Assuntos
Cartilagem Articular/patologia , Condrócitos/patologia , Matriz Extracelular/patologia , Microscopia de Força Atômica/métodos , Osteoartrite/patologia , Elasticidade , Humanos
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