RESUMO
Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.
Assuntos
Técnicas de Cultura de Células , Módulo de Elasticidade , Microscopia de Força Atômica/métodos , Fenômenos BiomecânicosRESUMO
Classical treatments of shoulder instability are associated with recurrence. To determine whether the modification of the capsule properties may be an alternative procedure, the effect of crosslinking treatment on the structure and mechanical properties of diseased human shoulder capsules was investigated. Joint capsules harvested from patients during shoulder surgery (n = 5) were treated or not with UV and/or riboflavin (0.1%, 1.0% and 2.5%). The structure and the mechanical properties of the capsules were determined by atomic force microscopy. The effect of treatments on cell death was investigated. Collagen fibrils were well-aligned and adjacent to each other with a D-periodicity of 66.9 ± 3.2 nm and a diameter of 71.8 ± 15.4 nm in control untreated capsules. No effect of treatments was observed on the organization of the collagen fibrils nor on their intrinsic characteristics, including D-periodicity or their mean diameter. The treatments also did not induce cell death. In contrast, UV + 2.5% riboflavin induced capsule stiffness, as revealed by the increased Young's modulus values (p < 0.0001 for each patient). Our results showed that the crosslinking procedure changed the biomechanics of diseased capsules, while keeping their structural organisation unchanged at the single fibril level. The UV/riboflavin crosslinking procedure may be a promising way to preserve the functions of collagen-based tissues and tune their elasticity for clinically relevant treatments.
Assuntos
Colágeno/química , Colágeno/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Articulação do Ombro/efeitos dos fármacos , Ombro/fisiologia , Fenômenos Biomecânicos/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Módulo de Elasticidade/efeitos dos fármacos , Elasticidade/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Humanos , Instabilidade Articular , Microscopia de Força Atômica/métodos , Riboflavina/química , Riboflavina/farmacologia , Raios UltravioletaRESUMO
At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical properties of muscle and tendon tissues. Further, there are no reports regarding characterization of mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting the ability to monitor changes in these tissues during disease progression or response to therapy. Therefore, we sought to develop novel protocols for the characterization of mechanical properties in musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of glue or chemical agents for muscle and tendon fiber immobilization during acquisition of transversal cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live animals, we have also developed an ultrasound elastography protocol using a new linear transducer, SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo. This protocol allows for the identification of changes in muscle and tendon elasticities. Such innovative technological approaches have no equivalent to date, promise to accelerate our understanding of musculotendinous mechanical properties and have numerous research and clinical applications.
Assuntos
Técnicas de Imagem por Elasticidade/métodos , Microscopia de Força Atômica/métodos , Músculo Esquelético/fisiologia , Tendões/fisiologia , Tendão do Calcâneo/fisiologia , Tendão do Calcâneo/ultraestrutura , Animais , Proteínas de Ligação a DNA/deficiência , Módulo de Elasticidade , Feminino , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Sarcômeros/fisiologia , Sarcômeros/ultraestrutura , Tendões/ultraestrutura , Fatores de Transcrição/deficiênciaRESUMO
Heparin-binding haemagglutinin (HBHA) is a surface-exposed virulence factor of Mycobacterium tuberculosis and is involved in the binding of mycobacteria to non-phagocytic cells, allowing for extra-pulmonary dissemination of the bacilli. Despite its surface exposure, HBHA is not produced as a pre-protein containing a typical cleavable N-terminal signal peptide and is thus likely secreted by a Sec-independent, as of yet unknown mechanism. Here, we used the bacterial adenylate cyclase two-hybrid system to identify the proteins encoded by rv0613c and mmpL14 as being able to interact with HBHA. Our study was focused on Rv0613c, as it showed more consistent interactions with HBHA than MmpL14. Deletion of its orthologous gene MSMEG_1285 in recombinant Mycobacterium smegmatis producing HBHA from M. tuberculosis resulted in the loss of proper surface exposure of HBHA, as evidenced by atomic force microscopy. Furthermore, the lack of MSMEG_1285 also abolished the clumping phenotype and rough colony morphology of the recombinant M. smegmatis and reduced its adherence to A549 epithelial cells. These phenotypes have previously been associated with surface-exposed HBHA. Thus, MSMEG_1285 is directly involved in the proper cell-surface exposure of HBHA. These observations identify MSMEG_1285/Rv0613c as the first accessory protein involved in the cell surface exposure of HBHA.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Mycobacterium tuberculosis/genética , Tuberculose/genética , Células A549 , Sequência de Aminoácidos/genética , Membrana Celular/genética , Células Epiteliais/metabolismo , Humanos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia , Fatores de Virulência/genéticaRESUMO
Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Proteoma/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Células CACO-2 , Cromatografia Líquida , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/ultraestrutura , Humanos , Intestinos/citologia , Intestinos/microbiologia , Lactococcus lactis/metabolismo , Lactococcus lactis/ultraestrutura , Microscopia Eletrônica , Anotação de Sequência Molecular , Dados de Sequência Molecular , Família Multigênica , Fragmentos de Peptídeos/análise , Plasmídeos , Probióticos/química , Proteólise , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
In yeasts, cell surface stresses are detected by a family of plasma membrane sensors. Among these, Wsc1 contains an extracellular cysteine-rich domain (CRD), which mediates sensor clustering and is believed to anchor the sensor in the cell wall. Although the formation of Wsc1 clusters and their interaction with the intracellular pathway components are important for proper stress signaling, the molecular mechanisms underlying clustering remain poorly understood. Here, we used the combination of single-molecule atomic force microscopy (AFM) with genetic manipulations to demonstrate that Wsc1 clustering involves disulfide bridges of the CRD. Using AFM tips carrying nitrilotriacetate groups, we mapped the distribution of individual His-tagged sensors on living yeast cells. While Wsc1 formed nanoscale clusters on native cells, clustering was no longer observed after treatment with the reducing agent dithiothreitol (DTT), indicating that intra- or intermolecular disulfide bridges are required for clustering. Moreover, DTT treatment resulted in a significant increase in cell surface roughness, suggesting that disulfide bridges between other cell-wall proteins are crucial for proper cell surface topology. The remarkable sensor properties unravelled here may well apply to other sensors and receptors with cysteine-rich domains throughout biology. Our combined method of AFM with genetic manipulations offers great prospects to explore the mechanisms underlying the clustering of cell surface proteins.
Assuntos
Membrana Celular/metabolismo , Parede Celular/metabolismo , Dissulfetos/metabolismo , Proteínas de Membrana/metabolismo , Organismos Geneticamente Modificados/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Dissulfetos/química , Ditiotreitol/metabolismo , Ditiotreitol/farmacologia , Expressão Gênica , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Força Atômica , Organismos Geneticamente Modificados/genética , Plasmídeos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Estresse Fisiológico , Transdução GenéticaRESUMO
Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains ("rafts") is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of approximately 200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.
Assuntos
Proteínas de Membrana/metabolismo , Microscopia de Força Atômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de AminoácidosRESUMO
A method is described for the site-directed manipulation of single filamentous bacteriophages, by using phage display technology and atomic force microscopy. f1 filamentous bacteriophages were genetically engineered to display His-tags on their pIX tail. Following adsorption on nitrilotriacetate-terminated surfaces, force spectroscopy with tips bearing monoclonal anti-pIII antibodies was used to pull on individual phages via their pIII head. Analysis of the force-extension profiles revealed that upon pulling, the phages are progressively straightened into an extended orientation until rupture of the anti-pIII/pIII complex. The single-virus manipulation technique presented here provides new opportunities for understanding the forces driving cell-virus and material-virus interactions, and for characterizing the binding properties of polypeptide sequences or proteins selected by the phage display technology.
Assuntos
Bacteriófagos/metabolismo , Análise Espectral/métodos , Adsorção , Bacteriófagos/genética , Bacteriófagos/fisiologia , Escherichia coli/virologia , Engenharia Genética , Microscopia de Força AtômicaRESUMO
Elucidating the molecular mechanisms behind the strength and mechanics of cell adhesion proteins is of central importance in cell biology and offers exciting avenues for the identification of potential drug targets. Here we use single-molecule force spectroscopy to investigate the adhesive and mechanical properties of the widely expressed Als5p cell adhesion protein from the opportunistic pathogen Candida albicans . We show that the forces required to unfold individual tandem repeats of the protein are in the 150-250 pN range, both on isolated molecules and on live cells. We also find that the unfolding probability increases with the number of tandem repeats and correlates with the level of cell adherence. We suggest that the modular and flexible nature of Als5p conveys both strength and toughness to the protein, making it ideally suited for cell adhesion. The single-molecule measurements presented here open new avenues for understanding the mechanical properties of adhesion molecules from mammalian and microbial cells and may help us to elucidate their potential implications in diseases such as inflammation, cancer, and infection.
RESUMO
Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.