RESUMO
Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin-cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins' native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins' homophilic bonds.
Assuntos
Microscopia de Força Atômica , Caderinas , Adesão Celular , Fenômenos Mecânicos , Ligação Proteica , Análise EspectralRESUMO
Extracellular matrix (ECM), as a dynamic component of the tissue, influences cell behavior and plays an important role in cell mechanics and tissue homeostasis. Reciprocally, this three-dimensional scaffold is dynamically, structurally and mechanically modified by cells. In the field of biophysics, the independent role of cell and ECM mechanics has been largely investigated; however, there is a lack of experimental data reporting the interdependent interplay between cell and ECM mechanics, measured simultaneously. Here, using Atomic Force Microscopy (AFM) we have characterized five different decellularized matrices diverse in their topography, ECM composition and stiffness and cultured them with normal and pathological fibroblasts (scar and Dupuytren's). We investigated the change in topography and elasticity of these matrices due to cell seeding, by using AFM peak force imaging and mechanical mapping, respectively. We found normal fibroblasts soften these matrices more than pathological fibroblasts, suggesting that pathological fibroblasts are profoundly influencing tissue stiffening in fibrosis. We detected different ECM composition of decellularized matrices used here influences fibroblast stiffness, thus highlighting that cell mechanics not only depends on ECM stiffness but also on their composition. We used confocal microscopy to assess fibroblasts invasion and found pathological fibroblasts were invading the matrices deeper than normal fibroblasts.
Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fenômenos Mecânicos , Microscopia de Força Atômica , Nanopartículas/química , Análise Espectral , Animais , Fenômenos Biomecânicos , Módulo de Elasticidade , Humanos , SuínosRESUMO
Tissue morphology and mechanics are crucial to the regulation of organ function. Investigating the exceptionally complex tissue of the brain at the sub-micron scale is challenging due to the complex structure and softness of this tissue, despite the large interest of biologists, medical engineers, biophysicists, and others in this topic. Atomic force microscopy (AFM) both as an imaging and as a mechanical tool provides an excellent opportunity to study soft biological samples such as live brain tissues. Here we review the principles of AFM, the performance of AFM in tissue imaging and mechanical mapping of cells and tissues, and finally opening the prospects and challenges of probing the biophysical properties of brain tissue using AFM.
RESUMO
Mechanical properties of myofibroblasts play a key role in Dupuytren's disease. Here, we used atomic force microscopy to measure the viscoelastic properties of 3 different types of human primary fibroblasts derived from a same patient: normal and scar dermal fibroblasts and palmar fascial fibroblasts from Dupuytren's nodules. Different stiffness hydrogels (soft ~1 kPa and stiff ~ 50 kPa) were used as cell culture matrix to mimic the mechanical properties of the natural tissues, and atomic force microscopy step response force curves were used to discriminate between elastic and viscous properties of cells. Since transforming growth factor-ß1 (TGF-ß1) is known to induce expression of α-smooth muscle actin positive stress fibers in myofibroblasts, we investigated the behavior of these fibroblasts before and after applying TGF-ß1. Finally, we performed an in vitro cell motility test, the wound healing or scratch assay, to evaluate the migratory properties of these fibroblasts. We found that (1) Dupuytren's fibroblasts are stiffer than normal and scar fibroblasts, the elastic modulus E ranging from 4.4, 2.1, to 1.8 kPa, for Dupuytren's, normal and scar fibroblasts, respectively; (2) TGF-ß1 enhances the level of α-smooth muscle actin expression and thus cell stiffness in Dupuytren's fibroblasts (E, ~6.2 kPa); (3) matrix stiffness influences cell mechanical properties most prominently in Dupuytren's fibroblasts; and (4) Dupuytren's fibroblasts migrate slower than the other fibroblasts by a factor of 3. Taking together, our results showed that mechanical and migratory properties of fibroblasts might help to discriminate between different pathological conditions, helping to identify and recognize specific cell phenotypes.