RESUMEN
Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake.
Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Orthoreovirus Mamífero 3/fisiología , Nanopartículas del Metal/química , Actinas/metabolismo , Animales , Avidina/química , Biotina/química , Cápside/química , Células Cultivadas , Fibroblastos/virología , Oro , Células HeLa , Humanos , Integrinas/metabolismo , Cinética , Orthoreovirus Mamífero 3/química , Orthoreovirus Mamífero 3/patogenicidad , Nanopartículas del Metal/virología , Modelos Teóricos , Miosinas/metabolismo , Ratas , Virión/patogenicidad , Virión/fisiologíaRESUMEN
Motility of eukaryotic cells or pathogens within tissues is mediated by the turnover of specific interactions with other cells or with the extracellular matrix. Biophysical characterization of these ligand-receptor adhesions helps to unravel the molecular mechanisms driving migration. Traction force microscopy or optical tweezers are typically used to measure the cellular forces exerted by cells on a substrate. However, the spatial resolution of traction force microscopy is limited to ~2 µm and performing experiments with optical traps is very time-consuming. Here we present the production of biomimetic surfaces that enable specific cell adhesion via synthetic ligands and at the same time monitor the transmitted forces by using molecular tension sensors. The ligands were coupled to double-stranded DNA probes with defined force thresholds for DNA unzipping. Receptor-mediated forces in the pN range are thereby semi-quantitatively converted into fluorescence signals, which can be detected by standard fluorescence microscopy at the resolution limit (~0.2 µm). The modular design of the assay allows to vary the presented ligands and the mechanical strength of the DNA probes, which provides a number of possibilities to probe the adhesion of different eukaryotic cell types and pathogens and is exemplified here with osteosarcoma cells and Plasmodium berghei Sporozoites.
RESUMEN
Coordination of the specific functions of α5ß1 and αvß3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvß3 and α5ß1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5ß1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvß3 ligand at 30 and 60 nm spacings. Analysis of αvß3 and α5ß1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5ß1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvß3 ligand. αvß3 integrin clusters are more pronounced on αvß3 ligand, though they can also be detected in cells adhering to α5ß1 ligand. Furthermore, α5ß1 integrin clusters are present in cells adhering to α5ß1 ligand, and often colocalize with αvß3 clusters. Taken together, these findings indicate that the activation of αvß3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions.
Asunto(s)
Movimiento Celular , Adhesiones Focales/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Adhesión Celular , Línea Celular Tumoral , Humanos , LigandosRESUMEN
Engineering of biomimetic interfaces has become a valuable tool for guiding cellular processes such as adhesion, spreading, motility, as well as proliferation, differentiation, and apoptosis. The interaction of cells with the extracellular matrix (ECM) or with other cells is involved in nearly every cellular response in vivo. Recent wide-ranging evidence shows that crosstalk between different environmental stimuli can have a tremendous impact on various cell functions. Therefore, the defined control of these stimuli in vitro can contribute to the understanding of the mechanisms underlying the ability of cells to perform "intelligent" missions like acquiring, processing, and responding to environmental information. This chapter summarizes recently developed nanopatterned biomimetic systems that allow independent control of different stimuli and illustrates their applications in cellular studies. Particular attention is devoted to nanopatterned 2D and 3D artificial ECM systems based on poly(ethylene glycol) materials. These allow independent control over the material elasticity and the nanoscale distribution of bioligands on the surface. In the case of engineering artificial cellular interfaces, additional attention has to be devoted to the critical functions of protein transport regulators, namely the cell membrane and the dynamic actin cytoskeleton; both are essential for the signaling activity of individual proteins and the entire cell.
Asunto(s)
Materiales Biomiméticos/química , Matriz Extracelular/química , Nanopartículas/química , Biología Celular , HumanosRESUMEN
Foreign body giant cells (FBGCs) are formed by fusion of mononucleated macrophages during the foreign body response to a nanoparticulate hydroxyapatite (HA) implanted in defects of mini-pig femura. The molecular mechanisms underlying the formation of FBGCs are still largely obscure. Here we propose connexin 43 (cx43) and CD44 as candidate molecules involved in the fusion process. Immunohistochemistry and ultrastructural immunogold labeling indicated that cx43 is present within the ruffled border of FBGCs and is the main component of gap junctions formed between fusing macrophages. CD44 was strongly expressed during clustering and fusion of mononucleated macrophages. FBGCs adhering apically at the implanted HA showed CD44 reactivity only along the basolateral aspects of the plasma membranes, while podosome formation was observed within the sealing zone and ruffled border. Taken together, these findings demonstrate that cx43 and CD44 are part of the fusion machinery responsible for the formation of FBGCs. Furthermore, the results of microfilament and cx43 labeling suggest a functional role for podosomes and hemi-channels in biomaterial degradation.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Conexina 43/metabolismo , Durapatita/efectos adversos , Reacción a Cuerpo Extraño/inducido químicamente , Células Gigantes de Cuerpo Extraño/patología , Receptores de Hialuranos/metabolismo , Nanopartículas/efectos adversos , Citoesqueleto de Actina/metabolismo , Animales , Western Blotting , Conexina 43/ultraestructura , Reacción a Cuerpo Extraño/metabolismo , Reacción a Cuerpo Extraño/patología , Células Gigantes de Cuerpo Extraño/efectos de los fármacos , Células Gigantes de Cuerpo Extraño/ultraestructura , Receptores de Hialuranos/ultraestructura , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Nanopartículas/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Porcinos , Porcinos EnanosRESUMEN
Focal adhesions are the anchoring points of cells to surfaces and are responsible for a large number of surface sensing processes. Nanopatterning studies have shown physiological changes in fibroblasts as a result of decreasing density of external binding ligands. The most striking of these changes is a decreased ability to form mature focal adhesions when lateral ligand distances exceed 76 nm. These changes are usually examined in the context of protein signaling and protein interactions. We show a physical explanation based on the balance between the forces acting on individual ligand connections and the reaction kinetics of those ligands. We propose three stability regimes for focal adhesions as a function of ligand spacing and applied stress: a stable regime, an unstable regime in which a large fraction of unbound protein causes adhesion disintegration, and a regime in which the applied force is too high to form an adhesion structure.