411 research outputs found

    An in vitro DNA sensor-based assay to measure receptor-specific adhesion forces of eukaryotic cells and pathogens

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    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

    Integrin α<sub>IIb</sub>β<sub>3</sub> activation and clustering in minimal synthetic cells

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    Platelet adhesion and activation are mediated by integrin αIIbβ3 clustering, which is crucial for the hemostatic function of platelets. In an activated state, integrins provide the connection between the extracellular matrix and the actin cytoskeleton through a variety of cytoplasmic proteins, such as talin. Here, droplet-based microfluidics is applied to generate cell-sized giant unilamellar vesicles (GUVs) with a defined molecular composition to quantify the adhesion of integrin αIIbβ3-containing protocells in relation to the number of integrin–talin head domain (THD) complexes. Furthermore, it is shown that THD induces integrin clustering in protocells adhering to fibrinogen. The formation of this molecular link, which has, so far, only been observed in vivo, is an essential step in synthetic cell design to recapitulate integrin-mediated bidirectional signaling across the membrane. These results pave the way for further quantitative investigations of protein–protein interactions between integrins and associated proteins and their assembly within such defined, but complex, synthetic cells. An essential future step to mimic the complex interaction between cells and their environment will be to combine synthetic approaches with peptide chemistry to guide the molecular mechanisms involved in integrin binding and activation

    A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis

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    Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease

    Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma

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    During wound repair, branching morphogenesis and carcinoma dissemination, cellular rearrangements are fostered by a solid-to-liquid transition, known as unjamming. The biomolecular machinery behind unjamming and its pathophysiological relevance remain, however, unclear. Here, we study unjamming in a variety of normal and tumorigenic epithelial two-dimensional (2D) and 3D collectives. Biologically, the increased level of the small GTPase RAB5A sparks unjamming by promoting non-clathrin-dependent internalization of epidermal growth factor receptor that leads to hyperactivation of the kinase ERK1/2 and phosphorylation of the actin nucleator WAVE2. This cascade triggers collective motility effects with striking biophysical consequences. Specifically, unjamming in tumour spheroids is accompanied by persistent and coordinated rotations that progressively remodel the extracellular matrix, while simultaneously fluidizing cells at the periphery. This concurrent action results in collective invasion, supporting the concept that the endo-ERK1/2 pathway is a physicochemical switch to initiate collective invasion and dissemination of otherwise jammed carcinoma

    Forces during cellular uptake of viruses and nanoparticles at the ventral side

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    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

    Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor

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    A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (vRGD) of integrin ligand RGD-motifs. vRGD was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 μm−2 (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels

    Quantitative Multicolor Compositional Imaging Resolves Molecular Domains in Cell-Matrix Adhesions

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    Background: Cellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution. Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined. Methodology/Principal Findings: We present here a compositional imaging approach for the analysis and display of multicomponent compositions. This methodology is based on microscopy-acquired multicolor data, multi-dimensional clustering of pixels according to their composition similarity and display of the cellular distribution of these composition clusters. We apply this approach for resolving the molecular complexes associated with focal-adhesions, and the time-dependent effects of Rho-kinase inhibition. We show here compositional variations between adhesion sites, as well as ordered variations along the axis of individual focal-adhesions. The multicolor clustering approach also reveals distinct sensitivities of different focaladhesion-associated complexes to Rho-kinase inhibition. Conclusions/Significance: Multicolor compositional imaging resolves ‘‘molecular signatures’ ’ characteristic to focaladhesions and related structures, as well as sub-domains within these adhesion sites. This analysis enhances the spatial information with additional ‘‘contents-resolved’ ’ dimensions. We propose that compositional imaging can serve as

    The Relative Importance of Topography and RGD Ligand Density for Endothelial Cell Adhesion

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    The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×102–6×1011 RGD/mm2. We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×105 RGD/mm2 on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×108 RGD/mm2 irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry

    Substrate Adhesion Regulates Sealing Zone Architecture and Dynamics in Cultured Osteoclasts

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    The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data
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