Quantifying the Influence of Pollen Aging on the Adhesive Properties of Hypochaeris radicata Pollen.

Although pollination is one of the most crucial biological processes that ensures plant reproduction, its mechanisms are poorly understood. Especially in insect-mediated pollination, a pollen undergoes several attachment and detachment cycles when being transferred from anther to insect and from insect to stigma. The influence of the properties of pollen, insect and floral surfaces on the adhesion forces that mediate pollen transfer have been poorly studied. Here, we investigate the adhesive properties of Hypochaeris radicata pollen and their dependence on pollen aging by quantifying the pull-off forces from glass slides using centrifugation and atomic force microscopy. We found that the properties of the pollenkitt-the viscous, lipid liquid on the surface of most pollen grains-influences the forces necessary to detach a pollen from hydrophilic surfaces. Our results show that aged H. radicata pollen form weaker adhesions to hydrophilic glass than fresh ones. On the other hand, when a pollen grain ages in contact with glass, the adhesion between the two surfaces increases over time. This study shows for the first time the pollen aging effect on the pollination mechanism.

Quantifying force transmission through fibroblasts: changes of traction forces under external shearing.

Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

Automated analysis of soft hydrogel microindentation: Impact of various indentation parameters on the measurement of Young's modulus.

Measurements of Young's moduli are mostly evaluated using strong assumptions, such as sample homogeneity and isotropy. At the same time, descriptions of measurement parameters often lack detailed specifications. Many of these assumptions are, for soft hydrogels especially, not completely valid and the complexity of hydrogel microindentation demands more sophisticated experimental procedures in order to describe their elastic properties more accurately. We created an algorithm that automates indentation data analysis as a basis for the evaluation of large data sets with consideration of the influence of indentation depth on the measured Young's modulus. The algorithm automatically determines the Young's modulus in indentation regions where it becomes independent of the indentation depth and furthermore minimizes the error from fitting an elastic model to the data. This approach is independent of the chosen elastic fitting model and indentation device. With this, we are able to evaluate large amounts of indentation curves recorded on many different sample positions and can therefore apply statistical methods to overcome deviations due to sample inhomogeneities. To prove the applicability of our algorithm, we carried out a systematic analysis of how the indentation speed, indenter size and sample thickness affect the determination of Young's modulus from atomic force microscope (AFM) indentation curves on polyacrylamide (PAAm) samples. We chose the Hertz model as the elastic fitting model for this proof of principle of our algorithm and found that all of these parameters influence the measured Young's moduli to a certain extent. Hence, it is essential to clearly state the experimental parameters used in microindentation experiments to ensure reproducibility and comparability of data.

3D Hydrogels Containing Interconnected Microchannels of Subcellular Size for Capturing Human Pathogenic Acanthamoeba Castellanii.

Porous hydrogel scaffolds are ideal candidates for mimicking cellular microenvironments, regarding both structural and mechanical aspects. We present a novel strategy to use uniquely designed ceramic networks as templates for generating hydrogels with a network of interconnected pores in the form of microchannels. The advantages of this new approach are the high and guaranteed interconnectivity of the microchannels, as well as the possibility to produce channels with diameters smaller than 7 µm. Neither of these assets can be ensured with other established techniques. Experiments using the polyacrylamide substrates produced with our approach have shown that the migration of human pathogenic Acanthamoeba castellanii trophozoites is manipulated by the microchannel structure in the hydrogels. The parasites can even be captured inside the microchannel network and removed from their incubation medium by the porous polyacrylamide, indicating the huge potential of our new technique for medical, pharmaceutical, and tissue engineering applications.

Impact of Cleaning Procedures on Adhesion of Living Cells to Three Abutment Materials.

PURPOSE: To test the adhesion properties of live gingival fibroblasts to three different implant abutment materials after five different cleaning procedures. MATERIALS AND METHODS: Highly polished discs of lithium disilicate (LS), zirconium dioxide (Zr), and titanium alloy (Ti) were fabricated. The specimens were cleaned by one of five different methods: steam (S), argon plasma (AP), ultrasound and disinfection (UD), ultrasound and sterilization in an autoclave (UA), or photofunctionalization with high-intensity ultraviolet light (PF). Cell detachment force (adhesion) was measured by single-cell force spectroscopy, which is a method to quantify cell adhesion at the single cell level. Data were statistically analyzed using parametric tests (analysis of variance [ANOVA], t tests). RESULTS: Cell detachment forces in the low nN regime were recorded in all experiments. Significant differences in cell adhesion on the different materials were found as a function of the cleaning method (P ≤ .0001). For LS abutments, no significant differences between the cleaning methods could be found (P > .05). For Zr specimens, the AP method showed the highest cell detachment forces, followed by UD, PF, S, and UA (S/UD, S/UA, S/PF, AP/UD, and UD/PF were not significantly different from each other). For Ti abutments, UD showed the highest cell detachment forces, followed by S, AP, and UA/PF (S/UD, S/UA, S/PF, AP/U, and UA/PF were not significantly different from each other). CONCLUSION: All cleaning methods provided comparable cell detachment forces for LS abutments. AP/PF or ultrasonic cleaning were the most suitable methods for strong cell adhesion on Zr. UD provided the best cell adhesion for Ti.

Adhesion forces and mechanics in mannose-mediated acanthamoeba interactions.

The human pathogenic amoeba Acanthamoeba castellanii (A. castellanii) causes severe diseases, including acanthamoeba keratitis and encephalitis. Pathogenicity arises from the killing of target-cells by an extracellular killing mechanism, where the crucial first step is the formation of a close contact between A. castellanii and the target-cell. This process is mediated by the glycocalix of the target-cell and mannose has been identified as key mediator. The aim of the present study was to carry out a detailed biophysical investigation of mannose-mediated adhesion of A. castellanii using force spectroscopy on single trophozoites. In detail, we studied the interaction of a mannose-coated cantilever with an A. castellanii trophozoite, as mannose is the decisive part of the cellular glycocalix in mediating pathogenicity. We observed a clear increase of the force to initiate cantilever detachment from the trophozoite with increasing contact time. This increase is also associated with an increase in the work of detachment. Furthermore, we also analyzed single rupture events during the detachment process and found that single rupture processes are associated with membrane tether formation, suggesting that the cytoskeleton is not involved in mannose binding events during the first few seconds of contact. Our study provides an experimental and conceptual basis for measuring interactions between pathogens and target-cells at different levels of complexity and as a function of interaction time, thus leading to new insights into the biophysical mechanisms of parasite pathogenicity.

Reinforcement of integrin-mediated T-Lymphocyte adhesion by TNF-induced Inside-out Signaling.

Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic in vitro assay for cell-surface contact times of about 1.5 seconds, whereas for larger contact times the effect disappears. Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery.

Tailoring substrates for long-term organotypic culture of adult neuronal tissue.

Organotypic tissue cultures are highly promising for performing in vivo type studies in vitro. Currently, however, very limited survival times of only a few days for adult tissue often severely limit their application. Here, superhydrophilic nanostructured substrates with ideal material properties ensure tissue adhesion, essential for organotypic culture, while migration of single cells out of the tissue is hampered. Tuning substrate properties, for the first time, adult neuronal tissue could be cultured for 14 days with no indications of degeneration.