Scientific publications

In CTLs: High glucose-culture enhances thapsigargin-induced SOCE but decreases target recognition-induced Ca2+ influx. High glucose-culture regulates expression of ORAIs and STIMs without affecting glucose uptake. More high glucose-cultured CTLs are prone to necrosis after execution of killing.

CytVs tether with lysosomes in T cells.Lysosome-tethered CytVs are preferentially transported to their desired destination.Lysosome tethering can mediate directionally distinctive CytV transport.Delivery of vesicles to their desired destinations plays a central role in maintaining proper cell functionality. In certain scenarios, depending on loaded cargos, the vesicles have spatially distinct destinations. For example, in T cells, some cytokines (e.g., IL-2) are polarized to the T cell–target cell interface, whereas the other cytokines are delivered multidirectionally (e.g., TNF-α). In this study, we show that in primary human CD4+ T cells, both TNF-α+ and IL-2+ vesicles can tether with endocytic organelles (lysosomes/late endosomes) by forming membrane contact sites. Tethered cytokine-containing vesicle (CytV)–endocytic organelle pairs are released sequentially. Only endocytic organelle-tethered CytVs are preferentially transported to their desired destination. Mathematical models suggest that endocytic organelle tethering could regulate the direction of cytokine transport by selectively attaching different microtubule motor proteins (such as kinesin and dynein) to the corresponding CytVs. These findings establish the previously unknown interorganelle tethering to endocytic organelles as a universal solution for directional cytokine transport in CD4+ T cells. Modulating tethering to endocytic organelles can, therefore, coordinately control directionally distinct cytokine transport.

Micropatterned dry adhesives rely mainly on van der Waals interactions. In this paper, we explore the adhesion strength increase that can be achieved by superimposing an electrostatic field through interdigitated subsurface electrodes. Micropatterns were produced by replica molding in silicone. The adhesion forces were characterized systematically by means of experiments and numerical modeling. The force increased with the square of the applied voltage for electric fields up to 800 V. For larger fields, a less-than-quadratic scaling was observed, which is likely due to the small, field-dependent electrical conductivity of the materials involved. The additional adhesion force was found to be up to twice of the field-free adhesion. The results suggest an alternative method for the controlled handling of fragile or miniaturized objects.

In underwater adhesion of a topographically patterned surface with a very soft material such as human skin, the elastic deformation can be large enough to achieve solid-on-solid contact not only on top of the hills but also in the valleys of the substrate topography. In this context, we have studied the dynamics of dewetting of a thin liquid film confined between a rigid, periodic micropillar array and a soft, elastic sphere. In our experiments, we observed two very distinct dewetting morphologies. For large ratios of array period to micropillar height and width, the dewetted areas tend to have a diamond-like shape and expand with a rate similar to a flat, unpatterned substrate. When the array period is reduced, the morphology of the dry spot becomes irregular and its expansion rate is significantly reduced. We developed a fully coupled numerical model of the dewetting process that reproduces the key features observed in experiments. Moreover, we performed contact mechanics simulations to characterize the deformation of the elastomer and the shape of the dewetted area in a unit cell of the micropillar array.

Abstract The transfer of biological concepts into synthetic micropatterned adhesives has recently enabled a new generation of switchable, reversible handling devices. Over the last two decades, many design principles have been explored that helped to understand the underlying mechanics and to optimize such adhesives for certain applications. An aspect that has been overlooked so far is the influence of longer hold times on the adhesive contacts. Exemplarily, the pull-off stress and work of separation of a micropatterned adhesive specimen are enhanced by factors 3 and 6, respectively, after 1000 min in contact with a glass substrate. In addition to such global measures, the increase of adhesion of all individual micropillars is analyzed. It is found that contact aging varied across the microarray, as it drastically depends on local conditions. Despite great differences on the micropillar scale, the adhesion of entire specimens increased with very similar power laws, as this is determined by the mean contact ageing of the individual structures. Overall, contact aging must be critically evaluated before using micropatterned adhesives, especially for long-term fixations and material combinations that are chemically attractive to each other.

ORAI1 proteins form highly selective Ca2+ channels in the plasma membrane. Crystallographic data point towards a hexameric stoichiometry of ORAI1 channels, whereas optical methods postulated ORAI1 channels to reside as dimers at rest, and other data suggests that they have a tetrameric configuration. Here, liquid-phase scanning transmission electron microscopy (STEM) and quantum dot (QD) labeling was utilized to study the conformation of ORAI1 proteins at rest. To address the question of whether ORAI1 was present as a dimer, experiments were designed using single ORAI1 monomers and covalently linked ORAI1 dimers with either one or two label-binding positions. The microscopic data was statistically analyzed via the pair correlation function. Label pairs were found in all cases, even for concatenated dimers with one label-binding position, which is only possible if a significant fraction of ORAI1 was assembled in larger order oligomers than dimers, binding at least two QDs. This interpretation of the data was consistent with Blue Native PAGE analysis showing that ORAI1 is mainly present as a complex of an apparent molecular mass larger than that calculated for a dimer.

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Abstract Innovations in liquid-phase electron microscopy (LP-EM) have made it possible to perform experiments at the optimized conditions needed to examine soft matter. The main obstacle is conducting experiments in such a way that electron beam radiation can be used to obtain answers for scientific questions without changing the structure and (bio)chemical processes in the sample due to the influence of the radiation. By overcoming these experimental difficulties at least partially, LP-EM has evolved into a new microscopy method with nanometer spatial resolution and sub-second temporal resolution for analysis of soft matter in materials science and biology. Both experimental design and applications of LP-EM for soft matter materials science and biological research are reviewed, and a perspective of possible future directions is given.

Three-dimensional (3D) printing technologies are widely applied in various industries and research fields and are currently the subject of intensive investigation and development. However, high-performance materials that are suitable for 3D printing are still in short supply, which is a major limitation for 3D printing, particularly for biomedical applications. The physicochemical properties of single constituent materials may not be sufficient to meet the needs of modern biotechnology development and production. To enhance the materials’ performance and broaden their applications, this work designed and tested a series of titanate nanofiller (nanowire and nanotube)-enhanced polycaprolactone (PCL) composites that were 3D-printable and provided superior mechanical properties. By grafting two different functional groups (phenyl- and thiol-terminated ligands), the nanofiller surface showed improved hydrophobicity, which significantly improved their dispersion in the PCL matrix. After characterizing the surface modification, we evaluated the significance of the homogeneity of the ceramic nanofiller in terms of printability, formability, and mechanical strength. Melt electrowriting additive manufacturing was used to fabricate microfibers of PCL and PCL/nanofiller composites. Improved nanofiller dispersion enabled intact and uniform sample morphology, and in contrast, nanofiller aggregation greatly varied the viscosity during the printing process, which could result in poorly printed structures. Importantly, the modified ceramic/PCL composite delivered enhanced and stable mechanical properties, where its Young’s modulus was measured to be 1.67 GPa, which is more than 7 times higher compared to that of pristine PCL (0.22 GPa). Retaining the cell safety properties (comparable to PCL), the concept of enhancing biocompatible polymers with modified nanofillers shows great potential in the field of customized 3D printing for biomedicine.