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- ItemFormation mechanism for stable hybrid clusters of proteins and nanoparticles(Washington D.C. : American Chemical Society, 2015) Moerz, Sebastian T.; Kraegeloh, Annette; Chanana, Munish; Kraus, TobiasCitrate-stabilized gold nanoparticles (AuNP) agglomerate in the presence of hemoglobin (Hb) at acidic pH. The extent of agglomeration strongly depends on the concentration ratio [Hb]/[AuNP]. Negligible agglomeration occurs at very low and very high [Hb]/[AuNP]. Full agglomeration and precipitation occur at [Hb]/[AuNP] corresponding to an Hb monolayer on the AuNP. Ratios above and below this value lead to the formation of an unexpected phase: stable, microscopic AuNP–Hb agglomerates. We investigated the kinetics of agglomeration with dynamic light scattering and the adsorption kinetics of Hb on planar gold with surface-acoustic wave-phase measurements. Comparing agglomeration and adsorption kinetics leads to an explanation of the complex behavior of this nanoparticle–protein mixture. Agglomeration is initiated either when Hb bridges AuNP or when the electrostatic repulsion between AuNP is neutralized by Hb. It is terminated when Hb has been depleted or when Hb forms multilayers on the agglomerates that stabilize microscopic clusters indefinitely.
- ItemSurviving the surf: The tribomechanical properties of the periostracum of Mytilus sp(Amsterdam : Elsevier, 2014) Wählisch, Felix C.; Peter, Nicolas J.; Torrents Abad, Oscar; Oliveira, Mariana V.G.; Schneider, Andreas S.; Schmahl, Wolfgang; Griesshaber, Erika; Bennewitz, RolandWe investigated the friction and wear behavior as well as the mechanical properties of the periostracum of Mytilus sp. Tribological properties were determined with a reciprocal sliding microtribometer, while mechanical characterization was performed using a nanoindenter. Measurements were performed in dry and wet conditions. On the dry periostracum we found a low friction coefficient of 0.078 ± 0.007 on the young parts and a higher one of 0.63 ± 0.02 on the old parts of the shell. Under wet, saline, conditions we only observed one average coefficient of friction of 0.37 ± 0.01. Microscopic ex situ analysis indicated that dry periostracum wore rather rapidly by plowing and fatigue, while it exhibited a high wear resistance when immersed in salt water. The Young’s modulus and hardness of the periostracum were also investigated in both dry and wet conditions. Under dry conditions the Young’s modulus of the periostracum was 8 ± 3 GPa, while under wet conditions it was 0.21 ± 0.05 GPa. The hardness of dry periostracum samples was 353 ± 127 MPa, whereas the hardness of wet samples was 5 ± 2 MPa. It was found that, in the wet state, viscous behavior plays a significant role in the mechanical response of the periostracum. Our results strongly indicate that the periostracum can provide an important contribution to the overall wear resistance of Mytilus sp. shell.
- ItemReal-time monitoring of calcium carbonate and cationic peptide deposition on carboxylate-SAM using a microfluidic SAW biosensor(Frankfurt am Main : Beilstein-Institut, 2014) Pohl, Anna; Weiss, Ingrid M.A microfluidic biosensor with surface acoustic wave technology was used in this study to monitor the interaction of calcium carbonate with standard carboxylate self-assembled monolayer sensor chips. Different fluids, with and without biomolecular components, were investigated. The pH-dependent surface interactions of two bio-inspired cationic peptides, AS8 and ES9, which are similar to an extracellular domain of the chitin synthase involved in mollusc shell formation, were also investigated in a biological buffer system. A range of experimental conditions are described that are suitable to study non-covalent molecular interactions in the presence of ionic substances, such as, mineral precursors below the solubility equilibrium. The peptide ES9, equal to the mollusc chitin synthase epitope, is less sensitive to changes in pH than its counterpart AS8 with a penta-lysine core, which lacks the flanking acidic residues. This study demonstrates the extraordinary potential of microfluidic surface acoustic wave biosensors to significantly expand our experimental capabilities for studying the principles underlying biomineralization in vitro.
- ItemQuantification of internalized silica nanoparticles via STED microscopy(London : Hindawi, 2015) Peuschel, Henrike; Ruckelshausen, Thomas; Cavelius, Christian; Kraegeloh, AnnetteThe development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2 x 10^10 particles mL^-1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20–27 of the particles sedimented. In comparison, 102-103 NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 10^12 particles mL^-1) of the smaller particles induced cytotoxicity.
- ItemPhotoactivatable Hsp47: A tool to control and regulate collagen secretion & assembly(Hoboken, NJ : Wiley, 2018) Khan, Essak; Sankaran, Shrikrishnan; Paez, Julieta; Muth, Christina; Han, Mitchell; Del Campo, AránzazuCollagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen-specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non-natural light-responsive tyrosine (o-nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor-mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.
- ItemDatasets from a vapor diffusion mineral precipitation protocol for Dictyostelium stalks(Amsterdam : Elsevier, 2016) Eder, Magdalena; Muth, Christina; Weiss, Ingrid M.Datasets from a slow carbonate vapor diffusion and mineral precipitation protocol for Dictyostelium ECM and cellulose stalks show examples for composite materials obtained by an in vitro approach, which differs substantially from the in vivo approach reported in The Journal of Structural Biology, doi: 10.1016/j.jsb.2016.03.015 [1]. Methods for obtaining the datasets include bright field transmitted light microscopy, fluorescence microscopy, LC-PolScope birefringence microscopy, variable pressure scanning electron microscopy (VP-SEM/ESEM), and Raman imaging spectroscopy.
- ItemVimentin levels and serine 71 phosphorylation in the control of cell-matrix adhesions, migration speed, and shape of transformed human fibroblasts(Basel : MDPI, 2017) Terriac, Emmanuel; Coceano, Giovanna; Mavajian, Zahra; Hageman, Tijmen A.G.; Christ, Andreas F.; Testa, Ilaria; Lautenschläger, Franziska; Gad, Annica K.G.Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30–50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix adhesions. Long vimentin filaments were found in the proximity of large FAs. Vimentin expression in these cells caused a reduction in FAs size and an elongated cell shape, but did not affect FA lifetime, or the speed or directionality of cell migration. Expression of a phospho-mimicking mutant (S71D) of vimentin increased the speed of cell migration. Taken together, our results suggest that in highly migratory, transformed mesenchymal cells, vimentin levels control the cell shape and FA size, but not cell migration, which instead is linked to the phosphorylation status of S71 vimentin. These observations are consistent with the possibility that not only levels, but also the assembly status of vimentin control cell migration.
- ItemSurface structure influences contact killing of bacteria by copper(Hoboken, NJ : Wiley, 2014) Zeiger, Marco; Solioz, Marc; Edongué, Hervais; Arzt, Eduard; Schneider, Andreas S.Copper kills bacteria rapidly by a mechanism that is not yet fully resolved. The antibacterial property of copper has raised interest in its use in hospitals, in place of plastic or stainless steel. On the latter surfaces, bacteria can survive for days or even weeks. Copper surfaces could thus provide a powerful accessory measure to curb nosocomial infections. We here investigated the effect of the copper surface structure on the efficiency of contact killing of Escherichia coli, an aspect which so far has received very little attention. It was shown that electroplated copper surfaces killed bacteria more rapidly than either polished copper or native rolled copper. The release of ionic copper was also more rapid from electroplated copper compared to the other materials. Scanning electron microscopy revealed that the bacteria nudged into the grooves between the copper grains of deposited copper. The findings suggest that, in terms of contact killing, more efficient copper surfaces can be engineered.
- ItemThe analysis of arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses(Rockville, MD : American Society of Plant Biologists, 2009) Fink-Straube, Claudia; Klatte, Marco; Schuler, Mara; Wirtz, Markus; Hell, Rüdiger; Bauer, PetraNicotianamine chelates and transports micronutrient metal ions in plants. It has been speculated that nicotianamine is involved in seed loading with micronutrients. A tomato (Solanum lycopersicum) mutant (chloronerva) and a tobacco (Nicotiana tabacum) transgenic line have been utilized to analyze the effects of nicotianamine loss. These mutants showed early leaf chlorosis and had sterile flowers. Arabidopsis (Arabidopsis thaliana) has four NICOTIANAMINE SYNTHASE (NAS) genes. We constructed two quadruple nas mutants: one had full loss of NAS function, was sterile, and showed a chloronerva-like phenotype (nas4x-2); another mutant, with intermediate phenotype (nas4x-1), developed chlorotic leaves, which became severe upon transition from the vegetative to the reproductive phase and upon iron (Fe) deficiency. Residual nicotianamine levels were sufficient to sustain the life cycle. Therefore, the nas4x-1 mutant enabled us to study late nicotianamine functions. This mutant had no detectable nicotianamine in rosette leaves of the reproductive stage but low nicotianamine levels in vegetative rosette leaves and seeds. Fe accumulated in the rosette leaves, while less Fe was present in flowers and seeds. Leaves, roots, and flowers showed symptoms of Fe deficiency, whereas leaves also showed signs of sufficient Fe supply, as revealed by molecular-physiological analysis. The mutant was not able to fully mobilize Fe to sustain Fe supply of flowers and seeds in the normal way. Thus, nicotianamine is needed for correct supply of seeds with Fe. These results are fundamental for plant manipulation approaches to modify Fe homeostasis regulation through alterations of NAS genes.
- ItemA correlative analysis of gold nanoparticles internalized by A549 cells(Hoboken, NJ : Wiley, 2014) Böse, Katharina; Koch, Marcus; Cavelius, Christian; Kiemer, Alexandra K.; Kraegeloh, AnnetteFluorescently labeled nanoparticles are widely used to investigate nanoparticle cell interactions by fluorescence microscopy. Owing to limited lateral and axial resolution, nanostructures (<100 nm) cannot be resolved by conventional light microÂscopy techniques. Especially after uptake into cells, a common fate of the fluorescence label and the particle core cannot be taken for granted. In this study, a correlative approach is presented to image fluorescently labeled gold nanoparticles inside whole cells by correlative light and electron microscopy (CLEM). This approach allows for detection of the fluorescently labeled particle shell as well as for the gold core in one sample. In this setup, A549 cells are exposed to 8 nm Atto 647N-labeled gold nanoparticles (3.3 × 109 particles mL−1, 0.02 μg Au mL−1) for 5 h and are subsequently imaged by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Eight fluorescence signals located at different intracellular positions are further analyzed by TEM. Five of the eight fluorescence spots are correlated with isolated or agglomerated gold nanoparticles. Three fluorescence signals could not be related to the presence of gold, indicating a loss of the particle shell.