Hauke Clausen-Schaumann

Mechanical Stability of Single DNA Molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (</=10 mM NaCl), while high ionic strength buffers (1 M NaCl) stabilize the double-helical conformation. The mechanical energy that can be deposited in the DNA double helix before force induced melting occurs was found to decrease with increasing temperature. This energy correlates with the base-pairing free enthalpy DeltaG(bp)(T) of DNA. Experiments with pure poly(dG-dC) and poly(dA-dT) DNA sequences again revealed a close correlation between the mechanical energies at which these sequences melt with base pairing free enthalpies DeltaG(bp)(sequence): while the melting transition occurs between 65 and 200 pN in lambda-phage DNA, depending on the loading rate, the melting transition is shifted to approximately 300 pN for poly(dG-dC) DNA, whereas poly(dA-dT) DNA melts at a force of 35 pN.

Force spectroscopy with single bio-molecules.

For many biological molecules, force is an important functional and structural parameter. With the rapidly growing knowledge about the relation between structure, function, and force, single-molecule force spectroscopy has become a versatile analytical tool for the structural and functional investigation of single bio-molecules in their native environments. Within the past year, detailed insights into binding potentials of receptor ligand pairs, protein folding pathways, molecular motors, DNA mechanics and the functioning of DNA-binding agents (such as proteins and drugs), as well as the function of molecular motors, have been obtained.

Atomic Force Microscope Imaging of Phospholipid Bilayer Degradation by Phospholipase A2

We have investigated the time course of the degradation of a supported dipalmitoylphosphatidylcholine bilayer by phospholipase A2 in aqueous buffer with an atomic force microscope. Contact mode imaging allows visualization of enzyme activity on the substrate with a lateral resolution of less than 10 nm. Detailed analysis of the micrographs reveals a dependence of enzyme activity on the phospholipid organization and orientation in the bilayer. These experiments suggest that it is possible to observe single enzymes at work in small channels, which are created by the hydrolysis of membrane phospholipids. Indeed, the measured rate of hydrolysis of phospholipids corresponds very well with the enzyme activity found in kinetic studies. It was also possible to correlate the number of enzymes at the surface, as calculated from the binding constant to the number of starting points of the hydrolysis. In addition, the width of the channels was found to be comparable to the diameter of a single phospholipase A2 and thus further supports the single-enzyme hypothesis.

Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy

Within the bone lie several different cell types, including osteoblasts (OBs) and mesenchymal stem cells (MSCs). The MSCs are ideal targets for regenerative medicine of bone due to their differentiation potential towards OBs. Human MSCs exhibit two distinct morphologies: rapidly self‐renewing cells (RS) and flat cells (FC) with very low proliferation rates. Another cell type found in pathological bone conditions is osteosarcoma. In this study, we compared the topographic and morphometric features of RS and FC cells, human OBs and MG63 osteosarcoma cells by atomic force microscopy (AFM). The results demonstrated clear differences: FC and hOB cells showed similar ruffled topography, whereas RS and MG63 cells exhibited smoother surfaces. Furthermore, we investigated how selected substrates influence cell morphometry. We found that RS and MG63 cells were flatter on fibrous substrates such as polystyrene and collagen I, but much more rounded on glass, the smoothest surface. In contrast, cells with large area, namely FC and hOB cells, did not exhibit pronounced changes in flatness with regards to the different substrates. They were, however, remarkably flatter in comparison to RS and MG63 cells. We could explain the differences in flatness by the extent of adhesion. Indeed, FC and hOB cells showed much higher content of focal adhesions. Finally, we used the AFM to determine the cellular Youngs modulus. RS, FC and hOB cells showed comparable stiffness on the three different substrates, while MG63 cells demonstrated the unique feature of increased elasticity on collagen I. In summary, our results show, for the first time, a direct comparison between the morphometric and biophysical features of different human cell types derived from normal and pathological bone. Our study manifests the opinion that along with RNA, proteomic and functional research, morphological and biomechanical characterization of cells also reveals novel cell features and interrelationships.

Dynamic Strength of the Silicon-Carbon Bond Observed over Three Decades of Force-Loading Rates

The mechanical strength of individual Si-C bonds was determined as a function of the applied force-loading rate by dynamic single-molecule force spectroscopy, using an atomic force microscope. The applied force-loading rates ranged from 0.5 to 267 nN/s, spanning 3 orders of magnitude. As predicted by Arrhenius kinetics models, a logarithmic increase of the bond rupture force with increasing force-loading rate was observed, with average rupture forces ranging from 1.1 nN for 0.5 nN/s to 1.8 nN for 267 nN/s. Three different theoretical models, all based on Arrhenius kinetics and analytic forms of the binding potential, were used to analyze the experimental data and to extract the parameters fmax and D(e) of the binding potential, together with the Arrhenius A-factor. All three models well reproduced the experimental data, including statistical scattering; nevertheless, the three free parameters allow so much flexibility that they cannot be extracted unambiguously from the experimental data. Successful fits with a Morse potential were achieved with fmax = 2.0-4.8 nN and D(e) = 76-87 kJ/mol, with the Arrhenius A-factor covering 2.45 x 10(-10)-3 x 10(-5) s(-1), respectively. The Morse potential parameters and A-factor taken from gas-phase density functional calculations, on the other hand, did not reproduce the experimental forces and force-loading rate dependence.

A force-based protein biochip

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand–receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein–protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells.

Despite of intensive research efforts, the precise mechanism of prostate cancer metastasis in bone is still not fully understood. Several studies have suggested that specific matrix production by the bone cells, such as collagen I, supports cancer cell invasion. The aim of this study was to investigate the effect of collagen I (COL1) and fibronectin (FN) on cell adhesion, cell elasticity and cytoskeletal organization of prostate cancer cells. Two cell lines, bone marrow- (PC3) and lymph node-derived (LNCaP) were cultivated on COL1 and FN (control protein). By using a quantitative adhesion assay and time-lapse analysis, it was found that PC3, but not LNCaP, adhered strongly and were more spread on COL1. Next, PC3 and LNCaP were evaluated by atomic force microscopy (AFM) and flatness shape factor and cellular Youngs modulus were calculated. The shape analysis revealed that PC3 were significantly flatter when grown on COL1 in comparison to LNCaP. In general, PC3 were also significantly stiffer than LNCaP and furthermore, their stiffness increased upon interaction with COL1. Since cell stiffness is strongly dependent on actin organization, phalloidin-based actin staining was performed and revealed that, of the two cell types as well as the two different matrix proteins, only PC3 grown on COL1 formed robust actin cytoskeleton. In conclusion, our study showed that PC3 cells have a strong affinity towards COL1. On this matrix protein, the cells adhered strongly and underwent a specific cell flattening. Moreover, with the establishment of PC3 contact to COL1 a significant increase of PC3 stiffness was observed due to a profound cytoskeletal rearrangement.

Increased stemness and migration of human mesenchymal stem cells in hypoxia is associated with altered integrin expression

Human mesenchymal stem cells (hMSCs) are regularly cultured and characterised under normoxic (21% O(2)) conditions, although the physiological oxygen tension in the stem cell niche is known to be as low as 1-2%. Oxygen itself is an important signalling molecule, but the distinct impact on various stem cell characteristics is still unclear. Therefore, the aim of this study was to evaluate the influence of oxygen concentration on the hMSC subpopulation composition, cell morphology and migration on different surfaces (polystyrene, collagen I, fibronectin, laminin) as well as on the expression of integrin receptors. Bone marrow-derived hMSCs were cultured either in normoxic (21% O(2)) or hypoxic (2% O(2)) conditions. The hMSC subpopulations were assessed by aspect ratio and cell area. Hypoxia promoted a more homogeneous cell population with a significantly higher fraction of rapidly self-renewing cells which are believed to be the true stem cells. Under hypoxic conditions hMSC volume and height were significantly decreased on all surfaces as measured by white light confocal microscopy. Furthermore, low oxygen tension led to a significant increase in cell velocity and Euclidian distance on all matrixes, which was evaluated by time-lapse microscopy. With regard to cell-matrix contacts, expression of several integrin subunits was evaluated by semi-quantitative RT-PCR. Increased expression of the subunits α(1), α(3), α(5,) α(6), α(11), α(v), β(1) and β(3) was observed in hypoxic conditions, while α(2) was higher expressed in normoxic cultured hMSCs. Taken together, our results indicate that hypoxic conditions promote stemness and migration of hMSC along with altering their integrin expression.

Changes in water chemistry can disable plankton prey defenses

The effectiveness of antipredator defenses is greatly influenced by the environment in which an organism lives. In aquatic ecosystems, the chemical composition of the water itself may play an important role in the outcome of predator–prey interactions by altering the ability of prey to detect predators or to implement defensive responses once the predator’s presence is perceived. Here, we demonstrate that low calcium concentrations (<1.5 mg/L) that are found in many softwater lakes and ponds disable the ability of the water flea, Daphnia pulex to respond effectively to its predator, larvae of the phantom midge, Chaoborus americanus. This low-calcium environment prevents development of the prey’s normal array of induced defenses, which include an increase in body size, formation of neck spines, and strengthening of the carapace. We estimate that this inability to access these otherwise effective defenses results in a 50–186% increase in the vulnerability of the smaller juvenile instars of Daphnia, the stages most susceptible to Chaoborus predation. Such a change likely contributes to the observed lack of success of daphniids in most low-calcium freshwater environments, and will speed the loss of these important zooplankton in lakes where calcium levels are in decline.

Mechanically activated rupture of single covalent bonds: evidence of force induced bond hydrolysis.

We have used temperature-dependent single molecule force spectroscopy to stretch covalently anchored carboxymethylated amylose (CMA) polymers attached to an amino-functionalized AFM cantilever. Using an Arrhenius kinetics model based on a Morse potential as a one-dimensional representation of covalent bonds, we have extracted kinetic and structural parameters of the bond rupture process. With 35.5 kJ mol(-1), we found a significantly smaller dissociation energy and with 9.0 × 10(2) s(-1) to 3.6 × 10(3) s(-1) also smaller Arrhenius pre-factors than expected for homolytic bond scission. One possible explanation for the severely reduced dissociation energy and Arrhenius pre-factors is the mechanically activated hydrolysis of covalent bonds. Both the carboxylic acid amide and the siloxane bond in the amino-silane surface linker are in principle prone to bond hydrolysis. Scattering, slope and curvature of the scattered data plots indicate that in fact two competing rupture mechanisms are observed.