Biomedical subjects

Christoph F Schmidt

Publications and source records attributed to Christoph F Schmidt.

10 recordsLinked to original sources

PubMed2006

Failure of viral shells.

We report a combined theoretical and experimental study of the structural failure of viral shells under mechanical stress. We find that discontinuities in the force-indentation curve associated with failure should appear when the so-called Föppl-von Kármán (FvK) number exceeds a critical value. A nanoindentation study of a viral shell subject to a soft-mode instability, where the stiffness of the shell decreases with increasing pH, confirms the predicted onset of failure as a function of the FvK number.

Biomechanical Phenomena↗

PubMed2006

Allosteric inhibition of kinesin-5 modulates its processive directional motility.

Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell division, represent alternatives to antimitotic agents that target tubulin. While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol, act through poorly understood allosteric mechanisms, not competing with ATP binding. Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays. We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.

Allosteric Regulation↗

PubMed2006

Elastic response, buckling, and instability of microtubules under radial indentation.

We tested the mechanical properties of single microtubules by lateral indentation with the tip of an atomic force microscope. Indentations up to approximately 3.6 nm, i.e., 15% of the microtubule diameter, resulted in an approximately linear elastic response, and indentations were reversible without hysteresis. At an indentation force of around 0.3 nN we observed an instability corresponding to an approximately 1-nm indentation step in the taxol-stabilized microtubules, which could be due to partial or complete rupture of a relatively small number of lateral or axial tubulin-tubulin bonds. These indentations were reversible with hysteresis when the tip was retracted and no trace of damage was observed in subsequent high-resolution images. Higher forces caused substantial damage to the microtubules, which either led to depolymerization or, occasionally, to slowly reannealing holes in the microtubule wall. We modeled the experimental results using finite-element methods and find that the simple assumption of a homogeneous isotropic material, albeit structured with the characteristic protofilament corrugations, is sufficient to explain the linear elastic response of microtubules.

Computer Simulation↗

PubMed2006

Optical trap stiffness in the presence and absence of spherical aberrations.

Optical traps are commonly constructed with high-numerical-aperture objectives. Oil-immersion objectives suffer from spherical aberrations when used for imaging in aqueous solutions. The effect of spherical aberrations on trapping strength has been modeled by approximation, and only a few experimental results are available in the case of micrometer-sized particles. We present an experimental study of the dependence of lateral and axial optical-trap stiffness on focusing depth for polystyrene and silica beads of 2 microm diameter by using oil- and water-immersion objectives. We demonstrate a strong depth dependence of trap stiffness with the oil-immersion objective, whereas no depth dependence was observed with the water-immersion objective.

Journal Article↗

PubMed2006

Bio imaging of intracellular NO production in single bone cells after mechanical stimulation.

UNLABELLED: We show the intracellular upregulation of NO production after mechanical stimulation, an essential chemical signal in bone remodeling. This is done in real time using the fluorescent chromophore DAR-4M AM. Differences in cellular response to mechanical stimulation of different regions of a single cell were observed. INTRODUCTION: Osteocytes are the most abundant bone cells that are believed to be the mechanosensors of bone, responding to mechanical stresses in interstitial fluid flow through the canaliculi. Under mechanical load, chemical signals such as NO play a key role in the activity of osteoblasts/osteoclasts that regulate bone remodeling. Despite the importance of NO in signaling, its real-time detection has proved challenging. This is largely because of the short NO half-life (typically approximately 0.1-5 s). Here, we show the upregulation of intracellular NO production in single osteocytes under localized mechanical stimulation. MATERIALS AND METHODS: We used the chromophore DAR-4M AM for NO detection. This is loaded into surface-attached MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells that are subjected to a localized mechanical stimulation using optical tweezers or a microneedle tip. DAR-4M AM is membrane-permeable and chelates NO, forming a stable, fluorescent compound, which is visible with a rhodamine filter. RESULTS: Nonstimulated MLO-Y4 and MC3T3-E1 cells showed basal NO production levels, as indicated by a gradual increase in their fluorescence intensity. Localized mechanical stimulation of single MC3T3-E1 cells and MLO-Y4 cells by optical tweezers (150-550 pN, 0.5-3 Hz, 1 minute) showed a nearly 15-30% increase, whereas MLO-Y4 cells stimulated by a microneedle (10-20 nN, 1 minute) showed nearly 15-16% increase relative to their nonstimulated state. Furthermore, stimulation of a single cell process by a microneedle resulted in a 2-10% increase in the fluorescence intensity. CONCLUSIONS: NO is essential for mechanically induced bone remodeling and is a meaningful parameter for measuring bone cell activation after mechanical loading. Here we show NO upregulation in individual bone cells after a localized mechanical stimulation. We also show that both the cell body and the cell processes might be involved in mechanosensing. This technique allows characterization of the mechanosensitivity of different parts of a single osteocyte. This opens up the possibility to uncover the complexities and function of single osteocytes in the dynamic process of bone remodeling.

Animals↗

PubMed2005

The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks.

During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family. Hypotheses for bipolar spindle formation include the 'push-pull mitotic muscle' model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at approximately 20 nm s(-1) towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at approximately 40 nm s(-1), comparable to spindle pole separation rates in vivo. Furthermore, we found that Eg5 can tether microtubule plus-ends, suggesting an additional microtubule-binding mode for Eg5. Our results demonstrate how members of the kinesin-5 family are likely to function in mitosis, pushing apart interpolar microtubules as well as recruiting microtubules into bundles that are subsequently polarized by relative sliding.

Animals↗

PubMed2004

Microrheology of solutions of semiflexible biopolymer filaments using laser tweezers interferometry.

Semiflexible polymers are of great biological importance in determining the mechanical properties of cells. Techniques collectively known as microrheology have recently been developed to measure the viscoelastic properties of solutions of submicroliter volumes. We employ one such technique, which uses a focused laser beam to trap a micron-sized silica bead and interferometric photodiode detection to measure passively the position fluctuations of the trapped bead with nanometer resolution and high bandwidth. The frequency-dependent complex shear modulus G*(f) can be extracted from the position fluctuations via the fluctuation-dissipation theorem and the generalized Stokes-Einstein relation. Using particle tracking microrheology, we report measurements of shear moduli of solutions of fd viruses, which are filamentous, semiflexible, and monodisperse bacteriophages, each 0.9 microm long, 7 nm in diameter, and having a persistence length of 2.2 microm. Recent theoretical treatments of semiflexible polymer dynamics provide quantitative predictions of the rheological properties of such a model system. The fd samples measured span the dilute, semidilute, and concentrated regimes. In the dilute regime G*(f) is dominated by (rigid rod) rotational relaxation, whereas the high-frequency regime reflects single-semiflexible filament dynamics consistent with the theoretical prediction. Due to the short length of fd viruses used in this study, the intermediate regime does not exhibit a well-developed plateau. A dynamic scaling analysis gives rise to a concentration scaling of c(1.36) (r=0.99) in the transition regime and a frequency scaling of f(0.63) (r=0.98) at high frequencies.

Journal Article↗

PubMed2004

Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy.

We have imaged microtubules, essential structural elements of the cytoskeleton in eukaryotic cells, in physiological conditions by scanning force microscopy. We have achieved molecular resolution without the use of cross-linking and chemical fixation methods. With tip forces below 0.3 nN, protofilaments with approximately 6 nm separation could be clearly distinguished. Lattice defects in the microtubule wall were directly visible, including point defects and protofilament separations. Higher tip forces destroyed the top half of the microtubules, revealing the inner surface of the substrate-attached protofilaments. Monomers could be resolved on these inner surfaces.

Animals↗

PubMed2003

Laser-induced heating in optical traps.

In an optical tweezers experiment intense laser light is tightly focused to intensities of MW/cm(2) in order to apply forces to submicron particles or to measure mechanical properties of macromolecules. It is important to quantify potentially harmful or misleading heating effects due to the high light intensities in biophysical experiments. We present a model that incorporates the geometry of the experiment in a physically correct manner, including heat generation by light absorption in the neighborhood of the focus, balanced by outward heat flow, and heat sinking by the glass surfaces of the sample chamber. This is in contrast to the earlier simple models assuming heat generation in the trapped particle only. We find that in the most common experimental circumstances, using micron-sized polystyrene or silica beads, absorption of the laser light in the solvent around the trapped particle, not in the particle itself, is the most important contribution to heating. To validate our model we measured the spectrum of the Brownian motion of trapped beads in water and in glycerol as a function of the trapping laser intensity. Heating both increases the thermal motion of the bead and decreases the viscosity of the medium. We measured that the temperature in the focus increased by 34.2 +/- 0.1 K/W with 1064-nm laser light for 2200-nm-diameter polystyrene beads in glycerol, 43.8 +/- 2.2 K/W for 840-nm polystyrene beads in glycerol, 41.1 +/- 0.7 K/W for 502-nm polystyrene beads in glycerol, and 7.7 +/- 1.2 K/W for 500-nm silica beads and 8.1 +/- 2.1 K/W for 444-nm silica beads in water. Furthermore, we observed that in glycerol the heating effect increased when the bead was trapped further away from the cover glass/glycerol interface as predicted by the model. We show that even though the heating effect in water is rather small it can have non-negligible effects on trap calibration in typical biophysical experimental circumstances and should be taken into consideration when laser powers of more than 100 mW are used.

Computer Simulation↗

PubMed2002

Optical trapping near resonance absorption.

Expressions for radiation-induced forces are presented for the case of a Rayleigh particle near the focus of a Gaussian laser beam at near-resonant conditions. Classical electromagnetic theory was used to obtain the dependence of the scattering and gradient forces on the incident laser frequency, the beam convergence angle, and the spatial position of the particle with respect to the focus. Approximative numerical analysis performed for particles with a single resonant absorption peak demonstrates the occurrence of up to 50-fold enhanced trapping forces at near-resonant frequencies. The use of this technique of gradient force enhancement may provide optical tweezers with enhanced trapping strengths and a degree of specificity.

Models, Theoretical↗