Rapid changes in flagellar rotation induced by external electric pulses.

The bacterial flagellar motor is the only molecular rotary machine found in living organisms, converting the protonmotive force, i.e., the membrane voltage and proton gradients across the cell membrane, into the mechanical force of rotation (torque). We have developed a method for holding a bacterial cell at the tip of a glass micropipette and applying electric pulses through the micropipette. This method has enabled us to observe the dynamical responses of flagellar rotation to electric pulses that change the membrane voltage transiently and repeatedly. We have observed that acceleration and deceleration of motor rotation are induced by application of these electric pulses. The change in the rotation rate occurred within 5 ms after pulse application.     

A look at membrane patches with a scanning force microscope.

We combined scanning force microscopy with patch-clamp techniques in the same experimental setup and obtained images of excised membrane patches spanning the tip of a glass pipette. These images indicate that cytoskeleton structures are still present in such membrane patches and form a strong connection between the membrane and the glass wall. This gives the membrane patch the appearance of a tent, stabilized by a scaffold of ropes. The lateral resolution of the images depends strongly on the observed structures and can reach values as low as 10 nm on the cytoskeleton elements of a (inside-out) patch. The observations suggest that measurements of membrane elasticity can be made, opening the way for further studies on mechanical properties of cell membranes.     

Mechanical Response of Single Plant Cells to Cell Poking： A Numerical Simulation Model

Cell poking is an experimental technique that is widely used to study the mechanical properties of plant cells. A full understanding of the mechanical responses of plant cells to poking force Is helpful for experimental work. The aim of this study was to numerically investigate the stress distribution of the cell wall, cell turgor, and deformation of plant cells in response to applied poking force. Furthermore, the locations damaged during poking were analyzed. The model simulates cell poking, with the cell treated as a spherical, homogeneous, isotropic elastic membrane, filled with incompressible, highly viscous liquid. Equilibrium equations for the contact region and the non-contact regions were determined by using membrane theory. The boundary conditions and continuity conditions for the solution of the problem were found. The forcedeformation curve, turgor pressure and tension of the cell wall under cell poking conditions were obtained. The tension of the cell wall circumference was larger than that of the meridian. In general, maximal stress occurred at the equator around. When cell deformation increased to a certain level, the tension at the poker tip exceeded that of the equator. Breakage of the cell wall may start from the equator or the poker tip, depending on the deformation. A nonlinear model is suitable for estimating turgor, stress, and stiffness, and numerical simulation is a powerful method for determining plant cell mechanical properties.     

Dynamic dorsoventral stiffness assessment of the ovine lumbar spine

Posteroanterior spinal stiffness assessments are common in the evaluating patients with low back pain. The purpose of this study was to determine the effects of mechanical excitation frequency on dynamic lumbar spine stiffness. A computer-controlled voice coil actuator equipped with a load cell and LVDT was used to deliver an oscillatory dorsoventral (DV) mechanical force to the L3 spinous process of 15 adolescent Merino sheep. DV forces (48 N peak, approximately 10% body weight) were randomly applied at periodic excitation frequencies of 2.0, 6.0, 11.7 and a 0.5-19.7 Hz sweep. Force and displacement were recorded over a 13-22 s time interval. The in vivo DV stiffness of the ovine spine was frequency dependent and varied 3.7-fold over the 0.5-19.7 Hz mechanical excitation frequency range. Minimum and maximum DV stiffness (force/displacement) were 3.86+/-0.38 and 14.1+/-9.95 N/mm at 4.0 and 19.7 Hz, respectively. Stiffness values based on the swept-sine measurements were not significantly different from corresponding periodic oscillations (2.0 and 6.0 Hz). The mean coefficient of variation in the swept-sine DV dynamic stiffness assessment method was 15%, which was similar to the periodic oscillation method (10-16%). The results indicate that changes in mechanical excitation frequency and animal body mass modulate DV spinal stiffness.     

Flicker in erythrocytes. I. Theoretical models and registration techniques

The phenomenon of stochastic low-frequency oscillations of erythrocyte cell membrane, termed usually the flicker of erythrocytes, is reviewed. The first part describes the theoretical models of erythrocyte flickering and the registration techniques. The relations are given and analyzed which connect the shape of both the frequency and the spatial spectra of stochastic membrane oscillations with geometrical and mechanical parameters of the erythrocyte and with the ambient physical characteristics. The existing concepts of excitation mechanisms of the membrane flickering are presented.     

Identification of TrkA on living PC12 cells by atomic force microscopy

In neural cells, nerve growth factor (NGF) initiates its survival signal through the binding to its cell surface receptor tyrosine kinase A (TrkA). Understanding the pattern of TrkA distribution and association in living cells can provide a fingerprint for the diagnostic comparison with alterations underlying ligand-receptor dysfunction seen in various neurological diseases. In this study, we use the NGF-TrkA-specific interaction as a probe to identify TrkA on living PC12 cell by atomic force microscopy (AFM). An NGF-modified AFM tip was used to perform force volume (FV) imaging, generating a 2D force map to illustrate the distribution and association of TrkA on PC12 cell membrane. It is found that TrkA is highly aggregated at local regions of the cell. This unique protein association may be required to promote its function as a receptor of NGF. The methodology that we developed in this study can be adapted by other systems, thus providing a general tool for investigating protein association in its natural environment.     

Identification of TrkA on living PC12 cells by atomic force microscopy

In neural cells, nerve growth factor (NGF) initiates its survival signal through the binding to its cell surface receptor tyrosine kinase A (TrkA). Understanding the pattern of TrkA distribution and association in living cells can provide a fingerprint for the diagnostic comparison with alterations underlying ligand-receptor dysfunction seen in various neurological diseases. In this study, we use the NGF-TrkA-specific interaction as a probe to identify TrkA on living PC12 cell by atomic force microscopy (AFM). An NGF-modified AFM tip was used to perform force volume (FV) imaging, generating a 2D force map to illustrate the distribution and association of TrkA on PC12 cell membrane. It is found that TrkA is highly aggregated at local regions of the cell. This unique protein association may be required to promote its function as a receptor of NGF. The methodology that we developed in this study can be adapted by other systems, thus providing a general tool for investigating protein association in its natural environment.     

Modeling electrically active viscoelastic membranes

The membrane protein prestin is native to the cochlear outer hair cell that is crucial to the ear's amplification and frequency selectivity throughout the whole acoustic frequency range. The outer hair cell exhibits interrelated dimensional changes, force generation, and electric charge transfer. Cells transfected with prestin acquire unique active properties similar to those in the native cell that have also been useful in understanding the process. Here we propose a model describing the major electromechanical features of such active membranes. The model derived from thermodynamic principles is in the form of integral relationships between the history of voltage and membrane resultants as independent variables and the charge density and strains as dependent variables. The proposed model is applied to the analysis of an active force produced by the outer hair cell in response to a harmonic electric field. Our analysis reveals the mechanism of the outer hair cell active (isometric) force having an almost constant amplitude and phase up to 80 kHz. We found that the frequency-invariance of the force is a result of interplay between the electrical filtering associated with prestin and power law viscoelasticity of the surrounding membrane. Paradoxically, the membrane viscoelasticity boosts the force balancing the electrical filtering effect. We also consider various modes of electromechanical coupling in membrane with prestin associated with mechanical perturbations in the cell. We consider pressure or strains applied step-wise or at a constant rate and compute the time course of the resulting electric charge. The results obtained here are important for the analysis of electromechanical properties of membranes, cells, and biological materials as well as for a better understanding of the mechanism of hearing and the role of the protein prestin in this mechanism.     

Force volume and stiffness tomography investigation on the dynamics of stiff material under bacterial membranes

The determination of the characteristics of micro-organisms in clinical specimens is essential for the rapid diagnosis and treatment of infections. A thorough investigation of the nanoscale properties of bacteria can prove to be a fundamental tool. Indeed, in the latest years, the importance of high resolution analysis of the properties of microbial cell surfaces has been increasingly recognized. Among the techniques available to observe at high resolution specific properties of microscopic samples, the Atomic Force Microscope (AFM) is the most widely used instrument capable to perform morphological and mechanical characterizations of living biological systems. Indeed, AFM can routinely study single cells in physiological conditions and can determine their mechanical properties with a nanometric resolution. Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work, we exploit the capabilities and peculiarities of AFM to analyze the mechanical properties of Escherichia coli in order to evidence with a high spatial resolution the mechanical properties of its structure. In particular, we will show that the bacterial membrane is not mechanically uniform, but contains stiffer areas. The force volume investigations presented in this work evidence for the first time the presence and dynamics of such structures. Such information is also coupled with a novel stiffness tomography technique, suggesting the presence of stiffer structures present underneath the membrane layer that could be associated with bacterial nucleoids.     

Effects of vibration on differentiation of cultured PC12 cells

Different types of physiological‐mechanical stress, such as shear stress in vascular endothelial cells or hydrostatic pressure in chondrocytes are well known as regulators of cell function. In this study, the effects of vibration, a type of non‐physiological mechanical stimulation, on differentiation of rat pheochromocytoma (PC12) cells are reported. A nano‐vibration system was designed to produce nanometer‐scale vibration. The frequency and amplitude of the nano‐vibrations were monitored by a capacitance displacement sensor connected to an oscilloscope. When PC12 cells exposed to nerve growth factor were subjected to vibration at 10 kHz, differentiation and elongation of their neurites were promoted earlier in the culture. Vibration promoted differentiation of PC12 cells. This approach could therefore also be promising for determining of the effects of the physical environment on cell differentiation. Biotechnol. Bioeng. 2011; 108:592–599. © 2010 Wiley Periodicals, Inc.     

Comparison of atomic force microscopy interaction forces between bacteria and silicon nitride substrata for three commonly used immobilization methods

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.     

Comparison of Atomic Force Microscopy Interaction Forces between Bacteria and Silicon Nitride Substrata for Three Commonly Used Immobilization Methods

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.     

Imaging material properties of biological samples with a force feedback microscope

Mechanical properties of biological samples have been imaged with a force feedback microscope. Force, force gradient, and dissipation are measured simultaneously and quantitatively, merely knowing the atomic force microscopy cantilever spring constant. Our first results demonstrate that this robust method provides quantitative high resolution force measurements of the interaction. The small oscillation imposed on the cantilever and the small value of its stiffness result in vibrational energies much smaller than the thermal energy, reducing interaction with the sample to a minimum. We show that the observed mechanical properties of the sample depend on the force applied by the tip and consequently on the sample indentation. Copyright © 2013 John Wiley & Sons, Ltd.     

Nano-mechanical exploration of the surface and sub-surface of hydrated cells of Staphylococcus epidermidis

The surface of hydrated cells of Staphylococcus epidermidis has been probed using an atomic force microscope. While local force measurements over the surface of bacteria reveal a heterogeneous chemical surface, with heterogeneous mechanical properties, different kinds of force curves appear with high frequency, and are thought to provide information on features contributing strongly to the overall mechanical and surface behaviour of the cell. Force curves often present two different mechanical regimes, being the first one (outer) of about 48 nm thick, and presenting a local relative elasticity of about 0.08 N/m, which is about a third of the relative elasticity of the inner part of the cell wall, harder, with a relative elasticity of about 0.24 N/m, in water. Both regimes appears as straight lines in the force versus distance curves (the ‘corresponding’ stress–strain curves in contact mechanics), but hysteresis is observed between the approach and the retraction line in the inner regime, indicating a degree of viscoelasticity. No viscoelasticity is observed in the outer regime, however, which presents quite linear and juxtaposed approach-retraction lines. These kinds of force curves do not present measurable pull-off forces nor snap-in forces, which indicates an almost null interaction between tip and bacterial surface, which could be in agreement with the measured very high hydrophobicity of this strain. Another kind of force curve has been observed recurrently, showing peaks in the retraction curves. Adhesive pull-off forces were measured giving an average of about 2 nN. Interestingly, however, these force curves appear only when quite irregular and wavy retraction curves are present, from the very beginning of its trace (maximum indentation). This leads us to think that these pull-off forces measured by our AFM do not give information on surface forces-unbinding events at the surface of the bacteria, but could be related to events at the sub-surface of the cell surface. Oscillations seen in the retraction curve in the portion corresponding to the contact with the bacteria surface could be due to rupture phenomena within the multilayered cell wall architecture expected in Gram-positive bacteria as Staphylococcus epidermidis, which could result in local irreversible deformations of the cell surface. Imaging with a sharp tip in contact mode sometimes leads to surface damage. Force curves recorded over damaged parts of the cell surface showed a completely different behaviour, in many cases with two well-defined high-adhesion peaks, and also interestingly, with snap-in forces of about 0–2 nN, which seems to indicate a completely different electrical/hydrophobicity state only a few nanometers down from the surface. Similar indentation effects can occur in the contact of a bacterial cell with a solid surface, even when showing only atomic-molecular-scale roughness, thus interacting not only with the very surface of the cell, especially when soft layers are present in the outer. Our results highlight the importance of the cell surface mechanical properties and their interplay with purely surface properties when analyzing cell–material interaction, and show the AFM as a useful method for investigating this.     

Boron-11 pure quadrupole resonance investigation of peptide boronic acid inhibitors bound to alpha-lytic protease

Pure quadrupole resonance is a potentially useful spectroscopic approach to study the coordination of quadrupolar nuclei in biological systems. We used a field-cycling NMR method to observe boron pure quadrupole resonance of two peptide boronic acid inhibitors bound to alpha-lytic protease. The method is similar to our earlier field-cycling experiment [Ivanov, D., and Redfield, A. R. (1998) Z. Naturforsch. A 53, 269-272] but uses a simple Hartmann-Hahn transfer from proton to (11)B before field cycle and direct (11)B observe after it. Pure quadrupole resonance is sensitive to the boron coordination geometry. For example, trigonal boron in neutral phenylboronic acid, which was used as a model compound, resonates at 1450 kHz, while the resonance of the tetrahedral phenylboronic acid anion appears at approximately 600 kHz. In the complex of the MeOSuc-Ala-Ala-Pro-boroVal inhibitor with the enzyme the quadrupole resonance signal was observed at 600-650 kHz, which indicates tetrahedral boron coordination in the active site. The quadrupole frequency of the MeOSuc-Ala-Ala-Pro-boroPhe enzyme-inhibitor complex, in which a boron-histidine bond is known to be formed, was found to be the same within experimental error as in the MeOSuc-Ala-Ala-Pro-boroVal enzyme-inhibitor adduct, suggesting that the boron coordination geometry in the enzyme-MeOSuc-Ala-Ala-Pro-boroPhe adduct is also close to tetrahedral.     

Mechanism of ethanol enhancement of apoptosis and caspase activation in serum-deprived PC12 cells

Neuronal death is one of the most prominent consequences of alcohol exposure during development. Ethanol-induced neuronal death appears to involve apoptosis. The objective of the present study was to characterize the effect of ethanol on neuronal cell viability and to determine the mechanism by which ethanol enhances apoptosis in neural cells. For these studies the rat pheochromocytoma (PC12) cells were used. PC12 cells were incubated for 24 h in the presence or absence of 100 mM ethanol. Apoptosis was induced by serum withdrawal. Ethanol in the presence of serum-containing media did not alter cell viability, while incubation of PC12 cells in serum-free media resulted in a significant increase in cell death that was further significantly increased by 35% in cells exposed to ethanol. The temporal response of the PC12 cells to serum withdrawal was studied over a period of 22 h. At least 18 h of ethanol exposure was necessary to observe a significant increase in death for cells incubated in serum-free media. An increase in the caspase-3 activity in PC12 cells deprived of serum was observed that was further increased by ethanol exposure. This increase of caspase-3 activity was correlated with an enhancement of caspase-9 activity. Ethanol exposure increased the amount of cytosolic cytochrome c in PC12 cells incubated in serum-free media but did not alter the level of cytochrome c in cells incubated in serum. Finally, a 26% increase was observed in the number of cells with depolarized mitochondria due to ethanol treatment. The present study implicates an increase in the mitochondrial outer membrane permeability as a potential mechanism of enhancement of apoptosis in serum-deprived PC12 cells by ethanol.     

Responses of cat auditory cortical neurons to tones of different frequencies and electrical stimulation of corresponding regions of the cochlea

Characteristic frequencies of neurons in the cat auditory cortex (area AI) whose receptive fields are located in different parts of the basilar membrane of the cochlea were determined in cats anesthetized with pentobarbital. The higher the characteristic frequency of a neuron in area AI, the nearer its receptive field lies to the base of the cochlea. Receptive fields of neurons with a characteristic frequency higher than 4 kHz lie on the first 10 mm of the basilar membrane. Receptive fields of neurons with a characteristic frequency below 4 kHz lie on the remaining 11–12 mm of the membrane. The effect of electrical stimulation of the center of the receptive field of a neuron corresponds to its response to a tone of characteristic frequency. The more the frequency of the acting tone differs from the characteristic frequency, or the further the point of stimulation from the center of the receptive field of the neuron, the less likely is the neuron to respond with an action potential. Neurons with a low characteristic frequency have wider receptive fields than neurons with a high characteristic frequency. Receptive fields of neurons with close characteristic frequencies on the basilar membrane overlap considerably. It was shown by the method of paired stimulation that excitation evoked in neurons in area AI by the action of a tone of a particular frequency is followed by long-lasting inhibition. This inhibition lasts longest and is most effective if a tone of the characteristic frequency is used.     

Experimental model for stimulation of cultured human osteoblast-like cells by high frequency vibration

Reliable and reproducible experimental methods for studying enhancement of osteoblast proliferation and metabolic activity in vitro provide invaluable tools for the research of biochemical processes involved in bone turnover in vivo. Some of the current methods used for this purpose are based on the ability of the osteoblasts to react metabolically to mechanical stimulation. These methods are based on the hypothesis that intracellular metabolic pathways could be influenced by the excitation of cytoskeletal components by mechanical cell deformation. Based on the same assumptions we developed a new experimental approach of biomechanical stimulation of cultured osteoblast-like cells by vibration. This method is based on the use of a specially designed vibration device that consists of an electric shaker with horizontally mounted well plate containing cell cultures. We used a first passage explant outgrowth of human osteoblast-like cell cultures, originating from samples of cancelous bone, collected from femoral necks of six donors during surgical arthroplasties of osteoarthritic hips. Well plates with replicates of cultured cells were exposed to a sine shaped vibration protocol in a frequency range of 20–60 Hz with displacement amplitude of 25 (±5) μm. We found that vibration at a distinct set of mechanical parameters of 20 Hz frequency and peak to peak acceleration of 0.5 ± 0.1 m/sec² is optimal for cell proliferation, and at 60 Hz frequency with peak to peak acceleration of 1.3 ± 0.1 m/sec² for metabolic activity. The presented easily reproducible experimental model should improve and simplify further research on the interactions between mechanical stimuli and intracellular biochemical pathways in osteoblasts. This revised version was published online in September 2006 with corrections to the Cover Date.     

Force as a probe of membrane protein structure and function

Force measurement techniques are being used increasingly to explore the mechanical properties of proteins, as well as the structural origins of intermolecular forces. Developments in instrumentation and the increasing availability of engineered and purified membrane proteins have widely expanded the range of biological systems that can be addressed. Within the past year, force measurements have identified novel mechanisms of binding between cell-surface proteins, as well as the mechanical properties of integral membrane proteins and the intramolecular interactions that stabilize their structures.     

Dynamic force spectroscopy on supported lipid bilayers: effect of temperature and sample preparation

Biological membranes are constantly exposed to forces. The stress-strain relation in membranes determines the behavior of many integral membrane proteins or other membrane related-proteins that show a mechanosensitive behavior. Here, we studied by force spectroscopy the behavior of supported lipid bilayers (SLBs) subjected to forces perpendicular to their plane. We measured the lipid bilayer mechanical properties and the force required for the punch-through event characteristic of atomic force spectroscopy on SLBs as a function of the interleaflet coupling. We found that for an uncoupled bilayer, the overall tip penetration occurs sequentially through the two leaflets, giving rise to two penetration events. In the case of a bilayer with coupled leaflets, penetration of the atomic force microscope tip always occurred in a single step. Considering the dependence of the jump-through force value on the tip speed, we also studied the process in the context of dynamic force spectroscopy (DFS). We performed DFS experiments by changing the temperature and cantilever spring constant, and analyzed the results in the context of the developed theories for DFS. We found that experiments performed at different temperatures and with different cantilever spring constants enabled a more effective comparison of experimental data with theory in comparison with previously published data.