A new view of mechanotransduction and strain amplification in cells with microvilli and cell processes

In this paper we shall describe new mechanical models for the deformation of the actin filament bundles in kidney microvilli and osteocytic cell processes to see whether these cellular extensions, like the stereocilia on hair cells in the inner ear, can function as mechanotransducers when subject to physiological flow. In the case of kidney microvilli we show that the hydrodynamic drag forces at the microvilli tip are <0.01 pN, but there is a 38-fold force amplification on the actin filaments at the base of the microvilli due to the resisting moment in its terminal web. This leads to forces that are more than sufficient to deform the terminal web complex of the microvillus where ezrin has been shown to couple the actin cytoskeleton to the Na(+)/H(+) exchanger. In the case of bone cell processes we show that the actin filament bundles have an effective Young's modulus that is 200 times > the measured modulus for the actin gel in the cell body. It is, therefore, unlikely that bone cell processes respond in vivo to fluid shear stress, as proposed in [59]. However, we show that the fluid drag forces on the pericellular matrix which tethers the cell processes to the canalicular wall can produce a 20-100 fold amplification of bone tissue strains in the actin filament bundle of the cell process.     

A 3D Motile Rod-Shaped Monotrichous Bacterial Model

We introduce a 3D model for a motile rod-shaped bacterial cell with a single polar flagellum which is based on the configuration of a monotrichous type of bacteria such as Pseudomonas aeruginosa. The structure of the model bacterial cell consists of a cylindrical body together with the flagellar forces produced by the rotation of a helical flagellum. The rod-shaped cell body is composed of a set of immersed boundary points and elastic links. The helical flagellum is assumed to be rigid and modeled as a set of discrete points along the helical flagellum and flagellar hook. A set of flagellar forces are applied along this helical curve as the flagellum rotates. An additional set of torque balance forces are applied on the cell body to induce counter-rotation of the body and provide torque balance. The three-dimensional Navier–Stokes equations for incompressible fluid are used to describe the fluid dynamics of the coupled fluid–microorganism system using Peskin’s immersed boundary method. A study of numerical convergence is presented along with simulations of a single swimming cell, the hydrodynamic interaction of two cells, and the interaction of a small cluster of cells.     

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. II. Tether flow terminated by P-selectin dissociation from PSGL-1

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip most often by one precipitous step of P-selectin:PSGL-1 dissociation. Analyzing the initial elastic response and membrane unbinding from the cytoskeleton in our companion article I, we focus in this article on the regime of tether extrusion that nearly always occurred before release of the extracellular adhesion bond at pulling speeds > or =1 microm/s. The force during tether growth appeared to approach a plateau at long times. Examined over a large range of pulling speeds up to 150 microm/s, the plateau force exhibited a significant shear thinning as indicated by a weak power-law dependence on pulling speed, f(infinity) = 60 pN(nu(pull)/microm/s)(0.25). Using this shear-thinning response to describe the viscous element in a nonlinear Maxwell-like fluid model, we show that a weak serial-elastic component with a stiffness of approximately 0.07 pN/nm provides good agreement with the time course of the tether force approach to the plateau under constant pulling speed.     

A mechanism for nuclear positioning in fission yeast based on microtubule pushing

The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane. In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process. Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells. We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell. The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites. When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus. After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip. Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.     

Modulation of Kinesin’s Load-Bearing Capacity by Force Geometry and the Microtubule Track

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule-associated proteins, and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional single-bead assay, we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule, the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration <0.2 s) to a persistent motor that sustains attachment (median attachment duration >3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice, which has implications for a range of cellular activities, including cell division and organelle transport.     

Improved resolution of tertiary structure elasticity in muscle protein

Rearrangement of tertiary structure in response to mechanical force (termed tertiary structure elasticity) in the tandem Ig chain is the first mode of elastic response for muscle protein titin. Tertiary structure elasticity occurs at low stretching forces (few tens of pN), and was described at atomic resolution in a recent molecular dynamics study, in which an originally crescent-shaped six-Ig chain was stretched into a linear chain. However, the force-extension profile that resulted from this explicit solvent simulation was dominated by the hydrodynamic drag force, and effects of tertiary structure elasticity only manifested for stretching forces above 20 pN. Here we report a slow pulling 100-ns simulation (along with other auxiliary simulations), in which hydrodynamic drag force is seen to reduce to near 0 pN, such that tertiary structure elasticity could be characterized over a 0–200 pN range. Statistical mechanical analysis showed that the stretching velocity was sufficiently low such that the protein remained significantly relaxed during the major part of its extension.     

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane unbinding forces for rates of loading (force/time) in the elastic regime from 240 pN/s to 38,000 pN/s, we discovered that the force distributions agreed well with the theory for kinetically limited failure of a weak bond. The kinetic rate for membrane unbinding was found to increase as an exponential function of the pulling force, characterized by an e-fold scale in force of approximately 17 pN and a preexponential factor, or apparent unstressed off rate, of approximately 1/s. The rheological properties of tether growth subsequent to the membrane unbinding events are presented in a companion article II.     

Basal body positioning is controlled by flagellum formation in Trypanosoma brucei

To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.     

Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2 ~ 5 microm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of alpha(5)- and beta(3)-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800 ~ 1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to alpha(5)beta(1)- and alpha(v)beta(3)-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces.     

Biophysical characterization of a binding site for TLQP-21, a naturally occurring peptide which induces resistance to obesity

Recently, we demonstrated that TLQP-21 triggers lipolysis and induces resistance to obesity by reducing fat accumulation [1]. TLQP-21 is a 21 amino acid peptide cleavage product of the neuroprotein VGF and was first identified in rat brain. Although TLQP-21 biological activity and its molecular signaling is under active investigation, a receptor for TLQP-21 has not yet been characterized. We now demonstrate that TLQP-21 stimulates intracellular calcium mobilization in CHO cells. Furthermore, using Atomic Force Microscopy (AFM), we also provide evidence of TLQP-21 binding-site characteristics in CHO cells. AFM was used in force mapping mode equipped with a cantilever suitably functionalized with TLQP-21. Attraction of this functionalized probe to the cell surface was specific and consistent with the biological activity of TLQP-21; by contrast, there was no attraction of a probe functionalized with biologically inactive analogues. We detected interaction of the peptide with the binding-site by scanning the cell surface with the cantilever tip. The attractive force between TLQP-21 and its binding site was measured, statistically analyzed and quantified at approximately 40 pN on average, indicating a single class of binding sites. Furthermore we observed that the distribution of these binding sites on the surface was relatively uniform.     

Visualizing single DNA-bound proteins using DNA as a scanning probe

Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions.     

Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.     

Force-induced focal adhesion translocation: effects of force amplitude and frequency

Vascular endothelial cells rapidly transduce local mechanical forces into biological signals through numerous processes including the activation of focal adhesion sites. To examine the mechanosensing capabilities of these adhesion sites, focal adhesion translocation was monitored over the course of 5 min with GFP-paxillin while applying nN-level magnetic trap shear forces to the cell apex via integrin-linked magnetic beads. A nongraded steady-load threshold for mechanotransduction was established between 0.90 and 1.45 nN. Activation was greatest near the point of forcing (<7.5 µm), indicating that shear forces imposed on the apical cell membrane transmit nonuniformly to the basal cell surface and that focal adhesion sites may function as individual mechanosensors responding to local levels of force. Results from a continuum, viscoelastic finite element model of magnetocytometry that represented experimental focal adhesion attachments provided support for a nonuniform force transmission to basal surface focal adhesion sites. To further understand the role of force transmission on focal adhesion activation and dynamics, sinusoidally varying forces were applied at 0.1, 1.0, 10, and 50 Hz with a 1.45 nN offset and a 2.25 nN maximum. At 10 and 50 Hz, focal adhesion activation did not vary with spatial location, as observed for steady loading, whereas the response was minimized at 1.0 Hz. Furthermore, applying the tyrosine kinase inhibitors genistein and PP2, a specific Src family kinase inhibitor, showed tyrosine kinase signaling has a role in force-induced translocation. These results highlight the mutual importance of force transmission and biochemical signaling in focal adhesion mechanotransduction. mechanotransduction; endothelial cell; paxillin; viscoelastic model     

Multiple membrane tethers probed by atomic force microscopy

Using the atomic force microscope to locally probe the cell membrane, we observed the formation of multiple tethers (thin nanotubes, each requiring a similar pulling force) as reproducible features within force profiles recorded on individual cells. Forces obtained with Chinese hamster ovary cells, a malignant human brain tumor cell line, and human endothelial cells (EA hy926) were found to be 28 +/- 10 pN, 29 +/- 9 pN, and 29 +/- 10 pN, respectively, independent of the nature of attachment to the cantilever. The rather large variation of the tether pulling forces measured at several locations on individual cells points to the existence of heterogeneity in the membrane properties of a morphologically homogeneous cell. Measurement of the summary lengths of the simultaneously extracted tethers provides a measure of the size of the available membrane reservoir through which co-existing tethers are associated. As expected, partial disruption of the actin cytoskeleton and removal of the hyaluronan backbone of the glycocalyx were observed to result in a marked decrease (30-50%) in the magnitude and a significant sharpening of the force distribution indicating reduced heterogeneity of membrane properties. Taken together, our results demonstrate the ability of the plasma membrane to locally produce multiple interdependent tethers-a process that could play an important role in the mechanical association of cells with their environment.     

Pulling,failing, and adaptive mechanotransduction of macrophage filopodia

Macrophages use filopodia to withdraw particles toward the cell body for phagocytosis. This can require substantial forces, which the cell generates after bio-mechanical stimuli are transmitted to the filopodium. Adaptation mechanisms to mechanical stimuli are essential for cells, but can a cell iteratively improve filopodia pulling? If so, the underlying mechanic adaptation principles organized on the protein level are unclear. Here, we tackle this problem using optically trapped 1 μm beads, which we tracked interferometrically at 1 MHz during connection to the tips of dorsal filopodia of macrophages. We observe repetitive failures while the filopodium tries to pull the bead out of the optical trap. Analyses of mean bead motions and position fluctuations on the nano-meter and microsecond scale indicate mechanical ruptures caused by a force-dependent actin-membrane connection. We found that beads are retracted three times slower under any load between 5 and 40 pN relative to the no-load transport, which has the same speed as the actin retrograde flow obtained from fluorescent speckle tracking. From this duty ratio of pulling velocities, we estimated a continuous on/off binding with τ_off = 2?τ_on, with measured off times τ_off = 0.1–0.5 s. Remarkably, we see a gradual increase of filopodia pulling forces from 10 to 30 pN over time and after failures, which points toward an unknown adaptation mechanism. Additionally, we see that the attachment strength and friction between the bead and filopodium tip increases under load and over time. All observations are typical for catch-bond proteins such as integrin-talin complexes. We present a mechanistic picture of adaptive mechanotransduction, which formed by the help of mathematical models for repetitive tip ruptures and reconnections. The analytic mathematical model and the stochastic computer simulations, both based on catch-bond lifetimes, confirmed our measurements. Such catch-bond characteristics could also be important for other immune cells taking up counteracting pathogens.     

The fluid mechanics of bolus ejection from the oral cavity

The squeezing action of the tongue against the palate provides driving forces to propel swallowed material out of the mouth and through the pharynx. Transport in respose to these driving forces, however, is dependent on the material properties of the swallowed bolus. Given the complex geometry of the oral cavity and the unsteady nature of this process, the mechanics governing the oral phase of swallowing are not well understood. In the current work, the squeezing flow between two approaching parallel plates is used as a simplified mathematical model to study the fluid mechanics of bolus ejection from the oral cavity. Driving forces generated by the contraction of intrinsic and extrinsic lingual muscles are modeled as a spatially uniform pressure applied to the tongue. Approximating the tongue as a rigid body, the motion of tongue and fluid are then computed simultaneously as a function of time. Bolus ejection is parameterized by the time taken to clear half the bolus from the oral cavity, t_1/2. We find that t_1/2 increases with increased viscosity and density and decreases with increased applied pressure. In addition, for low viscosity boluses (μapproximately 1000 cP), viscosity dominates. A transition region between these two regimes is found in which both properties affect the solution characteristics. The relationship of these results to the assessment and treatment of swallowing disorders is discussed.     

Biofunctionalization of a generic collagenous triple helix with the α2β1 integrin binding site allows molecular force measurements

The integrin α2β1 plays an important role in force-transmitting cell-matrix interactions. It recognizes the peptide sequence GFOGER (O=4-hydroxy-proline) presented as trimer within a collagenous triple-helical framework. We produced the recombinant non-hydroxylated mini-collagen, termed FC3, which harbors the α2β1 integrin recognition site. FC3 consists of a foldon-stabilized host triple helix of three chains with 10 GPP-repeats, into which the integrin binding motif was inserted. The triple-helical structure could further be stabilized by covalently cross-linking the three chains. Unlike collagen-I, FC3 lacks binding sites for matrix proteins and cellular receptors other than the collagen-binding integrins. It showed a preference for α2β1 over α1β1 integrin, especially when the chains were neither cross-linked nor prolyl-hydroxylated. Using FC3 as substratum for primary skin fibroblasts, we showed that the loss of α2β1 integrin could not be compensated by other collagen-binding integrins, suggesting a major role of α2β1 integrin in exerting sufficient mechanical force to induce or sustain cell spreading. Atomic force microscopy revealed that a single α2β1 integrin can withstand tensile forces of up to approximately 160pN before it releases FC3. Moreover, FC3 is fully competent to agonistically elicit α2β1 integrin-induced cell reactions, such as recruitment of α2β1 integrin into focal adhesions and lamellipodia formation. The biofunctionalized mini-collagen sheds light on the molecular forces of the α2β1 integrin-collagen interaction, which affects tissue homeostasis by contracting the connective tissue and by contributing to interstitial tissue pressure regulation. Additionally, biofunctionalized mini-collagens can be useful in force-resistant cell attachment to biomedical materials.     

Changes in the phosphorylation and distribution of vinculin during nerve growth factor induced neurite outgrowth

The mechanism of neurite initiation and elongation was studied using nerve growth factor (NGF) treatment of PC12 cells. The distribution of focal adhesion sites and of the cytoskeletal protein vinculin was determined in large, fused, multinucleated PC12 cells. In the absence of NGF, focal adhesion sites as seen by interference reflection microscopy were restricted to the cell periphery in a regular distribution. Vinculin assemblies (foci), observed by indirect immunofluorescence microscopy using affinity purified anti-vinculin antibodies, were restricted to the cell periphery at focal adhesion sites. Within 4 hr after NGF treatment of the cells, the distribution of both vinculin and focal adhesion sites began to change. Focal adhesion sites became restricted to discrete protruding portions of the cell periphery. Larger, brighter vinculin foci appeared at the tips of the cell margin extensions, concomitant with the loss of foci at locations between the protrusions. As neurites elongated focal adhesion sites and vinculin foci remained with the tips of the growth cone extensions. Both focal adhesion sites and vinculin foci were rarely seen in the perikarya of cells with elongating neurites, and these were always confined to extended portions of the cell body margin. Occasionally, vinculin foci could be seen at the proximal portion of the neurite, at bending elbows, and at discrete expansions along the length. By immunoprecipitation of vinculin from 32P-labeled cells, vinculin phosphorylation was found to be increased within 1 hr of NGF treatment. The role of vinculin phosphorylation and assembly in the formation and directional elongation of neuritic processes in response to NGF is discussed.     

An engineering analysis of rotating sieves for hybridoma cell retention ins stirred tank bioreactors

The use of internal rotating sieves for perfused hybridoma culture offers unique advantages but has been up to now largely empirical. Calculations have been performed on a 15 l spinfilter stirred tank in order to have an idea of hydrodynamic conditions inside and outside the rotating sieve. The large peripheral velocity value, resulting from sieve rotation (compared to axial and radial velocities) is expected to affect strongly sieve surface colonization by cells; this is confirmed by lab scale experiments, showing that cell colonization is prevented providing sieve rotation exceeds a defined value (around 0.6 m.s.1 tip speed); the fluid removal force calculated under these conditions appears to be in the range of 10 pN, similar to the adhesion force already reported for mammalian cells attached to inorganic substrata.