Cochlear outer hair cell horizontal top connectors mediate mature stereocilia bundle mechanics.

Outer hair cell (OHC) stereocilia bundle deflection opens mechanoelectrical transduction channels at the tips of the stereocilia from the middle and short rows, while bundle cohesion is maintained owing to the presence of horizontal top connectors. Here, we used a quantitative noncontact atomic force microscopy method to investigate stereocilia bundle stiffness and damping, when stimulated at acoustic frequencies and nanometer distances from the bundle. Stereocilia bundle mechanics were determined in stereocilin-deficient mice lacking top connectors and with detached tectorial membrane ( / double knockout) and heterozygous littermate controls ( / ).

c-Src activity is differentially required by cancer cell motility modes.

Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal to ameboid transition, whereas a highly confining space will trigger a switch to "leader bleb-based" migration. To identify oncogenic signaling pathways mediating these transitions, we undertook a targeted screen using clinically useful inhibitors.

MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines.

Apical surface supracellular mechanical properties in polarized epithelium using noninvasive acoustic force spectroscopy.

Maintenance of epithelial tissue integrity requires coordination between cell-cell adherens junctions, tight junctions (TJ), and the perijunctional actomyosin cytoskeleton. Here we addressed the hypothesis that alterations in TJ structure and remodeling of the actomyosin cytoskeleton modify epithelial mechanics. Current methods to measure supracellular mechanical properties disrupt intact monolayers, therefore, we developed a novel method using noncontact acoustic frequency-modulation atomic force microscopy (FM-AFM) and tested it on MDCK polarized monolayers.

Actomyosin Cortical Mechanical Properties in Nonadherent Cells Determined by Atomic Force Microscopy.

The organization of filamentous actin and myosin II molecular motor contractility is known to modify the mechanical properties of the cell cortical actomyosin cytoskeleton. Here we describe a novel method, to our knowledge, for using force spectroscopy approach curves with tipless cantilevers to determine the actomyosin cortical tension, elastic modulus, and intracellular pressure of nonadherent cells. We validated the method by measuring the surface tension of water in oil microdrops deposited on a glass surface.

Relationship between cell stiffness and stress fiber amount, assessed by simultaneous atomic force microscopy and live-cell fluorescence imaging.

Actomyosin stress fibers, one of the main components of the cell's cytoskeleton, provide mechanical stability to adherent cells by applying and transmitting tensile forces onto the extracellular matrix (ECM) at the sites of cell-ECM adhesion. While it is widely accepted that changes in spatial and temporal distribution of stress fibers affect the cell's mechanical properties, there is no quantitative knowledge on how stress fiber amount and organization directly modulate cell stiffness. We address this key open question by combining atomic force microscopy with simultaneous fluorescence imaging of living cells, and combine for the first time reliable quantitative parameters obtained from both techniques.

Cytosolic pressure provides a propulsive force comparable to actin polymerization during lamellipod protrusion.

Does cytosolic pressure facilitate f-actin polymerization to push the leading edge of a cell forward during self-propelled motion? AFM force-distance (f-d) curves obtained from lamellipodia of live cells often exhibit a signal from which the tension, bending modulus, elastic modulus and thickness in the membrane-cortex complex can be estimated close to the contact point. These measurements permit an estimate of the cytosolic pressure via the canonical Laplace force balance. The deeper portion of the f-d curve allows estimation of the bulk modulus of the cytoskeleton after removal of the bottom effect artifact.

Erk regulation of actin capping and bundling by Eps8 promotes cortex tension and leader bleb-based migration.

Within the confines of tissues, cancer cells can use blebs to migrate. Eps8 is an actin bundling and capping protein whose capping activity is inhibited by Erk, a key MAP kinase that is activated by oncogenic signaling. We tested the hypothesis that Eps8 acts as an Erk effector to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells.

Phase of shear vibrations within cochlear partition leads to activation of the cochlear amplifier.

Since Georg von Bekesy laid out the place theory of the hearing, researchers have been working to understand the remarkable properties of mammalian hearing. Because access to the cochlea is restricted in live animals, and important aspects of hearing are destroyed in dead ones, models play a key role in interpreting local measurements. Wentzel-Kramers-Brillouin (WKB) models are attractive because they are analytically tractable, appropriate to the oblong geometry of the cochlea, and can predict wave behavior over a large span of the cochlea.

Stimulated acoustic emissions from coupled strings.

We consider traveling transverse waves on two identical uniform taut strings that are elastically coupled through springs that gradually decrease their stiffness over a region of finite length. The wave system can be decomposed into two modes: an in-phase mode ([Formula: see text]) that is transparent to the coupling springs, and an out-of-phase mode ([Formula: see text]) that engages the coupling springs and can resonate at a particular location depending on the excitation frequency. The system exhibits linear mode conversion whereby an incoming ([Formula: see text]) wave is reflected back from the resonance location both as a propagating ([Formula: see text]) wave and an evanescent ([Formula: see text]) wave, while both types emerge as propagating forward through the resonance location.

Thyroid hormone increases fibroblast growth factor receptor expression and disrupts cell mechanics in the developing organ of corti.

Background: Thyroid hormones regulate growth and development. However, the molecular mechanisms by which thyroid hormone regulates cell structural development are not fully understood. The mammalian cochlea is an intriguing system to examine these mechanisms, as cellular structure plays a key role in tissue development, and thyroid hormone is required for the maturation of the cochlea in the first postnatal week.

Fibroblast growth factor receptor 3 regulates microtubule formation and cell surface mechanical properties in the developing organ of Corti.

Fibroblast Growth Factor (Fgf) signaling is involved in the exquisite cellular patterning of the developing cochlea, and is necessary for proper hearing function. Our previous data indicate that Fgf signaling disrupts actin, which impacts the surface stiffness of sensory outer hair cells (OHCs) and non-sensory supporting pillar cells (PCs) in the organ of Corti. Here, we used Atomic Force Microscopy (AFM) to measure the impact of loss of function of Fgf-receptor 3, on cytoskeletal formation and cell surface mechanical properties.

Determination of the elastic moduli of thin samples and adherent cells using conical atomic force microscope tips.

The atomic force microscope can detect the mechanical fingerprints of normal and diseased cells at the single-cell level under physiological conditions. However, atomic force microscopy studies of cell mechanics are limited by the 'bottom effect' artefact that arises from the stiff substrates used to culture cells. Because cells adhered to substrates are very thin, this artefact makes cells appear stiffer than they really are.

Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea.

Correct patterning of the inner ear sensory epithelium is essential for the conversion of sound waves into auditory stimuli. Although much is known about the impact of the developing cytoskeleton on cellular growth and cell shape, considerably less is known about the role of cytoskeletal structures on cell surface mechanical properties. In this study, atomic force microscopy (AFM) was combined with fluorescence imaging to show that developing inner ear hair cells and supporting cells have different cell surface mechanical properties with different developmental time courses.

Nonpolarized signaling reveals two distinct modes of 3D cell migration.

We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized.

Dual traveling waves in an inner ear model with two degrees of freedom.

We calculate traveling waves in the mammalian cochlea, which transduces acoustic vibrations into neural signals. We use a WKB-based mechanical model with both the tectorial membrane (TM) and basilar membrane (BM) coupled to the fluid to calculate motions along the length of the cochlea. This approach generates two wave numbers that manifest as traveling waves with different modes of motion between the BM and TM.

Auditory mechanics of the tectorial membrane and the cochlear spiral.

Purpose Of Review: This review is timely and relevant because new experimental and theoretical findings suggest that cochlear mechanics from the nanoscale to the macroscale are affected by the mechanical properties of the tectorial membrane and the cochlea's spiral shape.

Recent Findings: Main tectorial membrane themes addressed in this review are composition and morphology, nanoscale mechanical interactions with the outer hair cell bundle, macroscale longitudinal coupling, fluid interaction with inner hair cell bundles, and macroscale dynamics and waves. Main cochlear spiral themes are macroscale, low-frequency energy focusing and microscale organ of Corti shear gain.

Simulation of the response of the inner hair cell stereocilia bundle to an acoustical stimulus.

Mammalian hearing relies on a cochlear hydrodynamic sensor embodied in the inner hair cell stereocilia bundle. It is presumed that acoustical stimuli induce a fluid shear-driven motion between the tectorial membrane and the reticular lamina to deflect the bundle. It is hypothesized that ion channels are opened by molecular gates that sense tension in tip-links, which connect adjacent stepped rows of stereocilia.

Noncontact microrheology at acoustic frequencies using frequency-modulated atomic force microscopy.

We report an atomic force microscopy (AFM) method for assessing elastic and viscous properties of soft samples at acoustic frequencies under non-contact conditions. The method can be used to measure material properties via frequency modulation and is based on hydrodynamics theory of thin gaps we developed here. A cantilever with an attached microsphere is forced to oscillate tens of nanometers above a sample.

Collagen-based mechanical anisotropy of the tectorial membrane: implications for inter-row coupling of outer hair cell bundles.

Background: The tectorial membrane (TM) in the mammalian cochlea displays anisotropy, where mechanical or structural properties differ along varying directions. The anisotropy arises from the presence of collagen fibrils organized in fibers of approximately 1 microm diameter that run radially across the TM. Mechanical coupling between the TM and the sensory epithelia is required for normal hearing.

High-Frequency Oscillations of a Sphere in a Viscous Fluid near a Rigid Plane.

High-frequency oscillations of a rigid sphere in an incompressible viscous fluid moving normal to a rigid plane are considered when the ratio of minimum clearance to sphere radius is small. Asymptotic expansions are constructed that permit an analytical estimate of the force acting on the sphere as a result of its motion. An inner expansion, valid in the neighborhood of the minimum gap, reflects the dominance of viscous effects and fluid inertia.

The influence of cochlear shape on low-frequency hearing.

The conventional theory about the snail shell shape of the mammalian cochlea is that it evolved essentially and perhaps solely to conserve space inside the skull. Recently, a theory proposed that the spiral's graded curvature enhances the cochlea's mechanical response to low frequencies. This article provides a multispecies analysis of cochlear shape to test this theory and demonstrates that the ratio of the radii of curvature from the outermost and innermost turns of the cochlear spiral is a significant cochlear feature that correlates strongly with low-frequency hearing limits.

Evidence and implications of inhomogeneity in tectorial membrane elasticity.

The motion of the tectorial membrane (TM) with respect to the reticular lamina subserves auditory function by bending the outer hair cell bundles and inducing fluid flows that shear the inner hair bundles in response to sound energy. Little is currently known about its intrinsic elasticity or about the relation between the mechanical properties and function of the membrane. Here we subdivide the TM into three longitudinal regions and five radial zones and map the shear modulus of the TM using atomic force microscopy, and present evidence that the TM elasticity varies radially, after the distribution of type A collagen fibrils.

Evidence of tectorial membrane radial motion in a propagating mode of a complex cochlear model.

Knowledge of vibratory patterns in the cochlea is crucial to understanding the stimulation of mechanosensory cells. Experiments to determine the motion of the cochlear partition and surrounding fluid are extremely challenging. As a result, the motion data are incomplete and often contradictory.