Exert Traction Forces Research Articles

Chapter Six - Molecular Mechanisms Underlying the Force-Dependent Regulation of Actin-to-ECM Linkage at the Focal Adhesions

The linkage of the actin cytoskeleton to extracellular matrices (ECMs) at focal adhesions provides a physical path for cells to exert traction forces on substrates during cellular processes such as migration and morphogenesis. Mechanical strength of the actin-to-ECM linkage increases in response to forces loaded at this linkage. This is achieved by local accumulations of actin filaments, as well as linker proteins connecting actins to integrins, at force-bearing adhesion sites, which leads to an increase in the number of molecular bonds between the actin cytoskeleton- and ECM-bound integrins. Zyxin-dependent actin polymerization and filamin-mediated actin bundling are seemingly involved in the force-dependent actin accumulation. Each actin-integrin link is primarily mediated by the linker protein talin, which is strengthened by another linker protein vinculin connecting the actin filaments to talin in a force-dependent manner. This eliminates slippage between the actin cytoskeleton and talin (clutch mechanism), thus playing a crucial role in creating cell membrane protrusions mediated by actin polymerization. Finally, each integrin-ECM bond is also strengthened when a force is loaded on it, which ensures force transmission at focal adhesions, contributing to stable cell-substrate adhesion in cell migration.

Cortical and Cytoskeletal Structural Network regulates the Three-Dimensional Traction Forces Exerted by Migrating Amoeboid Cells

Cells exert traction forces, which are necessary to perform their migration. There is still a lack of information regarding the mechanical aspects of cell migration and in particular the quantification of the three-dimensional traction forces exerted during migration and invasion.We use a novel traction force cytometry method to measure the three-dimensional traction forces exerted by Dictyostelium cells chemotaxing over flat elastic substrates. Our measurements of the 3-D traction forces show that there are two independent mechanisms in their generation, one that relies on the cytoskeletal crosslinking and contractility, and another based on the cortical stiffness. In particular, we examine the roles that specific cortical and cytoskeletal crosslinking proteins and those liking the cell's cortex and the cytoskeletal F-actin play in the development of the spatial and temporal distribution of three-dimensional traction forces during migration. We investigate mutants with cortical integrity defects, such as cells lacking the protein cortexillin to study the cortical function. We use mutants with cytoskeletal crosslinking defects, such as cells lacking the proteins Myosin II and Abp120 to quantify the function of the cytoskeletal crosslinking. In addition we examine the proteins Myosin IA and B, regarding their role liking the cell's cortex and the cytoskeletal F-actin network.The effects on the magnitude and distribution of the tangential and perpendicular components of the traction forces to the substrate are shown to be different depending on the specific cytoskeletal and cortical mutations. Our 3-D traction measurements indicate that the vertical and horizontal traction forces are regulated by the cortical and cytoskeletal crosslinking. These two mechanisms are interconnected since the time evolution of the vertical and horizontal forces is well correlated.

Complex Matrix Remodeling and Durotaxis Can Emerge From Simple Rules for Cell-Matrix Interaction in Agent-Based Models

Using a top-down approach, an agent-based model was developed within NetLogo where cells and extracellular matrix (ECM) fibers were composed of multiple agents to create deformable structures capable of exerting, reacting to, and transmitting mechanical force. At the beginning of the simulation, long fibers were randomly distributed and cross linked. Throughout the simulation, imposed rules allowed cells to exert traction forces by extending pseudopodia, binding to fibers and pulling them towards the cell. Simulated cells remodeled the fibrous matrix to change both the density and alignment of fibers and migrated within the matrix in ways that are consistent with previous experimental work. For example, cells compacted the matrix in their pericellular regions much more than the average compaction experienced for the entire matrix (696% versus 21%). Between pairs of cells, the matrix density increased (by 92%) and the fibers became more aligned (anisotropy index increased from 0.45 to 0.68) in the direction parallel to a line connecting the two cells, consistent with the "lines of tension" observed in experiments by others. Cells migrated towards one another at an average rate of ∼0.5 cell diameters per 10,000 arbitrary units (AU); faster migration occurred in simulations where the fiber density in the intercellular area was greater. To explore the potential contribution of matrix stiffness gradients in the observed migration (i.e., durotaxis), the model was altered to contain a regular lattice of fibers possessing a stiffness gradient and just a single cell. In these simulations cells migrated preferentially in the direction of increasing stiffness at a rate of ∼2 cell diameter per 10,000 AU. This work demonstrates that matrix remodeling and durotaxis, both complex phenomena, might be emergent behaviors based on just a few rules that control how a cell can interact with a fibrous ECM.

Myosin IIA deficient cells migrate efficiently despite reduced traction forces at cell periphery

SummaryCell motility is a cornerstone of embryogenesis, tissue remodeling and repair, and cancer cell invasion. It is generally thought that migrating cells grab and exert traction force onto the extracellular matrix in order to pull the cell body forward. While previous studies have shown that myosin II deficient cells migrate efficiently, whether these cells exert traction forces during cell migration in the absence of the major contractile machinery is currently unknown. Using an array of micron-sized pillars as a force sensor and shRNA specific to each myosin II isoform (A and B), we analyzed how myosin IIA and IIB individually regulate cell migration and traction force generation. Myosin IIA and IIB localized preferentially to the leading edge where traction force was greatest, and the trailing edge, respectively. When individual myosin II isoforms were depleted by shRNA, myosin IIA deficient cells lost actin stress fibers and focal adhesions, whereas myosin IIB deficient cells maintained similar actin organization and focal adhesions as wild-type cells. Interestingly, myosin IIA deficient cells migrated faster than wild-type or myosin IIB deficient cells on both a rigid surface and a pillar array, yet myosin IIA deficient cells exerted significantly less traction force at the leading edge than wild-type or myosin IIB deficient cells. These results suggest that, in the absence of myosin IIA mediated force-generating machinery, cells move with minimal traction forces at the cell periphery, thus demonstrating the remarkable ability of cells to adapt and migrate.

Plectin-containing, centrally-localized focal adhesions exert traction forces in primary lung epithelial cells

Receptor clustering upon cell attachment to the substrate induces assembly of cytoplasmic protein complexes termed focal adhesions (FAs), which connect, albeit indirectly, the extracellular matrix to the cytoskeleton. A subset of cultured primary alveolar epithelial cells (AEC) display a unique pattern of vinculin/paxillin/talin-rich FAs in two concentric circles when cultured on glass and micropatterned substrates: one ring of FAs located at the cell periphery (pFAs), and another FA ring located centrally in the cell (cFAs). Unusually, cFAs associate with an aster-like actin array as well as keratin bundles. Moreover, cFAs show rapid paxillin turnover rates following fluorescence recovery after photobleaching and exert traction forces similar to those generated by FAs at the cell periphery. The plakin protein plectin localizes to cFAs and is normally absent from pFAs, whereas tensin, a marker of mature/fibrillar adhesions, is found in both cFAs and pFAs. In primary AEC in which plectin expression is depleted, cFAs are largely absent, with an attendant reorganization of both the keratin and actin cytoskeletons. We suggest that the mechanical environment in the lung gives rise to the assembly of unconventional FAs in AEC. These FAs not only show a distinctive arrangement, but also possess unique compositional and functional properties.

Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo?

During metastasis, cancer cells breach the basement membrane and migrate through the stroma mostly composed of a network of collagen I fibers. Cell migration on 2D is initiated by protrusion of the cell membrane followed by formation of adhesions that link the actin cytoskeleton to the extracellular matrix (ECM). Cells then move forwards by exerting traction forces on the adhesions at its front and by disassembling adhesions at the rear. In 2D, only the ventral surface of a migrating cell is in contact with the ECM, where cell–matrix adhesions are assembled. In 3D matrices, even though the whole surface of a migrating cell is available for interacting with the ECM, it is unclear whether discrete adhesion structures actually exist. Using high-resolution confocal microscopy we imaged the endogenous adhesome proteins in three different cancer cell types embedded in non-pepsinized collagen type I, polymerized at a slow rate, to allow the formation of a network that resembles the organization of EMC observed in vivo. Vinculin aggregates were detected in the cellular protrusions, frequently colocalizing with collagen fibers, implying they correspond to adhesion structures in 3D. As the distance from the substrate bottom increases, adhesion aggregates become smaller and almost undetectable in some cell lines. Using intravital imaging we show here, for the first time, the existence of adhesome proteins aggregates in vivo. These aggregates share similarities with the ones found in 3D collagen matrices. It still remains to be determined if adhesions assembled in 3D and in vivo share functional similarities to the well-described adhesions in 2D. This will provide a major step forward in understanding cell migration in more physiological environments.

AnIn VitroModel System to Quantify Stress Generation, Compaction, and Retraction in Engineered Heart Valve Tissue

Autologous heart valve tissue engineering relies on extracellular matrix production by cells seeded into a degrading scaffold material. The cells naturally exert traction forces to their surroundings, and due to an imbalance between scaffold, tissue, and these traction forces, stress is generated within the tissue. This stress results in compaction during culture and retraction of the leaflets at release of constraints, causing shape loss of the heart valve leaflets. In the present study, an in vitro model system has been developed to quantify stress generation, compaction, and retraction during culture and after release of constraints. Tissue-engineered (TE) constructs based on polyglycolic acid/poly-4-hydroxybutyrate scaffolds seeded with human vascular-derived cells were cultured for 4 weeks. Compaction in width was measured during culture, stress generation was measured during culture and after release of constraints at week 4, and contraction was measured after release of constraints at week 4. Both compaction and stress generation started after 2 weeks of culture and continued up to week 4. TE constructs compacted up to half of their original width and reached an internal stress of 6-8 kPa at week 4, which resulted in a retraction of 36%. The model system has provided a useful tool to unravel and optimize the balance between the different aspects of TE constructs to develop functional TE leaflets.

Spatiotemporal Constraints on the Force-Dependent Growth of Focal Adhesions

Focal adhesions (FAs) are the predominant mechanism by which cells mechanically couple to and exert traction forces on their extracellular matrix (ECM). It is widely presumed that FA size is modulated by force to mediate changes in adhesion strength at different levels of cellular tension. However, previous studies seeking correlations between force and FA morphology have yielded variable and often conflicting results. Here we show that a strong correlation between adhesion size and traction force exists only during the initial stages of myosin-mediated adhesion maturation and growth. For mature adhesions, no correlation between traction stress and size is observed. Rather, the tension that is sustained at mature adhesions is more strongly influenced by proximity to the cell edge, with peripheral adhesions transmitting higher tension than adhesions near the cell center. Finally, we show that mature adhesions can withstand sixfold increases in tension without changes in size. Thus, although a strong correlation between adhesion size and mechanical tension is observed during the initial stages of myosin-mediated adhesion maturation, no correlation is observed in mature, elongated adhesions. This work places spatiotemporal constraints on the force-dependent growth of adhesions and provides insight into the mechanical regulation of cell-ECM adhesion.

Physical forces during collective cell migration

It has been thought that sheets of cells move by traction forces exerted by the cells at the leading edge of the sheet. Using traction microscopy to create a map of physical forces, it is now shown that in fact it is cells many rows from the front that do most of the work. Fundamental biological processes including morphogenesis, tissue repair and tumour metastasis require collective cell motions1,2,3, and to drive these motions cells exert traction forces on their surroundings4. Current understanding emphasizes that these traction forces arise mainly in ‘leader cells’ at the front edge of the advancing cell sheet5,6,7,8,9. Our data are contrary to that assumption and show for the first time by direct measurement that traction forces driving collective cell migration arise predominately many cell rows behind the leading front edge and extend across enormous distances. Traction fluctuations are anomalous, moreover, exhibiting broad non-Gaussian distributions characterized by exponential tails10,11,12. Taken together, these unexpected findings demonstrate that although the leader cell may have a pivotal role in local cell guidance, physical forces that it generates are but a small part of a global tug-of-war involving cells well back from the leading edge.

A multi-modular tensegrity model of an actin stress fiber

Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on the extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model can also explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors and represent a new handle on multi-scale modeling of living materials.

Living microlens arrays

Both individual cells and sheets of cells exert traction forces on the substrate and these forces have been investigated using a wide range of methods. Here we compare the mechanical properties of fibroblasts and epithelial cells using a novel surface geometry. Living cells are added to a thin film of polystyrene [PS] attached to a substrate of crosslinked poly(dimethyl siloxane) [PDMS] microwells. The contractile nature of the cells attached to the surface and the compliance of the PDMS surface geometry allows the PS thin film to buckle, forming arrays of convex microlenses. The resulting curvature of the microlenses allows us to determine the applied strain of growing cell sheets. We report that a monolayer of epithelial cells exerts more stress on the substrate than fibroblasts and attribute this to the collective behavior of the epithelium. By subsequently adding different chemical triggers to the system, the contractile nature of the cells changes, thus modifying the focal length of the microlenses. Together, these findings demonstrate the importance of studying the mechanics of cell sheets and also introduce a new design paradigm for advanced materials, offering great promise for a range of applications.

EFFECT OF DIFFERENT TYPES OF EXTRACELLULAR MATRIX ON SMOOTH MUSCLE CELL TRACTION FORCES

Adherent cells are known to exert traction forces to adhere to their extracellular matrix at focal adhesions, where integrins interact with the extracellular matrix and mediate various cellular signals, possibly leading to modulations of a variety of physiological functions in cell proliferation, migration, etc. Integrins are obligate heterodimers containing two distinct chains, called the and subunits, very specific to the types of extracellular matrix such as fibronectin, vitronectin, collagen, etc. It is also known that change in cellular traction forces may modulate cell physiological functions. Therefore, it is speculated that extracellular matrixmediated integrin expression may modulate cellular traction forces, resulted in alterations in cell physiology. So far, a lot of efforts have been made to estimate cellular traction forces using micropillar substrates [Tan et al. 2003]. In this study, we performed traction force measurements using a micropillar substrate coated with different types of extracellular matrix including fibronectin, vitronectin, and type 1 collagen. Fluorescence observations of actin filament structures were also performed to study their relevance to magnitude and directions of traction forces.

Molecular Dynamics and Forces of a Motile Cell Simultaneously Visualized by TIRF and Force Microscopies

Cells must exert traction forces onto the substratum for continuous migration. Molecular dynamics such as actin polymerization at the front of the cell and myosin II accumulation at the rear should play important roles in the exertion of forces required for migration. Therefore, it is important to reveal the relationships between the traction forces and molecular dynamics. Traction forces can be calculated from the deformation of the elastic substratum under a migrating cell. A transparent and colorless elastic substratum with a high refractive index (1.40) and a low Young's modulus (1.0 kPa) were made from a pair of platinum-catalyzed silicones. We used this substratum to develop a new method for simultaneous recording of molecular dynamics and traction forces under a migrating cell in which total internal refractive fluorescence (TIRF) and force microscopies were combined. This new method allows the detection of the spatiotemporal distribution of traction forces produced by individual filopodia in migrating Dictyostelium cells, as well as simultaneous visualization of these traction forces and the dynamics of filamentous myosin II.

Viscoelastic Retraction of Single Living Stress Fibers and Its Impact on Cell Shape, Cytoskeletal Organization, and Extracellular Matrix Mechanics

Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale.

The Dynamics and Mechanics of Endothelial Cell Spreading

Cell adhesion to extracellular matrix is mediated by receptor-ligand interactions. When a cell first contacts a surface, it spreads, exerting traction forces against the surface and forming new bonds as its contact area expands. Here, we examined the changes in shape, actin polymerization, focal adhesion formation, and traction stress generation that accompany spreading of endothelial cells over a period of several hours. Bovine aortic endothelial cells were plated on polyacrylamide gels derivatized with a peptide containing the integrin binding sequence RGD, and changes in shape and traction force generation were measured. Notably, both the rate and extent of spreading increase with the density of substrate ligand. There are two prominent modes of spreading: at higher surface ligand densities cells tend to spread isotropically, whereas at lower densities of ligand the cells tend to spread anisotropically, by extending pseudopodia randomly distributed along the cell membrane. The extension of pseudopodia is followed by periods of growth in the cell body to interconnect these extensions. These cycles occur at very regular intervals and, furthermore, the extent of pseudopodial extension can be diminished by increasing the ligand density. Measurement of the traction forces exerted by the cell reveals that a cell is capable of exerting significant forces before either notable focal adhesion or stress fiber formation. Moreover, the total magnitude of force exerted by the cell is linearly related to the area of the cell during spreading. This study is the first to monitor the dynamic changes in the cell shape, spreading rate, and forces exerted during the early stages (first several hours) of endothelial cell adhesion.

A mechanochemical model of angiogenesis and vasculogenesis

Vasculogenesis and angiogenesis are two different mechanisms for blood vessel formation. Angiogenesis occurs when new vessels sprout from pre-existing vasculature in response to external chemical stimuli. Vasculogenesis occurs via the reorganization of randomly distributed cells into a blood vessel network. Experimental models of vasculogenesis have suggested that the cells exert traction forces onto the extracellular matrix and that these forces may play an important role in the network forming process. In order to study the role of the mechanical and chemical forces in both of these stages of blood vessel formation, we present a mathematical model which assumes that (i) cells exert traction forces onto the extracellular matrix, (ii) the matrix behaves as a linear viscoelastic material, (iii) the cells move along gradients of exogenously supplied chemical stimuli (chemotaxis) and (iv) these stimuli diffuse or are uptaken by the cells. We study the equations numerically, present an appropriate finite difference scheme and simulate the formation of vascular networks in a plane. Our results compare very well with experimental observations and suggest that spontaneous formation of networks can be explained via a purely mechanical interaction between cells and the extracellular matrix. We find that chemotaxis alone is not a sufficient force to stimulate formation of pattern. Moreover, during vessel sprouting, we find that mechanical forces can help in the formation of well defined vascular structures.

Keratocytes pull with similar forces on their dorsal and ventral surfaces.

As cells move forward, they pull rearward against extracellular matrices (ECMs), exerting traction forces. However, no rearward forces have been seen in the fish keratocyte. To address this discrepancy, we have measured the propulsive forces generated by the keratocyte lamella on both the ventral and the dorsal surfaces. On the ventral surface, a micromachined device revealed that traction forces were small and rearward directed under the lamella, changed direction in front of the nucleus, and became larger under the cell body. On the dorsal surface of the lamella, an optical gradient trap measured rearward forces generated against fibronectin-coated beads. The retrograde force exerted by the cell on the bead increased in the thickened region of the lamella where myosin condensation has been observed (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G. G. Borisy. 1997. J. Cell Biol. 139:397-415). Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts. The correlation between the level of traction force and the density of myosin suggests a model for keratocyte movement in which myosin condensation in the perinuclear region generates rearward forces in the lamella and forward forces in the cell rear.

Arginine Kinase Expression and Localization in Growth Cone Migration

Migrating neuronal growth cones exert traction forces that are generated by ATP-driven F-actin/myosin interactions. Sustained generation of these forces may require an energy supply mediated by the guanidino kinases, creatine kinase and arginine kinase. We cloned and sequenced grasshopper arginine kinase and examined its expression during embryogenesis and its subcellular localization in vivo and in vitro. During the first half of embryogenesis, arginine kinase is expressed selectively in a small percentage of ectodermal cells (dorsal closure cells), in a small percentage of mesodermal cells (muscle pioneers), and throughout the developing CNS. Most of these cell types are motile, including nascent neurons, muscle pioneers, dorsal closure cells, and many CNS glia. Neuroblasts also strongly express arginine kinase; they are nonmotile but are undergoing repeated rounds of (ATP-dependent) mitosis. Arginine kinase is colocalized with F-actin in a narrow band along the leading edges of lamellipodia of migrating glia. In neurons undergoing axonogenesis, arginine kinase is concentrated in growth cones and extends to the tips of filopodia. The amount of arginine kinase varies widely between growth cones, even between different growth cones of the same neuron. Energy for growth cone migration appears to be mobilized by (1) selective expression of arginine kinase by neurons, (2) localization of arginine kinase within growth cones, and (3) concentration of arginine kinase within specific growth cones, depending on the traction forces being generated. Mobilization of guanidino kinases may participate in the selective growth of specific growth cones.

Mechanistic Model of Wound Contraction

The interplay between cellular, biochemical, and biomechanical phenomena which result in wound contraction is examined with the aid of a mathematical model. The model accounts for fibroblasts repopulating the wound extracellular matrix (ECM) by a combination of migration and proliferation and, in the course of exerting traction forces associated with their motility, their deformation of the local ECM, which has a viscoelastic character. A base model, which assumes that cell and tissue properties are the same irrespective of position with respect to the wound center, is found to be inconsistent with wound contraction. Extensions of the base model which account for the possible influence of a concentration gradient of an inflammation-derived mediator on the traction, growth, or chemotactic properties of the fibroblasts do predict the qualitative features of a contracting wound. In particular, the case where traction is prescribed to increase as fibroblasts approach the wound center is shown to obey the commonly observed exponential rate contraction law and to be largely consistent with the findings of McGrath and Simon ( Plast. Reconstr. Surg. 72, 66, 1983) concerning the independence of the contraction rate constant and final extent of contraction from initial wound size and geometry.

Stereociliary bundles reorient during hair cell development and regeneration in the chick cochlea

We have examined changes in the orientation of Stereociliary bundles of hair cells in the cochlear sensory epithelium that occur during normal embryonic development and during the regeneration of hair cells that follows acoustic trauma. At the time when hair cell surfaces become recognizable in the embryonic cochlea, the bundles of stereocilia exhibit a range of orientations, as indicated by the position of the kinocilium and later, by the location of the tallest row of stereocilia. With time, the orientations of bundles on neighboring hair cells become more uniform, a condition that is maintained in the adult. Changes in stereocilia orientation are also observed during the regeneration of hair cells after acoustic trauma. When new hair cells first differentiate at sites of trauma in the recovering sensory epithelium, their Stereociliary bundles are not uniformly oriented. Then as the cells mature over a period of days, the bundles become aligned both with the neighboring bundles in the region of the previous lesion and with the pre-existing bundles that surround the site of regeneration. We conclude that the Stereociliary bundles of hair cells are reorienting as the cells differentiate. A common mechanism may guide reorientation both during embryonic development and during regeneration. Observations in living cochleae indicate that differentiating Stereociliary bundles establish asymmetric linkages to the extracellular matrix of the developing tectorial membrane. During the growth of the tectorial membrane, its progressive extension across the surface of the sensory epithelium may exert traction forces through those asymmetric linkages that pull the bundles of the hair cells into uniform alignment.