Physical cues from the extracellular environment that influence cell shape and directional migration are transduced into changes in cytoskeletal organization and biochemistry through integrin-based cell adhesions to extracellular matrix (ECM). Paxillin is a focal adhesion (FA) scaffold protein that mediates integrin anchorage to the cytoskeleton, and has been implicated in regulation of FA assembly and cell migration. To determine whether paxillin is involved in coupling mechanical distortion with directional movement, cell shape was physically constrained by culturing cells on square-shaped fibronectin-coated adhesive islands surrounded by non-adhesive barrier regions that were created with a microcontact printing technique. Square-shaped cells preferentially formed FAs and extended lamellipodia from their corner regions when stimulated with PDGF, and loss of paxillin resulted in loss of this polarized response. Selective expression of the N- and C-terminal domains of paxillin produced opposite, but complementary, effects on suppressing or promoting lamellipodia formation in different regions of square cells, which corresponded to directional motility defects in vitro. Paxillin loss or mutation was also shown to affect the formation of circular dorsal ruffles, and this corresponded to changes in cell invasive behavior in 3D. This commentary addresses the implications of these findings in terms of how a multifunctional FA scaffold protein can link physical cues to cell adhesion, protrusion and membrane trafficking so as to control directional migration in 2D and 3D. We also discuss how microengineered ECM islands and in vivo model systems can be used to further elucidate the functions of paxillin in directional migration.
paxillin; focal adhesion; lamellipodia; dorsal ruffles; migration; motility; microcontact printing; membrane trafficking; mechanotransduction
Polydimethylsiloxane (PDMS) has numerous desirable properties for fabricating microfluidic devices, including optical transparency, flexibility, biocompatibility, and fabrication by casting; however, partitioning of small hydrophobic molecules into the bulk of PDMS hinders industrial acceptance of PDMS microfluidic devices for chemical processing and drug development applications. Here we describe an attractive alternative material that is similar to PDMS in terms of optical transparency, flexibility and castability, but that is also resistant to absorption of small hydrophobic molecules.
In vitro 3D culture could provide an important model of tissues in vivo, but assessing the effects of chemical compounds on cells in specific regions of 3D culture requires physical isolation of cells, and thus currently relies mostly on delicate and low-throughput methods. This paper describes a technique (“cells-in-gels-in-paper” CiGiP) that permits rapid assembly of arrays of 3D cell cultures, and convenient isolation of cells from specific regions of these cultures. The 3D cultures were generated by stacking sheets of 200-μm-thick paper, each sheet supporting 96 individual “spots” (thin circular slabs) of hydrogels containing cells, separated by hydrophobic material (wax, PDMS) impermeable to aqueous solutions, and hydrophilic and most hydrophobic solutes. A custom-made 96-well holder isolated the cell-containing zones from each other. Each well contained media to which a different compound could be added. After culture, and disassembly of the holder, peeling the layers apart ‘sectioned’ the individual 3D cultures into 200-μm-thick sections which were easy to analyze using 2D imaging (e.g., with a commercial gel scanner). This 96-well holder brings new utilities to high-throughput, cell-based screening, by combining the simplicity of CiGiP with the convenience of a microtiter plate. This work demonstrated the potential of this type of assays by examining the cytotoxic effects of phenylarsine oxide (PAO) and cyclophosphamide (CPA) on human breast cancer cells positioned at different separations from culture media in 3D cultures.
Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. However, little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. Here we present a method to quantify cell-generated mechanical stresses that are exerted locally within living embryonic tissues using fluorescent, cell-sized, oil microdroplets with defined mechanical properties and coated with surface integrin or cadherin receptor ligands. After introducing a droplet between cells in a tissue, local stresses are determined from the droplet shape deformations, which are obtained via fluorescence microscopy and computerized image analysis. Using this method, we quantify the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates and confirm that these stresses (3.4 nN/µm2) are dependent on myosin II activity and more than two-fold larger than the stresses generated by cells of embryonic tooth mesenchyme when analyzed within similar cultured aggregates or in developing whole mouse mandibles.
This article is based on a lecture I presented as the recipient of the 2009 Pritzker Distinguished Lecturer Award at the Biomedical Engineering Society annual meeting in October 2009. Here, I review more than thirty years of research from my laboratory, beginning with studies designed to test the theory that cells use tensegrity (tensional integrity) architecture to stabilize their shape and sense mechanical signals, which I believed to be critical for control of cell function and tissue development. Although I was trained as a cell biologist, I found that the tools I had at my disposal were insufficient to experimentally test these theories, and thus I ventured into engineering to find critical solutions. This path has been extremely fruitful as it has led to confirmation of the critical role that physical forces play in developmental control, as well as how cells sense and respond to mechanical signals at the molecular level through a process known as cellular mechanotransduction. Many of the predictions of the cellular tensegrity model relating to cell mechanical behaviors have been shown to be valid, and this vision of cell structure led to discovery of the central role that transmembrane adhesion receptors, such as integrins, and the cytoskeleton play in mechanosensing and mechanochemical conversion. In addition, these fundamental studies have led to significant unexpected technology fallout, including development of micromagnetic actuators for non-invasive control of cellular signaling, microfluidic systems as therapeutic extracorporeal devices for sepsis therapy, and new DNA-based nanobiotechnology approaches that permit construction of artificial tensegrities that mimic properties of living materials for applications in tissue engineering and regenerative medicine.
Mechanotransduction; Tensegrity; Cell mechanics; Prestress; Cytoskeleton; Integrin; Biomimetic
Mechanical stresses that are preferentially transmitted across the cell surface via transmembrane integrin receptors activate gene transcription by triggering production of intracellular chemical second messengers, such as cAMP. Here we show that the sensitivity of the cAMP signaling pathway to mechanical stresses transferred across β1 integrins is mediated by force-dependent activation of the heterotrimeric G protein subunit Gαs within focal adhesions at the site of stress application. Gαs is recruited to focal adhesions that form within minutes following clustering of β1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and β1 integrin and Gαs co-precipitate when analyzed biochemically. Stress application to activated β1 integrins using magnetic twisting cytometry increases Gαs recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azido-anilido-GTP, whereas application of similar stresses to inactivated, integrins or control histocompatibility antigens has little effect. This response is relevant physiologically as application of mechanical strain to cells bound to flexible extracellular matrix-coated substrates induce translocation of phospho-CREB to the nucleus, which can be attenuated by inhibiting Gαs activity, either using the inhibitor melittin or suppressing its expression using siRNA. Although integrins are not typical G protein-coupled receptors, these results show that integrins focus mechanical stresses locally on heterotrimeric G proteins within focal adhesions at the site of force application, and transduce mechanical stimuli into an intracellular cAMP signaling response by activating Gαs at these membrane signaling sites.
mechanotransduction; G protein; integrin; focal adhesion; shear stress; mechanical strain; magnetic
Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels, and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response, however, the molecular basis remains unknown. Here we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 ion channels that, in turn, stimulate phosphatidyl inositol-3-kinase-dependent activation and binding of additional ·1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knockdown of TRPV4 channels using specific siRNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent ‘integrin to integrin’ signaling mechanism mediated by force-induced activation of mechanically-gated TRPV4 ion channels on the cell surface.
mechanical strain; integrin; TRPV4; endothelial cell; reorientation; cytoskeleton
To advance cancer research in a transformative way, we must redefine the problem. Although epithelial cancers, such as breast cancer, may be caused by random somatic gene mutations, the reality is that this is only one of many ways to induce tumor formation. Cancers also can be produced in experimental systems in vitro and in vivo, for example, by inducing sustained alterations of extracellular matrix (ECM) structure. Moreover, certain epithelial cancers can be induced to ‘reboot’ and regenerate normal tissue morphology when combined with embryonic mesenchyme or exogenous ECM scaffolds that are produced through epithelial-stromal interactions. At the same time, work in the field of Mechanical Biology has revealed that many cell behaviors critical for cancer formation (e.g., growth, differentiation, motility, apoptosis) can be controlled by physical interactions between cells and their ECM adhesions that alter the mechanical force balance in the ECM, cell and cytoskeleton. Epithelial tumor progression also can be induced in vitro by changing ECM mechanics or altering cytoskeletal tension generation through manipulation of the Rho GTPase signaling pathway. Mechanical interactions between capillary cells and ECM that are mediated by Rho signaling similarly mediate control of capillary cell growth and angiogenesis, which are equally critical for cancer progression and metastasis. These findings question basic assumptions in the cancer field, and raise the intriguing possibility that cancer may be a reversible disease that results from progressive deregulation of tissue architecture, which leads to physical changes in cells and altered mechanical signaling. This perspective raises the possibility of developing a tissue engineering approach to cancer therapy in which biologically-inspired materials that mimic the embryonic microenvironment are used to induce cancers to revert into normal tissues.
mechanical; extracellular matrix; stroma; cell traction; cytoskeleton; cancer therapy
Development of three dimensional (3D) microenvironments that direct stem cell differentiation into functional cell types remains a major challenge in the field of regenerative medicine. Here, we describe a new platform to address this challenge by utilizing a robotic microarray spotter for testing stem cell fates inside various miniaturized cell-laden gels in a systematic manner. To demonstrate the feasibility of our platform, we evaluated the osteogenic differentiation of human mesenchymal stem cells (hMSCs) within combinatorial 3D niches. We were able to identify specific combinations, that enhanced the expression of osteogenic markers. Notably, these ‘hit' combinations directed hMSCs to form mineralized tissue when conditions were translated to 3D macroscale hydrogels, indicating that the miniaturization of the experimental system did not alter stem cell fate. Overall, our findings confirmed that the 3D cell-laden gel microarray can be used for screening of different conditions in a rapid, cost-effective, and multiplexed manner for a broad range of tissue engineering applications.
Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP, controls capillary network formation in vitro and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors – TFII-I and GATA2 – that govern gene expression of the VEGF receptor, VEGFR2. Moreover, this novel angiogenesis signaling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.
VEGFR2; p190RhoGAP; TFII-I; GATA2; mechanotransduction; angiogenesis; capillary endothelial cell
Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on 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 also can 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.
Cell mechanics; cell structure; computer model; contractility; cytoskeleton; tensegrity
Anyone who is skilled in the art of physical therapy knows that the mechanical properties, behavior and movement of our bodies are as important for human health as chemicals and genes. However, only recently have scientists and physicians begun to appreciate the key role that mechanical forces play in biological control at the molecular and cellular levels. This article provides a brief overview of a lecture presented at the 1st International Fascia Research Congress that convened at Harvard Medical School in Boston, MA on October 4, 2007. (see figure 1) In this lecture, I described what we have learned over the past thirty years as a result of our research focused on the molecular mechanisms by which cells sense mechanical forces and convert them into changes in intracellular biochemistry and gene expression – a process called “mechanotransduction”. This work has revealed that molecules, cells, tissues, organs, and our entire bodies use “tensegrity” architecture to mechanically stabilize their shape, and to seamlessly integrate structure and function at all size scales. Through use of this tension-dependent building system, mechanical forces applied at the macroscale produce changes in biochemistry and gene expression within individual living cells. This structure-based system provides a mechanistic basis to explain how application of physical therapies might influence cell and tissue physiology.
tensegrity; mechanotransduction; cytoskeleton; integrins; cell tension; physical therapy
This article is a summary of a lecture on cellular mechanotransduction that was presented at a symposium on “Cardiac Mechano-Electric Feedback and Arrhythmias” that convened at Oxford, England in April 2007. Although critical mechanosensitive molecules and cellular components, such as integrins, stretch-activated ion channels, and cytoskeletal filaments, have been shown to contribute to the response by which cells convert mechanical signals into a biochemical response, little is known about how they function in the structural context of living cells, tissues and organs to produce orchestrated changes in cell behavior in response to stress. Here, studies are reviewed that suggest our bodies use structural hierarchies (systems within systems) composed of interconnected extracellular matrix and cytoskeletal networks that span from the macroscale to the nanoscale to focus stresses on specific mechanotransducer molecules. A key feature of these networks is that they are in a state of isometric tension (i.e., experience a tensile prestress), which ensures that various molecular-scale mechanochemical transduction mechanisms proceed simultaneously and produce a concerted response. These features of living architecture are the same principles that govern tensegrity (tensional integrity) architecture, and mathematical models based on tensegrity are beginning to provide new and useful descriptions of living materials, including mammalian cells. This article reviews how the use of tensegrity at multiple size scales in our bodies guides mechanical force transfer from the macro to the micro, as well as how it facilitates conversion of mechanical signals into changes in ion flux, molecular binding kinetics, signal transduction, gene transcription, cell fate switching and developmental patterning.
A mini-microscope was developed for in situ monitoring of cells by modifying off-the-shelf components of a commercial webcam. The mini-microscope consists of a CMOS imaging module, a small plastic lens and a white LED illumination source. The CMOS imaging module was connected to a laptop computer through a USB port for image acquisition and analysis. Due to its compact size, 8 × 10 × 9 cm, the present microscope is portable and can easily fit inside a conventional incubator, and enables real-time monitoring of cellular behaviour. Moreover, the mini-microscope can be used for imaging cells in conventional cell culture flasks, such as Petri dishes and multi-well plates. To demonstrate the operation of the mini-microscope, we monitored the cellular migration of mouse 3T3 fibroblasts in a scratch assay in medium containing three different concentrations of fetal bovine serum (5, 10, and 20%) and demonstrated differential responses depending on serum levels. In addition, we seeded embryonic stem cells inside poly(ethylene glycol) microwells and monitored the formation of stem cell aggregates in real time using the mini-microscope. Furthermore, we also combined a lab-on-a-chip microfluidic device for microdroplet generation and analysis with the mini-microscope and observed the formation of droplets under different flow conditions. Given its cost effectiveness, robust imaging and portability, the presented platform may be useful for a range of applications for real-time cellular imaging using lab-on-a-chip devices at low cost.
Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.
Developmental Biology; Issue 80; Mammary Glands; Animal; Drug Administration Routes; intraductal injection; local drug delivery; siRNA
Changes in extracellular matrix (ECM) structure or mechanics can actively drive cancer progression; however, the underlying mechanism remains unknown. Here we explore whether this process could be mediated by changes in cell shape that lead to increases in genetic noise, given that both factors have been independently shown to alter gene expression and induce cell fate switching. We do this using a computer simulation model that explores the impact of physical changes in the tissue microenvironment under conditions in which physical deformation of cells increases gene expression variability among genetically identical cells. The model reveals that cancerous tissue growth can be driven by physical changes in the microenvironment: when increases in cell shape variability due to growth-dependent increases in cell packing density enhance gene expression variation, heterogeneous autonomous growth and further structural disorganization can result, thereby driving cancer progression via positive feedback. The model parameters that led to this prediction are consistent with experimental measurements of mammary tissues that spontaneously undergo cancer progression in transgenic C3(1)-SV40Tag female mice, which exhibit enhanced stiffness of mammary ducts, as well as progressive increases in variability of cell-cell relations and associated cell shape changes. These results demonstrate the potential for physical changes in the tissue microenvironment (e.g., altered ECM mechanics) to induce a cancerous phenotype or accelerate cancer progression in a clonal population through local changes in cell geometry and increased phenotypic variability, even in the absence of gene mutation.
Here we report a proof-of-concept for development of pancreatic islet-targeting nanoparticles for immunomodulatory therapy of autoimmune Type 1 Diabetes. Modified with a unique islet-homing peptide, these polymeric nanomaterials exhibit 3-fold greater binding to islet endothelial cells and a 200-fold greater anti-inflammatory effect through targeted islet endothelial cell delivery of an immunosuppressant drug. Our findings also underscore the need to carefully tailor drug loading and nanoparticle dosage to achieve maximal vascular targeting and immunosuppression.
Diabetes; pancreatic islet; nanoparticle; drug delivery; vascular endothelium; inflammation; tissue targeting
This paper focuses on the development of magnetic cellular switches to enable magnetic control of intracellular functions in living mammalian cells, including receptor signal transduction and gene transcription. Our approach takes advantage of the mechanosensitivity of adenosine 3′,5′-monophosphate (cAMP) induction and downstream transcription controlled by the cAMP regulatory element (CRE) to engineer gene constructs that optically report gene expression in living cells. We activate transcription of these gene reporters by applying magnetic (mechanical) stress to magnetic microbeads bound to cell surface integrin receptors. In these gene reporter constructs, CRE motifs drive the expression of fluorescent proteins or enzymes that produce fluorescent products, such as DsRed and β-lactamase (BLA), respectively. We demonstrate that a chemical inducer of cAMP (forskolin) increases expression of CRE-DsRed in living cells. More importantly, a threefold increase in CRE-BLA expression is induced by application of mechanical stress to magnetic microbeads (4.5 µm) bound to cell surface integrin receptors. Induction of cAMP could be detected within 5 min using a protein fragment complementation assay involving interactions between the KID and KIX domains of the CRE binding protein linked to complementary halves of the BLA enzyme. These studies confirm that application of magnetic stress to integrins induces gene transcription by activating the cAMP-dependent transcription factor CREB. Ongoing studies focus on optimizing sensitivity and reducing signal-to-noise by establishing stable cell lines that express these gene reporters. These studies collectively demonstrate the feasibility of using magnetic technologies to control function in living mammalian cells and, hence, support the possibility of developing magnetically-actuated cellular components for use in future micro- and nanotechnologies.
Biological cells; biological control systems; biological signal transduction; biomagnetics
Mesenchymal condensation is critical for organogenesis, yet little is known about how this process is controlled. Here we show that Fgf8 and Sema3f produced by early dental epithelium respectively attract and repulse mesenchymal cells, which causes them to pack tightly together during mouse tooth development. Resulting mechanical compaction-induced changes in cell shape induce odontogenic transcription factors (Pax9, Msx1) and chemical cue (BMP4), and mechanical compression of mesenchyme is sufficient to induce tooth-specific cell fate switching. The inductive effects of cell compaction are mediated by suppression of the mechanical signaling molecule RhoA, and its over-expression prevents odontogenic induction. Thus, the mesenchymal condensation that drives tooth formation is induced by antagonistic epithelial morphogens that manifest their pattern-generating actions mechanically via changes in mesenchymal cell shape and altered mechanotransduction.
Mesenchymal condensation; organogenesis; epithelial-mesenchymal interactions; mechanical forces; cell shape; Fgf8; Sema3f; Nrp2; Pax9; RhoA; tooth
Angiogenesis is crucial for lung development. Although there has been considerable exploration, the mechanism by which lung vascular and alveolar formation is controlled is still not completely understood. Here we show that low-density lipoprotein receptor-related protein 5 (LRP5), a component of the Wnt ligand-receptor complex, regulates angiogenesis and alveolar formation in the lung by modulating expression of the angiopoietin (Ang) receptor, Tie2, in vascular endothelial cells (ECs). Vascular development in whole mouse lungs and in cultured ECs is controlled by LRP5 signaling, which is, in turn, governed by a balance between the activities of the antagonistic Tie2 ligands, Ang1 and Ang2. Under physiological conditions when Ang1 is dominant, LRP5 knockdown decreases Tie2 expression and thereby, inhibits vascular and alveolar development in the lung. Conversely, when Ang2 dominates under hyperoxia treatment in neonatal mice, high LRP5 and Tie2 expression suppress angiogenesis and lung development. These findings suggest that the LRP5-Tie2-Ang signaling axis plays a central role in control of both angiogenesis and alveolarization during postnatal lung development, and that deregulation of this signaling mechanism might lead to developmental abnormalities of the lung, such as are observed in bronchopulmonary dysplasia (BPD).
Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.
Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5–10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax−/− and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax−/− cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax−/− and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.
Integrins are ubiquitous transmembrane mechanoreceptors that elicit changes in intracellular biochemistry in response to mechanical force application, but these alterations generally proceed over seconds to minutes. Stress-sensitive ion channels represent another class of mechanoreceptors that are activated much more rapidly (within msec), and recent findings suggest that calcium influx through Transient Receptor Potential Vanilloid-4 (TRPV4) channels expressed in the plasma membrane of bovine capillary endothelial cells is required for mechanical strain-induced changes in focal adhesion assembly, cell orientation and directional migration. However, whether mechanically stretching a cell’s extracellular matrix (ECM) adhesions might directly activate cell surface ion channels remains unknown. Here we show that forces applied to β1 integrins result in ultra-rapid (within 4 msec) activation of calcium influx through TRPV4 channels. The TRPV4 channels were specifically activated by mechanical strain in the cytoskeletal backbone of the focal adhesion, and not by deformation of the lipid bilayer or submembranous cortical cytoskeleton alone. This early-immediate calcium signaling response required the distal region of the β1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and external force application to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response. Local direct strain-dependent activation of TRPV4 channels mediated by force transfer from integrins and CD98 may therefore enable compartmentalization of calcium signaling within focal adhesions that is critical for mechanical control of many cell behaviors that underlie cell and tissue development.
In vitro 3D culture is an important model for tissues in
vivo. Cells in different locations of 3D tissues are
physiologically different, because they are exposed to different concentrations
of oxygen, nutrients, and signaling molecules, and to other environmental
factors (temperature, mechanical stress, etc). The majority of high-throughput
assays based on 3D cultures, however, can only detect the
average behavior of cells in the whole 3D construct.
Isolation of cells from specific regions of 3D cultures is possible, but relies
on low-throughput techniques such as tissue sectioning and micromanipulation.
Based on a procedure reported previously (“cells-in-gels-in-paper”
or CiGiP), this paper describes a simple method for culture of arrays of thin
planar sections of tissues, either alone or stacked to create more complex 3D
tissue structures. This procedure starts with sheets of paper patterned with
hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells
suspended in extracellular matrix (ECM) gel onto the patterned paper creates an
array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose
fibers) containing cells. Stacking the sheets with zones aligned on top of one
another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D
culture, by peeling apart the sheets of paper, “sections” all 96
cultures at once. It is, thus, simple to isolate 200-micron-thick
cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D
cultures are assembled from multiple layers, the number of cells plated
initially in each layer determines the spatial distribution of cells in the
stacked 3D cultures. This capability made it possible to compare the growth of
3D tumor models of different spatial composition, and to examine the migration
of cells in these structures.
The actin cross-linking protein filamin A reduces migration, invasion, and metastasis of breast cancer cells.
The actin cross-linking protein filamin A (FLNa) functions as a scaffolding protein and couples cell cytoskeleton to extracellular matrix and integrin receptor signaling. In this study, we report that FLNa suppresses invasion of breast cancer cells and regulates focal adhesion (FA) turnover. Two large progression tissue microarrays from breast cancer patients revealed a significant decrease of FLNa levels in tissues from invasive breast cancer compared with benign disease and in lymph node–positive compared with lymph node–negative breast cancer. In breast cancer cells and orthotopic mouse breast cancer models, down-regulation of FLNa stimulated cancer cell migration, invasion, and metastasis formation. Time-lapse microscopy and biochemical assays after FLNa silencing and rescue with wild-type or mutant protein resistant to calpain cleavage revealed that FLNa regulates FA disassembly at the leading edge of motile cells. Moreover, FLNa down-regulation enhanced calpain activity through the mitogen-activated protein kinase–extracellular signal-regulated kinase cascade and stimulated the cleavage of FA proteins. These results document a regulation of FA dynamics by FLNa in breast cancer cells.