Focal Contacts Research Articles

Cell Adhesion on Cathodic Arc Plasma Deposited ZrAlSiN Thin Films

Interaction between ZrAlSiN thin films and osteoblasts cells was conducted in vitro. Ti and ZrAlSiN films were deposited on glass coverslips by cathodic arc deposition. Ti, ZrAlSiN thin films and the bare glass coverslips were cultured with human osteoblast cells (hFOB). Cell morphology was observed with a confocal microscope in the bright field mode. Cell viability was assayed by MTT. Cell proliferation was fluorescent visualized at 10 X magnification. Cell cytoskeletons were analyzed by observing actin stress fiber organization. Focal contact adhesion was studied by investigation of the density of the vinculin. Cell viability, cell proliferation, developed actin stress fibers and focal contact adhesion were found better on ZrAlSiN thin films than on Ti thin films or glass surfaces.

New peptide-based and animal-free coatings for animal cell culture in bioreactors

Background Anchorage dependent cells require an appropriate extracellular matrix for their survival, migration, proliferation, phenotyping and/or differentiation [1-3]. These cells interact with extracellular matrix proteins, primarily through integrins, which induces focal adhesion contacts assembly and activation of signalling pathways that regulate diverse cellular processes [4]. Culture supports usually include biochemical components allowing such cells to adhere and to reconstitute an extracellular environment close to that found in vivo. Currently, this artificial environment is achieved by extracellular matrix constituents deposition, adsorption or grafting; among them collagens, fibronectin, laminin, artificial lamina propria... [5]. However, such animal proteins used in cell culture may induce pro-inflammatory stress, be unstable against proteolysis or loose activity after adsorption [6,7]. Synthetic microenvironments should be more suitable for clinical purposes: (i) improved control of physicochemical and mechanical properties, (ii) limited risks of immunogenicity, (iii) increased biosafety (animal free) and (iv) facilitated scale-up [1]. In this framework, research has recently focused on synthetic peptides or peptidomimetics that can mimic the extracellular matrix. Such molecules can be immobilized as recognition motifs on the surface of culture supports with a greater stability and easier surfaces characterization [5]. Self-assembling peptide hydrogels could mimic the chemical and mechanical aspects of the natural extracellular matrix [8,9] by undergoing large deformations, as in mammalian tissues. They have an inherent biocompatibility and should be able to direct cell behaviour [10]. They also can be functionalized with various biologically active ligands constituting good candidates to a new range of smart biomaterials, able to ensure adhesion of different cell types [11-13]. The range of biomimetic peptides that direct cell adhesion and are recognized by integrins is large. Recognition sequences derived from different extracellular matrix proteins include RGD [1], which are specific to different cell lines [1,5,6]. In this context, this work aims at designing animal-free, chemically defined and industrially scalable coatings for animal cell culture, as an alternative to collagen, fibronectin or Matrigel for laboratory and industrial large scale applications. These are based on self-assembling short peptides bearing adhesion bioactive sequences like RGDderived or other adhesion sequences developed to coat polystyrene or polyethylene terephthalate surfaces. Adhesion sequences should be recognized by cells, which should favour their anchorage and spreading.

Revised role of interstitial cells of Cajal in cholinergic neurotransmission in the gut

Revised role of interstitial cells of Cajal in cholinergic neurotransmission in the gut

DAPK and cytoskeleton-associated functions

Death-associated protein kinase (DAPK) undergoes activation in response to various death stimuli, and they have been associated with an increase in DAPK catalytic activity. One of the most prominent features of DAPK-induced cell death is the effect on the cytoskeleton, including loss of matrix attachment, and membrane blebbing. One known cytoskeletal-associated substrate of DAPK is the myosin-II light chain, phosphorylated by DAPK on Ser(19), thus stabilizing actin stress fibres. Moreover, paxillin, a component of focal adhesions, was found to be localized in close proximity to the tips of the DAPK-positive filaments, indicating that stress fibres containing DAPK extend to focal contacts. Forced expression of DAPK in multiple cell types results in morphological changes such as cell rounding, membrane blebbing, shrinking and detachment. During directed migration, DAPK functions as a potent inhibitor of cell polarization, as evidenced by its perturbation of the formation of static protrusion at the leading edge. Furthermore, DAPK inhibits random migration by suppressing directional persistence. One of the studies considered DAPK as an anoikis inducer. Others showed that DAP-kinase inhibits the activities of cell surface integrins by converting them into an inactive conformation. Biochemical experiments have established the DAPK binding to Syntaxin1 and its subsequent phosphorylation at Ser(188) in a Ca(2+) dependent manner. This phosphorylation event has been shown to decrease the binding of Syntaxin to MUNC18-1, a protein critically involved in synaptic vesicle docking. Here, we have investigated the structural interactions that modulate DAPK phosphorylation with Syntaxin and its functional role in binding to the MUNC18-1 to regulate vesicle docking. This review will summarize our current knowledge of the role of DAPK on cytoskeleton reorganization and report the mechanisms that regulate these changes.

Adherent cells avoid polarization gradients on periodically poled LiTaO3 ferroelectrics

The response of fibroblast cells to periodically poled LiTaO3 ferroelectric crystals has been studied. While fibroblast cells do not show morphological differences on the two polarization directions, they show a tendency to avoid the field gradients that occur between polarization domains of the ferroelectric. The response to the field gradients is fully established after one hour, a time at which fibroblasts form their first focal contacts. If suspension cells, with a lower tendency to establish strong surface contacts are used, no influence of the field gradients is observed.

Proteomic Profiling of Endothelial Invasion Revealed Receptor for Activated C Kinase 1 (RACK1) Complexed with Vimentin to Regulate Focal Adhesion Kinase (FAK)

Angiogenesis is critical for many physiological and pathological processes. To identify molecules relevant to angiogenesis, we performed a proteomic screen comparing invading versus non-invading endothelial cells in three-dimensional collagen matrices. We found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein vimentin that correlated with increased endothelial cell invasion. Because both RACK1 and vimentin have been linked to focal adhesion kinase (FAK), we investigated whether this pathway regulated invasion. RACK1 depletion reduced invasion responses, and this was associated with attenuated activation of FAK. Knockdown of vimentin significantly decreased levels of phosphorylated and total FAK. Treatment with a pharmacological inhibitor of FAK dose-dependently reduced invasion, indicating a crucial role for FAK activity during invasion. Because RACK1 and vimentin were both up-regulated with sphingosine 1-phosphate treatment, required for invasion, and regulated FAK, we tested whether they complexed together. RACK1 complexed with vimentin, and growth factors enhanced this interaction. In addition, RACK1, vimentin, and FAK formed an intermolecular complex in invading endothelial cultures in three dimensions in response to stimulation by sphingosine 1-phosphate and growth factors. Moreover, depletion of RACK1 decreased the association of vimentin and FAK, suggesting that RACK1 was required for stabilizing vimentin-FAK interactions during sprouting. Silencing of vimentin and RACK1 decreased cell adhesion and focal contact formation. Taken together, these results demonstrate that proangiogenic signals converge to enhance expression and association of RACK1 and vimentin, which regulated FAK, resulting in successful endothelial sprout formation in three-dimensional collagen matrices.

In vitro biocompatibility assessment of Ti40Cu38Zr10Pd12 bulk metallic glass

The use of biocompatible materials has attained an increasing importance for tissue regeneration and transplantation. The excellent mechanical and corrosion properties of Ti40Cu38Zr10Pd12 bulk metallic glass (BMG) turn it into a potential candidate for its use in orthopaedic implants. Before being considered as a biomaterial, some biological parameters must be taken into account. In this study,mouse preosteoblasts were cultured in the presence or absence of the alloy at different times (24 h, 7 and 21 days) and no differences in cell viability were detected.Moreover, cells were able to adhere to the alloy surface by establishing focal contacts, and displayed a flattened polygonal morphology. After 14 days in culture, differentiation into osteoblasts was observed. Besides, the amount of Cu ions released and their potential toxic effects were analyzed, showing that the amount of Cu released did not increase cell death. Finally, the low levels of inflammatory cytokines secreted by THP-1 differentiated macrophages exposed to the alloy suggest the absence of an immunogenic response to the alloy. In conclusion, in vitro studies indicate that the Ti40Cu38Zr10Pd12 BMG could be considered as a biomaterial to be used in orthopaedic implants.

Protein Kinase D1-mediated Phosphorylations Regulate Vasodilator-stimulated Phosphoprotein (VASP) Localization and Cell Migration

Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP) protein family members link actin dynamics and cellular signaling pathways. VASP localizes to regions of dynamic actin reorganization such as the focal adhesion contacts, the leading edge or filopodia, where it contributes to F-actin filament elongation. Here we identify VASP as a novel substrate for protein kinase D1 (PKD1). We show that PKD1 directly phosphorylates VASP at two serine residues, Ser-157 and Ser-322. These phosphorylations occur in response to RhoA activation and mediate VASP re-localization from focal contacts to the leading edge region. The net result of this PKD1-mediated phosphorylation switch in VASP is increased filopodia formation and length at the leading edge. However, such signaling when persistent induced membrane ruffling and decreased cell motility.

Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1α in Cofilin Regulation and Cytoplasmic Actin Distribution

Cells can adapt to hypoxia by various mechanisms. Yet, hypoxia-induced effects on the cytoskeleton-based cell architecture and functions are largely unknown. Here we present a comprehensive analysis of the architecture and function of L929 fibroblasts under hypoxic conditions (1% O2). Cells cultivated in hypoxia showed striking morphological differences as compared to cells cultivated under normoxic conditions (20% O2). These changes include an enlargement of cell area and volume, increased numbers of focal contacts and loss of cell polarization. Furthermore the β- and γ-actin distribution is greatly altered. These hypoxic adjustments are associated with enhanced cell spreading and a decline of cell motility in wound closure and single cell motility assays. As the hypoxia-inducible factor-1α (HIF-1α) is stabilised in hypoxia and plays a pivotal role in the transcriptional response to changes in oxygen availability we used an shRNA-approach to examine the role of HIF-1α in cytoskeleton-related architecture and functions. We show that the observed increase in cell area, actin filament rearrangement, decrease of single cell migration in hypoxia and the maintenance of p-cofilin levels is dependent on HIF-1α stabilisation.

Genomics of corneal wound healing: a review of the literature

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.

Ovarian cancer ascites-derived vitronectin and fibronectin: Combined purification, molecular features and effects on cell response

BackgroundIntra-abdominal ascites is a complication of ovarian cancers and constitutes a permissive microenvironment for metastasis. Since fibronectin and vitronectin are key actors in ovarian cancer progression, we investigated their occurrence and molecular characteristics in various ascites fluids and the influence of these ascites-derived proteins on cell behavior. MethodsFibronectin and vitronectin were investigated by immunoblotting within various ascites fluids. A combined affinity-based protocol was developed to purify both proteins from the same sample. Each purified protein was characterized with regard to its molecular features (molecular mass of isoforms, tryptophan intramolecular environment, hydrodynamic radii), and its influence on cell adhesion. ResultsFibronectin and vitronectin were found in all tested ascites. Several milligrams of purified proteins were obtained from ascites of varying initial volumes. Molecular mass isoforms and conformational lability of proteins differed according to the ascites of origin. When incorporated into the cancer cell environment, ascites-derived fibronectin and vitronectin supported cell adhesion and migration with various degrees of efficiency, and induced the recruitment of integrins into focal contacts. ConclusionsTo our knowledge, this is the first combined purification of two extracellular matrix proteins from a single pathological sample containing a great variety of bioactive molecules. This study highlights that ascites-derived fibronectin and vitronectin exhibit different properties depending on the ascites. General significanceInvestigating the relationships between the molecular properties of ascites components and ovarian cancer cell phenotype according to the ascites may be critical for a better understanding of the recurrence of this lethal disease and for further biomarker identification.

Cadmium affects focal adhesion kinase (FAK) in mesangial cells: Involvement of CaMK‐II and the actin cytoskeleton

The toxic metal ion cadmium (Cd(2+)) induces pleiotropic effects on cell death and survival, in part through effects on cell signaling mechanisms and cytoskeletal dynamics. Linking these phenomena appears to be calmodulin-dependent activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II). Here we show that interference with the dynamics of the filamentous actin cytoskeleton, either by stabilization or destabilization, results in disruption of focal adhesions at the ends of organized actin structures, and in particular the loss of vinculin and focal adhesion kinase (FAK) from the contacts is a result. Low-level exposure of renal mesangial cells to CdCl2 disrupts the actin cytoskeleton and recapitulates the effects of manipulation of cytoskeletal dynamics with biological agents. Specifically, Cd(2+) treatment causes loss of vinculin and FAK from focal contacts, concomitant with cytoskeletal disruption, and preservation of cytoskeletal integrity with either a calmodulin antagonist or a CaMK-II inhibitor abrogates these effects of Cd(2+). Notably, inhibition of CaMK-II decreases the migration of FAK-phosphoTyr925 to a membrane-associated compartment where it is otherwise sequestered from focal adhesions in a Cd(2+)-dependent manner. These results add further insight into the mechanism of the CaMK-II-dependent effects of Cd(2+) on cellular function.

Abstract 542: Dedicator of Cytokinesis 2 (DOCK2) Promotes Vascular Lesion Formation through Stimulation of Smooth Muscle Cell Proliferation and Migration

The objective of this study is to determine the role of dedicator of cytokinesis 2 (DOCK2) in vascular lesion formation after experimental angioplasty. By using a rat carotid artery balloon-injury model, we demonstrate that neointima formation is closely associated with DOCK2 expression. DOCK2 is not expressed in normal arteries. However, its expression was observed in the developing neointima and media after injury as shown by immunohistochemistry staining, qPCR and Western blot analyses. shRNA knockdown of DOCK2 via adenovirus (Ad)-mediated gene delivery dramatically inhibited the neointimal formation by 65% as compared to those treated with control shRNA or saline. Similar results were obtained when intima/media area ratios were compared between DOCK2 knockdown and control groups. Loss of function studies in primary culture of rat aortic smooth muscle cells (RASMCs) indicated that DOCK2 is essential for both the proliferation and migration of RASMCs. PDGF-BB induced DOCK2 expression in RASMCs. shRNA knockdown of DOCK2 significantly inhibited PDGF-BB-induced proliferation and migration of RASMCs. DOCK2 appeared to stimulate RASMC migration via inducing focal adhesion contact and stress fiber formation as shown by the altered expression of vinculin and F-actin, respectively. Taken together, our studies demonstrate that DOCK2 plays an important role in vascular lesion formation following vascular injury. Therefore, targeting DOCK2 is a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.

Abstract 2632: SPARC modulates melanoma cell size, adherence, actin architecture and migration through Akt/S6K and Rac1 regulation.

Abstract Cancer cells display intermediate levels of adherence and a poorly organized actin cytoskeleton. These characteristics provide a favorable state for initiating dynamic processes -such as cell migration- that ultimately lead to invasion of the neighbor stroma and metastatic foci implantation.During the last years, it has become evident that the extracellular matrix (ECM) is the source of several molecules that regulate these processes, being the matricellular family of proteins one of the most important ones. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a matricellular protein whose overexpression in malignant or stromal cells was often associated with increased aggressiveness and bad prognosis in melanoma as well as in a wide range of other human cancers. In previous studies, we and others have reported that SPARC plays a key role in melanoma progression including the modulation of cell capacity to migrate. Here, we describe the underlying molecular mechanism through which SPARC modulates this process.SPARC knockdown (KD) in A375 and IIB-MEL-LES melanoma cells induced dramatic changes in cells’ size and actin architecture. SPARC-KD cells showed an enlarged size and an ordered actin CSK with actin bundles spanning all the cell body and ending with prominent focal contacts at the edge of the cell. These cells also showed lamellipodia formation and accelerated spreading in serum. On the other hand, SPARC-KD melanoma cells evidenced strongly restricted capacity to spread on type IV collagen and laminin that was associated with downregulation of α6 integrin and upregulation of β1 integrin. Both the re-expression of SPARC as well as the addition of the protein were able to reverse the cellular phenotype and integrin levels.Small GTPases are intermediate factors that regulate lamellipodia formation, cell spreading and integrins activity. Transient transfection of dominant negative (DN) or constitutively active sGTPases pointed to Rac1 as being involved in this process. G-ELISA activation assays confirmed increased Rac1 activity in SPARC-KD cells. The diminished capacity of SPARC-KD cells to migrate was restored by SPARC re-expression, exogenous addition and by expression of Rac1-DN. Interestingly Rac1-DN, expression also restored α6 and β1 integrin.Further studies on mediators of SPARC's effect demonstrated an increased phosphorylation of cSrc and S6K with inhibition of AKT phosphorylation in cells SPARC-KD. Remarkably, the exogenous addition of SPARC to SPARC-KD cells increased AKT phosphorylation and decreased S6K phosphorylation concomitantly with an augmented cell migration.Taken together, these results show that SPARC promotes cell migration through the modulation of an intracellular pathway that involves cSrc/AKT/S6K and Rac1 that affects cell shape, including cell size, adherence and actin architecture. Citation Format: Edgardo Salvatierra, Claudia Leishman, Mariano Alvarez, Osvaldo Podhajcer. SPARC modulates melanoma cell size, adherence, actin architecture and migration through Akt/S6K and Rac1 regulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2632. doi:10.1158/1538-7445.AM2013-2632

Structure activity relationship of synaptic and junctional neurotransmission

Chemical neurotransmission may include transmission to local or remote sites. Locally, contact between 'bare' portions of the bulbous nerve terminal termed a varicosity and the effector cell may be in the form of either synapse or non-synaptic contact. Traditionally, all local transmissions between nerves and effector cells are considered synaptic in nature. This is particularly true for communication between neurons. However, communication between nerves and other effectors such as smooth muscles has been described as nonsynaptic or junctional in nature. Nonsynaptic neurotransmission is now also increasingly recognized in the CNS. This review focuses on the relationship between structure and function that orchestrate synaptic and junctional neurotransmissions. A synapse is a specialized focal contact between the presynaptic active zone capable of ultrafast release of soluble transmitters and the postsynaptic density that cluster ionotropic receptors. The presynaptic and the postsynaptic areas are separated by the 'closed' synaptic cavity. The physiological hallmark of the synapse is ultrafast postsynaptic potentials lasting milliseconds. In contrast, junctions are juxtapositions of nerve terminals and the effector cells without clear synaptic specializations and the junctional space is 'open' to the extracellular space. Based on the nature of the transmitters, postjunctional receptors and their separation from the release sites, the junctions can be divided into 'close' and 'wide' junctions. Functionally, the 'close' and the 'wide' junctions can be distinguished by postjunctional potentials lasting ~1s and tens of seconds, respectively. Both synaptic and junctional communications are common between neurons; however, junctional transmission is the rule at many neuro-non-neural effectors.

Overexpression of Integrin-β1 in Leiomyoma Promotes Cell Spreading and Proliferation

Uterine leiomyoma, the most common tumors found in the women of the reproductive age, may cause abnormal uterine bleeding and be life threatening. Compared with myometrium, leiomyoma contains excessive extracellular matrix (ECM). However, the pathological roles of ECM in the development of leiomyoma remain largely unknown. Integrins are the major adhesion molecules on cell surface to interact with ECM. The interactions of ECM with integrins regulate cell adhesion and initiate signals for cell growth, differentiation, and migration. The aim of this study was to investigate the expression and functional role of integrin-β1 in leiomyoma pathogenesis. Levels of integrin-β1 protein were determined by Western blotting in paired normal and leiomyomal tissues (n = 15). Knockdown of integrin-β1 and inhibition of ECM-integrin interaction by disintegrin were used to evaluate the impact of integrin-β1 in cell adhesion, spreading, and proliferation. Levels of integrin-β1 were significantly up-regulated in leiomyomal cells compared with their normal counterparts. Knockdown of integrin-β1 did not affect cell adhesion on fibronectin or laminin matrix but significantly inhibits cell spreading ability. Consistent with this notion, the phosphorylation of focal adhesion kinase and the recruitment of paxillin to the focal contact were decreased in integrin-β1 knockdown cells, which attenuates contraction force. The inability of cell spreading leads to inhibition of cyclin D1 expression and impedes cell cycle progression. More importantly, disruption of ECM-integrin interaction by the small protein, disintegrin inhibited cyclin D1 expression and cell proliferation. These data demonstrate that integrin-β1 is a critical ligand to enhance cell-ECM contact force and thus promotes cell proliferation. Disruption of ECM-integrin-β1 signaling may serve as an option to inhibit the progression of leiomyoma.

Estimation of Joint Incongruence in Dysplastic Canine Elbows Before and After Dynamic Proximal Ulnar Osteotomy

To characterize joint incongruence in dysplastic canine elbows before and after dynamic proximal ulnar osteotomy (DPUO). Clinical, prospective study. Dogs (n = 10; 12 elbows) with ≥ 2 mm radioulnar incongruence (RUI), FCP, and/or OCD. Computed tomography in a nonload bearing position was used to generate in silico 3D models of the elbow joint before DPUO, and this was repeated after DPUO union (median 3 months postoperatively). On these models, RUI, humeroradial and humeroulnar subchondral joint space width (SJSW) as well as alignment of the proximal ulnar segment were investigated. RUI at the medial coronoid process (MCP) decreased (P = .001), while it increased at the lateral coronoid process (P = .0005), and remained unchanged at the level of the trochlear ridge (P = .25). SJSW at the MCP increased (P = .001), changing from focal joint space collapse preoperatively to a more homogeneous pattern of moderate SJSW at follow-up. The proximal ulnar segment rotated in all 3 planes with caudal tipping at the level of the osteotomy and varus deformity, while no axial translation could be noted. DPUO results in reduction of RUI and amelioration of focal contact area at the MCP. This effect is the result of a complex three-dimensional rotation of the proximal ulnar segment, rather than axial shift.

Adhesion and differentiation of neuronal cells on Zn-doped bioactive glasses

To verify the compatibility of rigid supports with neuronal cells for biomechanical application, we have evaluated the biocompatibility of Zn-doped bioglasses versus neuronal cell line SKNBE. Undifferentiated and retinoic acid-differentiated cells were used. We have observed that bioglasses doped with low concentration of Zn favored cell adhesion and proliferation of undifferentiated SKNBE neuronal cells, while the high Zn concentration strongly interfered with cell proliferation. Instead the high Zn concentration lightly stimulates the adhesive and strongly stimulates the phenotype characterization of differentiated SKNBE cells. Focal contact sites were observed in cells performing spread adhesive morphology, while they were down-regulated in cells performing differentiation behavior. GAP-43 and neurofilament were expressed in differentiated cells. However, GAP-43 was also found to be expressed in undifferentiated cells, where its expression seems related to proliferative behavior of cells. This work evidenced the importance of the biomaterial chemical structure in influencing proliferation or differentiation pathways of neuronal cells.

Force Mapping during the Formation and Maturation of Cell Adhesion Sites with Multiple Optical Tweezers

Focal contacts act as mechanosensors allowing cells to respond to their biomechanical environment. Force transmission through newly formed contact sites is a highly dynamic process requiring a stable link between the intracellular cytoskeleton and the extracellular environment. To simultaneously investigate cellular traction forces in several individual maturing adhesion sites within the same cell, we established a custom-built multiple trap optical tweezers setup. Beads functionalized with fibronectin or RGD-peptides were placed onto the apical surface of a cell and trapped with a maximum force of 160 pN. Cells form adhesion contacts around the beads as demonstrated by vinculin accumulation and start to apply traction forces after 30 seconds. Force transmission was found to strongly depend on bead size, surface density of integrin ligands and bead location on the cell surface. Highest traction forces were measured for beads positioned on the leading edge. For mouse embryonic fibroblasts, traction forces acting on single beads are in the range of 80 pN after 5 minutes. If two beads were positioned parallel to the leading edge and with a center-to-center distance less than 10 µm, traction forces acting on single beads were reduced by 40%. This indicates a spatial and temporal coordination of force development in closely related adhesion sites. We also used our setup to compare traction forces, retrograde transport velocities, and migration velocities between two cell lines (mouse melanoma and fibroblasts) and primary chick fibroblasts. We find that maximal force development differs considerably between the three cell types with the primary cells being the strongest. In addition, we observe a linear relation between force and retrograde transport velocity: a high retrograde transport velocity is associated with strong cellular traction forces. In contrast, migration velocity is inversely related to traction forces and retrograde transport velocity.

The small molecule genkwanine M induces single mode, mesenchymal tumor cell motility

Individual tumor cells utilize one of two modes of motility to invade the extracellular matrix, mesenchymal or amoeboid. We have determined that the diterpenoid genkwanine M (GENK) enhances the mesenchymal mode of cell motility that is intrinsic to HT-1080 osteosarcoma cells, stimulates a mesenchymal mode of motility in stationary MDA-MB-453 breast carcinoma cells, and induces a shift to a mesenchymal mode of cell motility in LS174T colorectal adenocarcinoma cells that normally utilize the alternate amoeboid mode of motility. The ability of GENK to stimulate or induce mesenchymal motility was preceded by a rapid cell spreading, elongation and polarization that did not require new gene expression. However, these initial morphologic changes were integrin dependent and they were associated with a reorganization of focal contacts and focal adhesions as well as an activation of the focal adhesion kinase. Therefore, GENK induces a mesenchymal mode of cell motility in a wide variety of tumor cell types that may be mediated, at least in part, by an activation of integrin-associated signaling.