Localization Of Paxillin Research Articles

Immunohistochemical expression of paxillin in potentially malignant disorders and squamous cell carcinoma patients.

Cell adhesion molecules are essential to maintain the integrity of stratified squamous epithelium but their expression has to be dynamic to aid the mobility and turnover of cells. Paxillin is one such multi-domain protein which integrates numerous signals from cell surface receptors, integrins and growth factors. It thus functions as a regulator of various physiological and pathological processes including tissue remodeling, cell motility, gene expression, matrix organization, cell proliferation, metastasis and survival. Hence, the assessment of paxillin expression in normal control, potentially malignant disorders and oral squamous cell carcinoma patients was carried out. The present retrospective study comprised of 20 each clinically and histologically confirmed case of normal control, potentially malignant disorders, and oral squamous cell carcinomas. All the slides were stained immunohistochemically using Paxillin antibody. The localization, staining intensity and percentage of positivity for paxillin expression was statistically significant among normal control and potentially malignant disorders, whereas oral squamous cell carcinoma showed a non-significant difference. Upon comparison of histopathological grading of potentially malignant disorders, mild versus severe and moderate versus severe epithelial dysplasia showed a statistical significant difference among all the parameters of paxillin expression. However, WDSCC & MDSCC a statistically significant difference among localization and staining intensity of paxillin. Paxillin may play an important role in pathogenesis of oral squamous cell carcinoma by altering the adhesive properties of the tumor cells interacting with the extracellular matrix which in turn affects their invasive behavior and histologic differentiation.

Locus-Conserved Circular RNA cZNF292 Controls Endothelial Cell Flow Responses.

Circular RNAs (circRNAs) are generated by back splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here, we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. Combining published endothelial RNA-sequencing data sets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localization and focal adhesion organization. Mechanistically, we identified the protein SDOS (syndesmos) to specifically interact with cZNF292 in endothelial cells by RNA-affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganization thereby recapitulating cZfp292 knockout phenotypes. Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

PAK4 methylation by the methyltransferase SETD6 attenuates cell adhesion

P21-activated kinase 4 (PAK4), a member of serine/threonine kinases family is over-expressed in numerous cancer tumors and is associated with oncogenic cell proliferation, migration and invasion. Our recent work demonstrated that the SET-domain containing protein 6 (SETD6) interacts with and methylates PAK4 at chromatin in mammalian cells, leading to activation of the Wnt/β-catenin signaling pathway. In our current work, we identified lysine 473 (K473) on PAK4 as the primary methylation site by SETD6. Methylation of PAK4 at K473 activates β-catenin transcriptional activity and inhibits cell adhesion. Specific methylation of PAK4 at K473 also attenuates paxillin localization to focal adhesions leading to overall reduction in adhesion-related features, such as filopodia and actin structures. The altered adhesion of the PAK4 wild-type cells is accompanied with a decrease in the migrative and invasive characteristics of the cells. Taken together, our results suggest that methylation of PAK4 at K473 plays a vital role in the regulation of cell adhesion and migration.

Structural Basis of Paxillin Recruitment by Kindlin-2 in Regulating Cell Adhesion

Activation of cell surface receptor integrin has been extensively studied as the first key step to trigger cell adhesion, but the subsequent events, widely regarded as integrin "outside-in" signaling to form supramolecular complexes (focal adhesions [FAs]) to promote dynamic cell adhesion, remain poorly elucidated. Integrin activator kindlin-2 was recently found to associate with paxillin in nascent FAs, implicating an early yet undefined integrin outside-in signaling event. Here we show structurally that kindlin-2 recognizes paxillin via a distinct interface involving the ubiquitin-like kindlin-2 F0 domain and the paxillin LIM4 domain. The interface is adjacent to the membrane binding site of kindlin-2 F0, suggesting a mechanism for kindlin-2 to recruit paxillin to the membrane-proximal site where FA assembly is initiated. Disruption of the interface impaired the localization of paxillin, causing strong defects in FA assembly and cell migration. These data unveil a structural basis of the kindlin-2/paxillin interaction in controlling dynamic cell adhesion.

Probing cellular response to topography in three dimensions

Biophysical aspects of in vivo tissue microenvironments include microscale mechanical properties, fibrillar alignment, and architecture or topography of the extracellular matrix (ECM). These aspects act in concert with chemical signals from a myriad of diverse ECM proteins to provide cues that drive cellular responses. Here, we used a bottom-up approach to build fibrillar architecture into 3D amorphous hydrogels using magnetic-field driven assembly of paramagnetic colloidal particles functionalized with three types of human ECM proteins found in vivo. We investigated if cells cultured in matrices comprised of fibrils of the same size and arranged in similar geometries will show similar behavior for each of the ECM proteins tested. We were able to resolve spatial heterogeneities in microscale mechanical properties near aligned fibers that were not observed in bulk tissue mechanics. We then used this platform to examine factors contributing to cell alignment in response to topographical cues in 3D laminin-rich matrices. Multiple human cell lines extended protrusions preferentially in directions parallel or perpendicular to aligned fibers independently of the ECM coating. Focal adhesion proteins, as measured by paxillin localization, were mainly diffuse in the cytoplasm, with few puncta localized at the protrusions. Integrin β1 and fascin regulated protrusion extension but not protrusion alignment. Myosin II inhibition did not reduce observed protrusion length. Instead, cells with reduced myosin II activity generated protrusions in random orientations when cultured in hydrogels with aligned fibers. Similarly, myosin II dependence was observed in vivo, where cells no longer aligned along the abluminal surfaces of blood vessels upon treatment with blebbistatin. These data suggest that myosin II can regulate sensing of topography in 3D engineered matrices for both normal and transformed cells.

On-Chip Studies of Magnetic Stimulation Effect on Single Neural Cell Viability and Proliferation on Glass and Nanoporous Surfaces.

Transcranial magnetic stimulation (TMS) is a noninvasive neuromodulation technique, an FDA-approved treatment method for various neurological disorders such as depressive disorder, Parkinson's disease, post-traumatic stress disorder, and migraine. However, information concerning the molecular/cellular-level mechanisms of neurons under magnetic simulation (MS), particularly at the single neural cell level, is still lacking, resulting in very little knowledge of the effects of MS on neural cells. In this paper, the effects of MS on the behaviors of neural cell N27 at the single-cell level on coverslip glass substrate and anodic aluminum oxide (AAO) nanoporous substrate are reported for the first time. First, it has been found that the MS has a negligible cytotoxic effect on N27 cells. Second, MS decreases nuclear localization of paxillin, a focal adhesion protein that is known to enter the nucleus and modulate transcription. Third, the effect of MS on N27 cells can be clearly observed over 24 h, the duration of one cell cycle, after MS is applied to the cells. The size of cells under MS was found to be statistically smaller than that of cells without MS after one cell cycle. Furthermore, directly monitoring cell division process in the microholders on a chip revealed that the cells under MS generated statistically more daughter cells in one averagecell cycle timethan those without MS. All these results indicate that MS can affect the behavior of N27 cells, promoting their proliferation and regeneration.

Toxoplasma gondii disrupts β1 integrin signaling and focal adhesion formation during monocyte hypermotility

The motility of blood monocytes is orchestrated by the activity of cell-surface integrins, which translate extracellular signals into cytoskeletal changes to mediate adhesion and migration. Toxoplasma gondii is an intracellular parasite that infects migratory cells and enhances their motility, but the mechanisms underlying T. gondii-induced hypermotility are incompletely understood. We investigated the molecular basis for the hypermotility of primary human peripheral blood monocytes and THP-1 cells infected with T. gondii Compared with uninfected monocytes, T. gondii infection of monocytes reduced cell spreading and the number of activated β1 integrin clusters in contact with fibronectin during settling, an effect not observed in monocytes treated with lipopolysaccharide (LPS) or Escherichia coli Furthermore, T. gondii infection disrupted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 (Tyr-397) and Tyr-925 and of the related protein proline-rich tyrosine kinase (Pyk2) at Tyr-402. The localization of paxillin, FAK, and vinculin to focal adhesions and the colocalization of these proteins with activated β1 integrins were also impaired in T. gondii-infected monocytes. Using time-lapse confocal microscopy of THP-1 cells expressing enhanced GFP (eGFP)-FAK during settling on fibronectin, we found that T. gondii-induced monocyte hypermotility was characterized by a reduced number of enhanced GFP-FAK-containing clusters over time compared with uninfected cells. This study demonstrates an integrin conformation-independent regulation of the β1 integrin adhesion pathway, providing further insight into the molecular mechanism of T. gondii-induced monocyte hypermotility.

Effects of substrate stiffness and actomyosin contractility on coupling between force transmission and vinculin-paxillin recruitment at single focal adhesions.

Focal adhesions (FAs) regulate force transfer between the cytoskeleton and ECM-integrin complexes. We previously showed that vinculin regulates force transmission at FAs. Vinculin residence time in FAs correlated with applied force, supporting a mechanosensitive model in which forces stabilize vinculin's active conformation to promote force transfer. In the present study, we examined the relationship between traction force and vinculin-paxillin localization to single FAs in the context of substrate stiffness and actomyosin contractility. We found that vinculin and paxillin FA area did not correlate with traction force magnitudes at single FAs, and this was consistent across different ECM stiffness and cytoskeletal tension states. However, vinculin residence time at FAs varied linearly with applied force for stiff substrates, and this was disrupted on soft substrates and after contractility inhibition. In contrast, paxillin residence time at FAs was independent of local applied force and substrate stiffness. Paxillin recruitment and residence time at FAs, however, were dependent on cytoskeletal contractility on lower substrate stiffness values. Finally, substrate stiffness and cytoskeletal contractility regulated whether vinculin and paxillin turnover dynamics are correlated to each other at single FAs. This analysis sheds new insights on the coupling among force, substrate stiffness, and FA dynamics.

Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway.

Background and objectivesResolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.MethodsVSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.ResultsTreatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.ConclusionsOur results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

Abstract 5051: Docosahexaenoic acid inhibits cancer cell invasion and migration by modifying focal adhesion complex structure and signaling

Abstract The extracellular matrix (ECM) is responsible for the regulation of many cellular processes, including survival, proliferation, migration and invasion. Cellular-ECM contact induces a coordinated process recruiting functional protein complexes, consisting of scaffold, adapter, linking and docking proteins, to form focal adhesion (FA) structures. Once formed, FA complexes then recruit signaling proteins to activate downstream cellular pathways. Aberrations in FA structure and signaling have shown to contribute to cancer initiation, progression and cellular motility. Studies investigating the molecular mechanisms accounting for FA structure and function in cancer cells have demonstrated unique growth behavior and migratory properties in vitro. While docosahexaenoic acid (DHA), an omega-3 PUFA, has been shown to inhibit growth of a number of cancer cell lines, the effects of DHA on FA dynamics and signaling have not been investigated. In the present study, we show that DHA induced changes in the morphological growth phenotype in cancer cell lines grown in Matrigel™. DHA treatment also inhibited planar cell movement (cell migration) and amoeboid motility (invasion) in breast and lung cancer cell lines. Associated with the observed morphological changes and inhibited growth, were changes in the expression of a number of proteins involved in the FA complex. Upon further characterization, cell line specific changes in protein expression levels and protein localization of FAK, e-cadherin, paxillin and vinculin were observed with DHA treatment, which suggests that DHA is modifying FA complex structure. Additionally, changes in the phosphorylation patterns of major signaling pathways associated with the FA complex, like EGFR, ERK and c-Myc, were also observed with DHA treatment. Overall, these results provide new insight into FA activation and signaling and demonstrate a new mechanism for DHA treatment in cancer. Citation Format: Michael Mouradian, Palvinder K. Bains, Ronald S. Pardini. Docosahexaenoic acid inhibits cancer cell invasion and migration by modifying focal adhesion complex structure and signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5051.

Nuclear transport of paxillin depends on focal adhesion dynamics and FAT domains.

ABSTRACTThe nuclear transport of paxillin appears to be crucial for paxillin function but the mechanism of transport remains unclear. Here, we show that the nuclear transport of paxillin is regulated by focal adhesion turnover and the presence of FAT domains. Focal adhesion turnover was controlled using triangular or circular fibronectin islands. Circular islands caused higher focal adhesion turnover and increased the nuclear transport of paxillin relative to triangular islands. Mutating several residues of paxillin had no effect on its nuclear transport, suggesting that the process is controlled by multiple domains. Knocking out FAK (also known as PTK2) and vinculin caused an increase in nuclear paxillin. This could be reversed by rescue with wild-type FAK but not by FAK with a mutated FAT domain, which inhibits paxillin binding. Expressing just the FAT domain of FAK not only brought down nuclear levels of paxillin but also caused a large immobile fraction of paxillin to be present at focal adhesions, as demonstrated by fluorescence recovery after photobleaching (FRAP) studies. Taken together, focal adhesion turnover and FAT domains regulate the nuclear localization of paxillin, suggesting a possible role for transcriptional control, through paxillin, by focal adhesions.

Abstract A51: Targeting mTOR metabolism for glioblastoma cancer therapy

Abstract Aerobic glycolysis (the Warburg effect), first recognized almost a century ago, by Otto Warburg is a core hallmark of cancer. The Warburg effect describes a switch in glucose metabolism from oxidative phosphorylation to glycolysis. Recently links have been established between the oncogenic pathways that drive tumorigenesis and the mechanistic basis of tumor cell metabolism. The kinase mTOR is a major driver of tumor metabolism and proliferation of cancer cells, acts downstream of numerous oncogenic pathways. Several drugs targeting the mTOR pathway are being developed, however the most common drug rapamycin does not inhibit mTOR complex-2. Therefore in the current study, we examined the potential benefit of MLN0128, a novel potent mTOR ATP competitive inhibitor, as a therapeutic strategy for glioblastoma (GBM), one of the deadliest brain tumors. We studied the action of MLN0128 in human GBM tumor cells and patient derived stem cells. We show the inhibitory effect of MLN0128 in nanomolar concentrations on tumor proliferation in multiple GBM cell lines via the blockage of phospho-AKT, -ribosomal protein, -4EBP1 inhibiting both mTORC1 and mTORC2 signaling in GBM. Reduction of invasive potential and disruption of paxillin localization to focal adhesions was observed in vitro. MLN0128 treatment decreased glucose consumption and lactate production. It inhibited rate of glycolysis, glycolytic capacity and glycolytic reserve. It also inhibited ATP production, mitochondrial respiration and spare respiratory capacity. MLN0128 was superior in inhibiting metabolism compared to rapamycin. On clonogenic survival assay MLN0128 decreased GBM cell survival and increased radiosensitivity. In vivo treatment of mouse xenografts with MLN0128 downregulated mTOR targets and significantly inhibited tumor volume compared to drug and irradiation alone. Combination treatment of GBM cells with MLN0128 and other chemotherapeutic agents like carboplatin, irinotecan and TMZ showed synergistic effects when combination index was calculated in GBM tumor cells and stem cells. However, the therapeutic efficacy of TMZ combination was greater than MLN0128 combination with either carboplatin or irinotecan. Moreover, similar TMZ chemosensitivity was observed in MGMT non-methylated, TMZ in-sensitive GBM stem cells and MGMT methylated GBM cells. These findings support the rationale for clinical testing of MLN0128 in patients with GBM to provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors with standard care. Citation Format: Anita Tandle, Tamalee Kramp, Philip Tofilon, Kevin Camphausen. Targeting mTOR metabolism for glioblastoma cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A51.

The Effect of Thymosin β4 for Osteoblast Adhesion on Titanium Surface.

Titanium (Ti) is the most widely used implant material in dentistry and orthopedics but the release of metal ions from Ti implants results in increased bone resorption by enhancing the production of inflammatory cytokines from the macrophages and facilitating osteoclast differentiation. Thymosin β4 (Tβ4) has several biological activities, such as promoting wound healing, angiogenesis, cell proliferation and migration in mammalian cells. This study examined the role of Tβ4 in osteoblasts via focal adhesions (FAs) and ERK1/2 signaling related to cell adhesion and proliferation for cell survival on the Ti surface. As a result, cell adhesion and proliferation increased in the Tβ4 treated cells (Tβ4/MC3T3-E1) but was significantly lower in the Tβ4 knock-down cells by Tβ4-siRNA (si-Tβ4/MC3T3-E1) than that of the untreated cells. The levels of FAK phosphorylation, paxillin expression, and paxillin localization were higher in the Tβ4/IMC3T3-E1 cells than that of the untreated cells but lower in the si-Tβ4/MC3T3-E1 cells. In addition, the levels of cell proliferation, Grb2 and Ras protein expression and phosphorylation of ERK1/2 were higher in the Tβ4/MC3T3-E1 cells than in the untreated cells but lower in the si-Tβ4/IMC3T3-E1 cells. These results suggest that Tβ4 might be a good nanomolecule that promotes osteoblast survival by facilitating adhesion and proliferation on the Ti surface.

Tyrosine phosphorylation of paxillin alpha is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells.

Temporal and spatial regulation of actin-based cytoskeletal organization and focal adhesion formation play an essential role in cell migration. Here, we show that tyrosine phosphorylation of a focal adhesion protein, paxillin, crucially participates in these regulations. We found that tyrosine phosphorylation of paxillin was a prominent event upon integrin activation during epithelial-mesenchymal trans-differentiation and cell migration. Four major tyrosine phosphorylation sites were identified, and two of them were highly inducible upon integrin activation. Paxillin exhibits three distinct subcellular localizations as follows: localization along the cell periphery colocalized with circumferential actin meshworks, macroaggregation at focal adhesions connected to actin stress fibers, and diffuse cytoplasmic distribution. Tyrosine phosphorylation of paxillin localized at the cell periphery and focal adhesions was shown using phosphorylation site-specific antibodies. Mutations in the phosphorylation sites affected the peripheral localization of paxillin and paxillin-containing focal adhesion formation during cell migration and cell-cell collision, accompanied by altered actin organizations. Our analysis indicates that phosphorylation of multiple tyrosines in paxillin alpha is necessary for the proper function of paxillin and is involved in the temporospatial regulation of focal adhesion formation and actin cytoskeletal organization in motile cells.

Hypoxia Affects Cell Migration and Wound Healing by Altering Basal Lamina Proteins and Focal Adhesion Turnover

Corneal disease and injury remain a major cause of blindness according to the World Health Organization. Certain conditions such as diabetes, corneal infection, and corneal ulceration may cause the cornea to be exposed to a hypoxic state for extended periods of time. Over 95% of the new cases of diabetes are Type II. In these patients, a simple wound can lead to complications which are compounded by the reduced ability to recover from hypoxic stress. An ex vivo corneal organ culture model was employed to examine cell migration. After a 24 hour exposure to hypoxia healing is delayed by 6 hours compared to control. In addition, there is a morphological change at the leading edge of cells. We hypothesize that hypoxia affects cell migration and wound healing by altering the regulation of basal lamina proteins and the turnover of focal adhesions. We determined that wounded rat corneas exposed to a hypoxic environment display decreased epithelial expression of syndecan‐1 and fibronectin. The localization of fibronectin throughout the epithelium and stroma is altered in the hypoxic cornea. Additional studies examined the alterations in expression and co‐localization of focal adhesion proteins. Hypoxia caused a change in the localization of vinculin and paxillin along the basal lamina and within the epithelium. Furthermore, there was a change in localization along the basal lamina of Y118 paxillin in the hypoxic cornea. By determining the changes in basal lamina composition and characterizing the changes in regulation and dynamics of focal adhesion proteins, we aim to improve the treatment of delayed wound healing associated with corneal hypoxia.

Deregulation of SLIT2-Mediated Cdc42 Activity Is Associated with Esophageal Cancer Metastasis and Poor Prognosis

SLIT2, a secreted protein, has been found to inactivate Cdc42 GTPase to modulate neural cell migration. However, alteration of SLIT2-mediated Cdc42 in terms of migration regulation remains undefined in esophageal squamous cell carcinoma (ESCC). We report here in ESCC cell, animal, and clinical models that SLIT2 acts as a migration suppressor and serves as a prognostic biomarker. The immunohistochemistry data indicated that 31.8% (49 of 154) of tumors from ESCC patients showed low expression of SLIT2 protein which correlated with poor overall survival and disease-free survival. DNA methylation analysis suggested that promoter hypermethylation is responsible for low expression of SLIT2 in ESCC. Knockdown of SLIT2 increased ESCC cell migration, while SLIT2 stable overexpression reduced cell migration. ESCC cells treated with conditioned media from cells overexpressing SLIT2 also suppressed cell migration. Importantly, silencing of SLIT2 decreased the complex formation, and thus induced Cdc42 activity and promoted membrane localization of focal adhesion kinase and Paxillin. Anti-metastatic effect of SLIT2 was confirmed in an experimental metastasis model of SLIT2 knockdown ESCC cells. Our results provide novel evidence that low expression of SLIT2 correlates with poor prognosis and promotes metastasis in ESCC, which may be regulated by the Cdc42-mediated pathways.

Mechanical force transmission at single integrin complexes in living cells (479.1)

Mechanical interactions between cells and the extracellular matrix (ECM) play central roles in directing cell migration, proliferation, and stem‐cell differentiation. However, fundamental aspects of how cells detect and generate mechanical forces at the cell‐ECM interface remain poorly understood. Here we describe the use of molecular tension sensors to visualize the forces experienced by single cellular adhesion molecules with nanometer, piconewton, and second resolutions. The molecular tension sensors consist of a Förster resonant energy transfer (FRET) donor and acceptor pair separated by a protein domain that acts as an extensible spring. Sensor molecules are immobilized to an avidin‐coated glass coverslip at one end and present an integrin‐binding site at the other. Cellular integrins transmit force to the FRET pairs, resulting in decreased FRET with increasing load. We find that human foreskin fibroblasts (HFFs) adhere to sensor‐functionalized surfaces and develop focal adhesions as evidenced by paxillin localization and actin stress fiber formation. We observe a bimodal distribution of FRET efficiency values for sensor molecules beneath HFFs, with one peak corresponding to zero load and the other indicating a distribution of forces between 1 and 4 pN. The relatively narrow range of forces that we observe suggests that mechanical tension at individual adhesion complexes is subject to active feedback and control. Ongoing work makes use of molecular‐scale force measurements to elucidate the mechanically activated signaling events that underlie cell migration and adhesion.

Focal Adhesion Axial Topography by the Z-Phasor Approach in Confocal Microscopy

The protein lateral and axial organization within focal adhesions has been studied by state of the art super resolution methods due to its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation requiring sample fixation that limits their application. In this report we present a phasor-based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that when about a total of 4000 photon counts are collected along a z-scan, axial localization precision close to 10 nm is achievable. We show at 10nm resolution that axial localization of paxillin, FAK and talin is similar at different focal adhesion sites, while F-actin shows a sharp increase in height towards the cell center. We further demonstrated that using line scans we can obtain axial resolution of 10 nm with a temporal resolution of less than a second of the changes of topography along the line. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials.Work supported in part by NIH grants P50 GM076516 and P41 GM103540.

Rsu1 contributes to regulation of cell adhesion and spreading by PINCH1‐dependent and ‐ independent mechanisms

Cell adhesion and migration are complex processes that require integrin activation, the formation and dissolution of focal adhesion (FAs), and linkage of actin cytoskeleton to the FAs. The IPP (ILK, PINCH, Parvin) complex regulates FA formation via binding of the adaptor protein ILK to β1 integrin, PINCH and parvin. The signaling protein Rsu1 is linked to the complex via binding PINCH1. The role of Rsu1 and PINCH1 in adhesion and migration was examined in non-transformed mammary epithelial cells. Confocal microscopy revealed that the depletion of either Rsu1 or PINCH1 by siRNA in MCF10A cells decreased the number of focal adhesions and altered the distribution and localization of β1 integrin, vinculin, talin and paxillin without affecting the levels of FA protein expression. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. In addition, constitutive phosphorylation of actin regulatory proteins occurred in the absence of PINCH1. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function by regulating levels of PINCH1. However, while both Rsu1- or PINCH1-depleted cells retained the ability to activate adhesion signaling in response to EGF stimulation, only Rsu1 was required for EGF-induced p38 Map Kinase phosphorylation and ATF2 activation, suggesting an Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with an Rsu1 mutant that does not bind to PINCH1 failed to restore FAs or migration but did promote spreading and constitutive p38 activation. These data show that Rsu1-PINCH1 association with ILK and the IPP complex is required for regulation of adhesion and migration but that Rsu1 has a critical role in linking integrin-induced adhesion to activation of p38 Map kinase signaling and cell spreading. Moreover, it suggests that Rsu1 may regulate p38 signaling from the IPP complex affecting other functions including survival.Electronic supplementary materialThe online version of this article (doi:10.1007/s12079-013-0207-5) contains supplementary material, which is available to authorized users.

Single Piconewton Forces at Individual Integrins Support Robust Cell Adhesion

Mechanical interactions between cells and the extracellular matrix (ECM) exert a profound influence on cell migration, proliferation, and stem cell differentiation. However, fundamental aspects of how cells detect and generate mechanical forces at the cell-ECM interface remain poorly understood. Here we describe a new technique, termed Molecular Force Microscopy (MFM) that visualizes the forces experienced by single cellular adhesion molecules with nanometer, piconewton, and sub-second resolutions. MFM uses a new class of FRET-based molecular tension sensors that bind to an avidin-coated glass coverslip at one end and present an integrin binding site at the other. Cellular integrins transmit force to the FRET pair, resulting in decreased FRET with increasing load. Unlike previously reported force sensors, MFM sensor molecules allow quantitative FRET imaging at the single molecule level. We found that human foreskin fibroblasts (HFFs) adhered to and spread on surfaces functionalized with the MFM probes, and developed mature focal adhesions as evidenced by paxillin localization and actin stress fiber formation. We observed a bimodal distribution of FRET efficiency values for MFM sensor molecules beneath HFFs, with one peak corresponding to zero load and the other indicating a distribution of forces between 1 and 4 pN. Despite evidence of robust adhesion, the forces we measured were ∼10-fold lower than the force necessary to break individual integrin-ECM bonds. Our data provide the first direct measurement of the tension per integrin molecule necessary to form stable contacts with the ECM. The relatively narrow range of forces that we observed suggests that mechanical tension at individual adhesion molecules is subject to exquisite feedback and control. Ongoing work uses the unique capabilities of MFM to elucidate the mechanical signal transduction mechanisms that underlie cell migration and adhesion.