Focal Adhesion Complexes Research Articles

Abstract 3794: The role of LMTK3 in breast cancer cell growth and invasion.

Abstract Abstract Title: The role of LMTK3 in breast cancer cell growth and invasion Background: We have identified Lemur tyrosine kinase-3 (LMTK3) as a novel regulator of estrogen receptor α (ERα) that is associated with disease free and overall survival and predicted response to endocrine therapies in breast cancer. In addition, inhibition of LMTK3 in tamoxifen (tam)-sensitive and tam-resistant xenograft mouse models led to tumor volume reduction. Moreover, a genome microarray analysis revealed various genes modulated by LMTK3 implicated in different canonical pathways. Herein we present evidence that further elucidates the mechanism of LMTK3 involvement in breast cancer growth and invasion. Methods: Stable over-expressed and depleted LMTK3 breast cancer cell lines were generated to study the effects of LMTK3 on proliferation and invasion. Various molecular and cellular biology techniques (including RT-PCR, immunofluorescence, immunoprecipitation and invasion assays) were carried out to elucidate the participation of LMTK3 in various signalling pathways. Stable isotope labelling by amino acids in cell culture (SILAC) was undertaken to identify and analyse novel LMTK3-regulated proteins. Results: Transwell and collagen 1 invasion assays results demonstrated that over-expression of LMTK3 in non-invasive MCF7 cells promote invasion while conversely knockdown of LMTK3 in MDA-MB-231 cells significantly inhibited invasion. Moreover, stable LMTK3 over-expressing cells form larger and looser acini structures compared to parental cells in 3D matrigel overlay assays. Wound healing assays demonstrated an increased motility of cells that over-express LMTK3 compared to LMTK3 knock-out cell lines. Cell adhesion assays revealed preferential binding of LMTK3 over-expressing cell lines to different ECM ligands. RT-qPCR analysis indicated up-regulation of an invasive gene signature (including MMPs, vimentin, and others) associated with LMTK3 over-expression. SILAC analysis identified various novel LMTK3-regulated proteins and phospho-proteins. In addition, modulation of LMTK3 affected the expression of specific members of the integrin family, as well as proteins involved in the focal adhesion complex. Co-immunoprecipitation and immunofluorescence experiments further helped us identify interacting partners of LMTK3 pertaining to the pathways conferring the observed pro-invasive phenotypic results. Finally, the involvement of LMTK3 in promoting tumorigenicity was confirmed in an in vivo xenograft mouse model. Conclusion: We show that LMTK3 is important for mediating breast cancer cell growth and invasion and propose a model of the mechanism of LMTK3 action in these processes. These data position LMTK3 in the map of well-known oncogenic signaling cascades. Citation Format: Yichen Xu, Aleksandra Filipovic, Hua Zhang, Lei Cheng Lit, Ylenia Lombardo, Justin Stebbing, Georgios Giamas. The role of LMTK3 in breast cancer cell growth and invasion. [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 3794. doi:10.1158/1538-7445.AM2013-3794

The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro

Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions—which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance.

Shape-dependent cell migration and focal adhesion organization on suspended and aligned nanofiber scaffolds

In the body, cells dynamically respond to chemical and mechanical cues from the extracellular matrix (ECM), yet precise mechanisms by which biophysical parameters (stiffness, topography and alignment) affect cell behavior remain unclear. Here, highly aligned and suspended multilayer polystyrene (PS) nanofiber scaffolds are used to study biophysical influences on focal adhesion complex (FAC) arrangement and associated migration behavior of mouse C2C12 cells arranged in specific shapes: spindle, parallel and polygonal. Furthermore, the role of cytoskeletal-altering drugs including blebbistatin, nocodazole and cytochalasin-D on FAC formation and migratory behavior is investigated. For the first time, this work reports that cells on suspended fiber networks, including cells with administered drugs, elongated along the fiber axes and developed longer (∼ 4×) and more concentrated FAC clusters compared to cells on flat PS control substrates. Additionally, substrate designs which topographically restrict sites of cell attachment and align adhesions were found to promote higher migration speeds (spindle: 52μmh−1, parallel: 39μmh−1, polygonal: 25μmh−1, flat: 32μmh−1). This work demonstrates that suspended fiber topography-induced concentration of FACs along fiber axes generates increased migration potential as opposed to flat surfaces, which diffuse and randomly orient adhesions.

Signal transduction in cerebral arteries after subarachnoid hemorrhage ‐ a phosphoproteomic approach

Following subarachnoid hemorrhage (SAH), cerebral vasculopathy contribute to delayed ischemia and poor outcome. We hypothesise that this is triggered by early SAH‐induced intracellular signals in cerebral arteries. To reveal these, protein extracts of rat cerebral arteries isolated 1h post‐SAH were digested, and labelled phosphopeptides were quantified by high resolution tandem mass spectrometry. Selected phosphorylations were validated by immunoblotting and monitored over a time‐course and pharmacological inhibition of key components was performed. We observed major SAH‐induced phosphorylations on focal adhesion complexes, extracellular regulated kinase 1/2 (ERK1/2), calcium calmodulin‐dependent kinase II, signal transducer and activator of transcription (STAT3) and c‐Jun, the latter two downstream of ERK1/2. Inhibition of ERK1/2 starting 6h post‐SAH prevented later increases in cerebrovascular constrictor receptors, matrix metalloprotease‐9 and wall thickness and improved neurological outcome. STAT3 inhibition partially prevented the vasculopathy but had no effect on neurological outcome. In conclusion, quantitative mass spectrometry is a strong approach to study in vivo signalling in vascular tissue. ERK1/2 inhibition after SAH prevents pathological cerebrovascular changes several days later and improves outcome.

Specific Integrin Expression Is Associated with Podosome-Like Structures on Mesodermal Progenitor Cells

Mesenchymal stromal cells (MSCs) are a heterogeneous cell population capable of differentiating toward several cell lines in vitro and, possibly, in vivo. Within cultured MSCs, we identified and purified a precursor cell population [mesodermal progenitor cells (MPCs)] retaining robust proliferation potential and ability to differentiate into endothelial or mesenchymal cells. MPC-derived MSCs retain the ability to further differentiate into osteoblasts, cartilage, or fat cells. Here we further characterized MPCs and MSCs by evaluating expression of integrins and adhesion molecules showing their ability to assemble the molecular machinery involved in endothelium adhesion. MPCs were shown to interact with activated and nonactivated endothelium, whereas MSCs exhibited activation of focal adhesion complexes, higher cell motility, and reduced or absent adhesiveness onto endothelial cells, suggesting a matrix remodeling vocation. We also reported a consistent expression of CXCR4 on the MPC cell surface, suggesting that the different phenotypic behavior could be related to specific functions of the cell in each differentiation stage.

Effects of Progesterone and Medroxyprogesterone on Actin Remodeling and Neuronal Spine Formation

Sex steroids are important regulators of neuronal cell morphology, and this is critical for gender differences in brain function and dysfunction. Neuronal morphology is controlled by multiprotein complexes including moesin (a member of the ezrin/radixin/moesin family), focal adhesion kinase (FAK), or the Wiskott-Aldrich syndrome protein-family verprolin homologous (WAVE1) protein, controlling dynamic remodeling of the cytoskeleton and cell membrane. We investigated the actions of natural progesterone (P) and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin remodeling, focal adhesion complex formation, and actin branching in rat cortical neurons. Treatment with P and, to a lesser extent, MPA, increases the number and density of dendritic spines. P increases the phosphorylation of moesin, FAK, and WAVE1, and their redistribution toward cell membrane sites where spines are formed. Signaling to moesin is achieved by PR via a Gα/Gβ-dependent signaling to the small GTPase Ras homolog gene family, member A and its related kinase, Rho-associated kinase-2. In parallel, WAVE1 recruitment is triggered by a Gαi/Gβ-dependent signaling of PR to c-Src, FAK, and Rac1 GTPase. Rac1 recruits cyclin-dependent kinase-5, which phosphorylates WAVE1. Silencing of moesin, FAK, or WAVE1 abrogates the increase in dendritic spines induced by progesterone. In all applications, MPA is found to act similar to P, albeit with a lower efficacy. In conclusion, our findings indicate that the control of actin polymerization and branching and focal adhesion complex formation via moesin, FAK, and WAVE1 is a key function of progesterone receptor in neurons, which may be relevant for the regulation of dendritic spine turnover and neuronal plasticity.

Focal Adhesion Dynamics Are Altered in Schizophrenia

Evidence from genetic association studies implicate genes involved in neural migration associated with schizophrenia risk. Neural stem/progenitor cell cultures (neurosphere-derived cells) from olfactory mucosa of schizophrenia patients have significantly dysregulated expression of genes in focal adhesion kinase (FAK) signaling, a key pathway regulating cell adhesion and migration. The aim of this study was to investigate whether olfactory neurosphere-derived cells from schizophrenia patients have altered cell adhesion, cell motility, and focal adhesion dynamics. Olfactory neurosphere-derived cells from nine male schizophrenia patients and nine male healthy control subjects were used. Cells were assayed for cell adhesion and cell motility and analyzed for integrins and FAK proteins. Focal adhesions were counted and measured in fixed cells, and time-lapse imaging was used to assess cell motility and focal adhesion dynamics. Patient-derived cells were less adhesive and more motile than cells derived from healthy control subjects, and their motility was reduced to control cell levels by integrin-blocking antibodies and by inhibition of FAK. Vinculin-stained focal adhesion complexes were significantly smaller and fewer in patient cells. Time-lapse imaging of cells expressing FAK tagged with green fluorescent protein revealed that the disassembly of focal adhesions was significantly faster in patient cells. The evidence for altered motility and focal adhesion dynamics in patient-derived cells is consistent with dysregulated gene expression in the FAK signaling pathway in these cells. Alterations in cell adhesion dynamics and cell motility could bias the trajectory of brain development in schizophrenia.

Topography and Coating of Platinum Improve the Electrochemical Properties and Neuronal Guidance

To improve neuronal-electrode interfaces, we analyzed the influence of surface topographies combined with coating on the electrochemistry of platinum and neuronal differentiation of PC-12 cells. Surface structuring on nanoscale was realized by femtosecond laser ablation. Additional coating with laminin (LA), collagen type I (COL) or poly-d-lysine (PDL) did not change the produced topography. We further demonstrated that impedance could be improved in all cases. The pre-requisites of differentiation - viability and attachment - were fulfilled on the topography. Cell attachment of non-differentiated and differentiated cells and their formation of focal adhesion complexes were even enhanced compared to unstructured platinum. However, without the nerve growth factor (NGF) no cellular outgrowth and differentiation were possible. The topography enabled cell elongation and reduced the amount of rounded cells, but less effective than coating. Differentiation was either comparable or increased on the structures when compared with unstructured coatings. For instance, microtubule associated protein (MAP2) was detected most on the topography alone. But a combination of surface structuring and coating had the strongest impact on differentiation: the usage of COL provoked best cell elongation and beta III tubulin expression, PDL best synaptophysin. LA-coating had no noteworthy effect. These findings point out that innovative electronic devices like cochlear implants include two aspects: (a) nanotopography to improve the transmission of electrical signals and neuronal attachment; and (b) an additional coating to stimulate neuronal differentiation.

Evaluation of Ni-free Zr–Cu–Fe–Al bulk metallic glass for biomedical implant applications

This study was conducted to investigate the surface characteristics, including the chemical composition, metal ion release, protein adsorption, and cell adhesion, of a Ni-free Zr-based (Zr62.5Cu22.5Fe5Al10) bulk metallic glass (BMG) with low elastic modulus for biomedical implant applications. X-ray photoelectron spectroscopy was used to identify the surface chemical composition and the protein (albumin and fibronectin) adsorption of the specimen. The metal ions released from the specimen in simulated blood plasma and artificial saliva solutions were measured using an inductively coupled plasma-mass spectrometer. The cell adhesion, in terms of the morphology, focal adhesion complex, and skeletal arrangement, of human bone marrow mesenchymal stem cells was evaluated using scanning electron microscope observations and immunofluorescent staining. For comparison purposes, the above-mentioned tests were also carried out on the widely used biomedical metal, Ti. The results showed that the main component on the outermost surface of the amorphous Zr62.5Cu22.5Fe5Al10 BMG was ZrO2 with small amounts of Cu, Al, and Fe oxides. The released metal ions from Zr62.5Cu22.5Fe5Al10 BMG were well below the critical concentrations that cause negative biological effects. The Zr62.5Cu22.5Fe5Al10 BMG had a greater adsorption capacity for albumin and fibronectin than that of commercial biomedical Ti. The Zr62.5Cu22.5Fe5Al10 BMG surface showed an attached cell number similar to the Ti surface but had better cell adhesion morphology and cytoskeletal arrangement. Based on the present results, the Ni-free Zr62.5Cu22.5Fe5Al10 BMG has the potential to be used for biomedical implant applications.

The C-terminal Peptide of Chondroadherin Modulates Cellular Activity by Selectively Binding to Heparan Sulfate Chains

Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH(359), now shown to bind to heparin with a K(D) of 13 μm. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparan sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their β(1) integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.

Tenascin-C and tenascin-W in whisker follicle stem cell niches: possible roles in regulating stem cell proliferation and migration

The whisker follicle has CD34-positive stem cells that migrate from their niche near the bulge along the glassy membrane to the whisker bulb, where they participate in the formation of the whisker shaft. Using immunohistochemistry, we found the glycoprotein tenascin-C in the fibrous capsule of mouse whisker follicles, along the glassy membrane and in the trabecular region surrounding keratin-15-negative, CD34-positive stem cells. The related glycoprotein tenascin-W is found in the CD34-positive stem cell niche, in nearby trabeculae and along the glassy membrane. Tenascin-W is also found in the neural stem cell niche of nearby hair follicles. The formation of stress fibers and focal adhesion complexes in CD34-positive whisker-derived stem cells cultured on fibronectin was inhibited by both tenascin-C and tenascin-W, which is consistent with a role for these glycoproteins in promoting the migration of these cells from the niche to the whisker bulb. Tenascin-C, but not tenascin-W, increased the proliferation of whisker follicle stem cells in vitro. Thus, the CD34-positive whisker follicle stem cell niche contains both tenascin-C and tenascin-W, and these glycoproteins might play a role in directing the migration and proliferation of these stem cells.

Substrate Topography and Stiffness affect Cardiomyocyte Anchorage, Shortening and Shortening Velocity

Substrate stiffness and topography that mimic healthy and unhealthy heart tissue were engineered for contractility and anchorage assessment of neonatal rat ventricular myocytes in vitro. Substrates were molded from PDMS with soft 10 kPa (normal) or rigid 1000 kPa (fibrotic) stiffness and either flat or topography with 15μm-high, 15μm-diameter posts, spaced 75μm apart. Data of living (line scans) and fixed (immunohistochemistry Z-stacks) cells was collected on a Zeiss 710 confocal microscope. Line scan data was analyzed with LSM Browser to determine cell shortening and shortening velocity; Z-stack images were analyzed with Volocity and MetaMorph to quantify distribution of the focal adhesion protein, paxillin and assess sarcomere remodeling. Shortening (displacement) was greater in the mid-region vs. the end of the cell, regardless of substrate topography or stiffness. Shortening in the mid-region was greater on rigid substrates, regardless of topography. Shortening velocity is fastest (∼10μm/sec) on rigid post substrata and slowest (∼5μm/sec) on soft substrates, regardless of topography. The majority of quantified paxillin was localized in the outer perimeter of the cell (over 50% on post and over 70% on flat substrates), regardless of stiffness. Paxillin was also localized on post topographies but in a smaller quantity (∼30% on rigid and ∼15% on soft substrates). This 3D-post environment, that permits paxillin distribution in the Z-axis, simultaneously reduces the cell surface area (x-y plane). The focal adhesion complex on the 3D post anchors and stabilizes deep layers of sarcomere formation, which extend to the cell perimeter. In conclusion, topography predominates in contractile shortening whereas the shortening velocity is more dependent on substrata stiffness. Thus, contractile dysfunction of cardiomyocytes in stiff diseased 3D tissue might affect velocity more than shortening. Funding: T32/HL07692, HL62426

Dynamics of Focal Adhesions

Focal adhesions(FA) are the cells machinery to sense the environment and help in migration. They are macromolecular complexes which link the actin to the integrins and are extremely dynamic in nature. The mechanism of FA movement in the cell is unclear. Treadmilling and sliding are the two modes in discussion. Recent experimental results for force velocity relationship in focal adhesions show a biphasic relationship between F-actin speed and traction force.In our study, an FA complex is modelled as a one dimensional array of point masses connected laterally to each other and to the substrate by springs. Binding and unbinding of molecules to the substrate corresponds to sliding and adding/deleting from the ends corresponds to treadmilling in the system. Through gillespie simulations, we sought to understand the mechanism of FA movement by comparing the force velocity curves generated to the experimental data. From our preliminary results, for a one layer model in a small range of forces, we see that there is a range of possible velocities for a given force depending on the ratio of rate constants for treadmilling and sliding mechanisms. Another observation is that on increasing the stiffness of the substrate, the slope of these curves decreases significantly only in the sliding dominated mechanisms.

The Role of Mechanical and Biochemical Interactions in Focal Adhesion and Cellular Shapes

We present findings from a two-dimensional mathematical model of a biological cell interacting with a soft substrate and focus on the role of the interaction of intracellular mechanical stresses and plaque protein concentrations on cell and focal adhesion shapes. The cell is treated as a hypoelastic actively-deforming continuum and the substrate is modeled as a linearly elastic continuum. The active deformation, captured by the addition of an active rate of deformation tensor, models local cytoskeletal reorganization. Focal adhesions connecting the cell and the substrate are modeled as a collection of discrete elastic springs. The model allows for the focal adhesion complexes to grow and shrink depending on mechanical forces that are acting on them and local concentrations of plaque protein. A model of stress-induced plaque protein dynamics, which is based on earlier work of Besser and Safran (2006), is coupled to the model of cell-substrate mechanics. Finite element simulations of the model allow us to explore the effects of original focal adhesion configuration, cytoskeletal dynamics, and focal adhesion strength on the shape evolution of individual focal adhesions and on overall cell shape.

A complex containing LPP and α-Actinin mediates TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells

Transforming growth factor β (TGFβ) is a potent modifier of the malignant phenotype in ErbB2-expressing breast cancers. We demonstrate that epithelial-derived breast cancer cells, which undergo a TGFβ-induced epithelial-to-mesenchymal transition (EMT), engage signaling molecules that normally facilitate cellular migration and invasion of mesenchymal cells. We identify lipoma preferred partner (LPP) as an indispensable regulator of TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. We show that LPP re-localizes to focal adhesion complexes upon TGFβ stimulation and is a critical determinant in TGFβ-mediated focal adhesion turnover. Finally, we have determined that the interaction between LPP and α-actinin, an actin cross-linking protein, is necessary for TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. Thus, our data reveal that LPP, which is normally operative in cells of mesenchymal origin, can be co-opted by breast cancer cells during an EMT to promote their migration and invasion.

Focal Adhesion Kinase as a Cellular Force Sensor

Focal adhesion kinase (FAK) is a crucial component of focal adhesion sites, transducing signals between the cytosol and the extracellular matrix. The translocation of FAK to focal adhesion sites and its functional activation by tyrosine phosphorylation leads to the formation of large multi-molecular focal adhesion complexes. The three-dimensional structure of FAK consists of a tyrosine kinase domain and two large non-catalytic regions. The N-terminal FERM domain is involved in auto-inhibition of the kinase by blocking a phosphorylation site (Tyr576/577)[1], and also includes a basic patch for anchoring FAK to membrane-embedded PIP2. The exposure of this phosphorylation site induces the maximum activity of FAK. We tested if mechanical forces as they are present at focal adhesion sites can induce an allosteric switch to an active state of FAK with an exposed phosphorylation site, using Molecular Dynamics simulations of membrane-bound and unbound FAK.We indeed find mechanical forces propagated onto FAK when tethered between the membrane and the cytoskeleton to remove the auto-inhibitory FERM domain from the kinase domain of FAK. We instead observe an only partial FAK activation upon anchoring FAK to the membrane in the absence of mechanical force. The mechano-transduction mechanism of FAK can explain how FAK acts as a force sensor, translating mechanical forces at the focal adhesion site into downstream biochemical signal events including the MAPK pathway.[1] D. Lietha, X. Cai, D.F.J. Ceccarelli, Y. Li, M. D. Schaller and M. J. Eck. Cell, 2007(129), 1177-1187

Prostate transglutaminase (TGase-4, TGaseP) enhances the adhesion of prostate cancer cells to extracellular matrix, the potential role of TGase-core domain

BackgroundTransglutaminase-4 (TGase-4), also known as the Prostate Transglutaminase, is an enzyme found to be expressed predominately in the prostate gland. The protein has been recently reported to influence the migration and invasiveness of prostate cancer cells. The present study aimed to investigate the influence of TGase-4 on cell-matrix adhesion and search for the candidate active domain[s] within the protein.MethodsHuman prostate cancer cell lines and prostate tissues were used. Plasmids that encoded different domains and full length of TGase-4 were constructed and used to generate sublines that expressed different domains. The impact of TGase-4 on in vitro cell-matrix adhesion, cell migration, growth and in vivo growth were investigated. Interactions between TGase-4 and focal adhesion complex proteins were investigated using immunoprecipitation, immunofluorescence and phosphospecific antibodies.ResultsTGase-4 markedly increased cell-matrix adhesion and cellular migration, and resulted in a rapid growth of prostate tumours in vivo. This effect resided in the Core-domain of the TGase-4 protein. TGase-4 was found to co-precipitate and co-localise with focal adhesion kinase (FAK) and paxillin, in cells, human prostate tissues and tumour xenografts. FAK small inhibitor was able to block the action mediated by TGase-4 and TGase-4 core domain.ConclusionTGase-4 is an important regulator of cell-matrix adhesion of prostate cancer cells. This effect is predominately mediated by its core domain and requires the participation of focal adhesion complex proteins.

The Efficacy of Complete Surgical Excision of Keloid and Piercing Sinus Tract on Earlobe Keloid

The Efficacy of Complete Surgical Excision of Keloid and Piercing Sinus Tract on Earlobe Keloid

Crosslinked PEG mats for peptide immobilization and stem cell adhesion.

We have designed a lightly crosslinked PEG based copolymer coating with compositional flexibility as well as extended stability for studying human mesenchymal stem cells (hMSCs). Copolymers contain a majority of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) as a cytophobic background with poly(ethylene glycol) methacrylate (PEGMA) for peptide coupling, and less than 10% glycidyl methacrylate (GMA) for crosslinking. Copolymer thin films were crosslinked into 30 nm thick mats by either thermal treatment or ultraviolet light and were stable for 35 days in water at 37 °C. The amount of PEGMA in the copolymer was optimized to ∼11% to minimize non-specific cell-protein interactions while maximizing the amount of total bound peptides. Following the binding of RGDSP to the mat, hMSCs were seeded. The hMSC adhesion, spreading and focal adhesion complex formation were promoted in a concentration dependent manner. Mats coupled with a non-adhesive scramble (RDGSP) maintained their cytophobicity. Competitive detachment experiments further demonstrated that cell adhesion was mediated by receptor binding to the RGDSP peptide. Cell culture experiments performed at 1 and 2 weeks show that mats can still resist cell adhesion after incubation in a serum containing medium. X-ray photoelectron spectroscopy (XPS) was effectively used to quantify the average total peptide concentration as 12.6 ± 6.14 pmol cm-2. A square 2.2 mm N (1s) element map shows an average value of 17.9 pmol cm-2 of RGDSP, which correlates well with the multipoint high resolution data. The stability of the copolymer, compositional flexibility, ease of application and the ability to precisely quantify bound peptides on the mats make these materials ideal for the study of cellular processes, where stability, functionality and topography of the biointerface are relevant.

Computational Studies of the Integrin Receptor in Complex Biologically Relevant Lipid Bilayers

Integrins are large heterodimeric (αβ) cell surface receptors which play a key role in the formation of focal adhesion complexes and are involved in various signal transduction pathways. They are located in the plasma membrane and they activated to a high affinity state by the formation of a complex between the integrin β-subunit tail, the membrane and talin. Despite the existence of structural data for all domains of integrins, the structure of the complete integrin receptor and details of its interactions with bilayers remain unclear. In this study, a model of the complete integrin receptor in complex with the talin F2-F3 domain was constructed using recent structural data. The complex was inserted in biologically relevant bilayers, whose lipid composition resembles that of the plasma membrane, containing phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect in bilayer structure and dynamic. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer in an asymmetric way, including a slowing-down of lipids in an annulus around the protein due to the interactions of the lipids with the integrin receptor.