Distribution Of Vinculin Research Articles

Quantitative Correlation of Nanotopography with Cell Spreading via Focal Adhesions Using Adipose-Derived Stem Cells.

Nanotopography mimicking extracellular environments reportedly impact cell morphological changes; however, elucidating this relationship has been challenging. To control cellular responses using nanostructures, in this study, the quantitative relationship between nanotopography and cell spreading mediated by focal adhesions (FAs) is demonstrated using adipose-derived stem cells (ASCs). The spreading of ASCs and area of FAs are analyzed for the distribution of filamentous actin and vinculin, respectively, using fluorescent images. FAs require a specific area for adhesion (herein defined as effective contact area [ECA]) to maintain cell attachment on nanopillar arrays. An ECA is the area of FAs supported by nanopillars, multiplying the area fraction (AF) of their top surface. Regarding the spreading of cells, the mean area of ASCs linearly decreases as the mean area of FAs increases. Because the area of FAs is inversely correlated to the AF of the nanopillar arrays, the spreading of cells can be quantitatively correlated with nanotopography. The results provide a conceptual framework for controlling cell behaviors to design artificial substrates for tissue-engineering applications.

Single-Cell Probe Force Studies to Identify Sox2 Overexpression-Promoted Cell Adhesion in MCF7 Breast Cancer Cells.

The replacement of the cantilever tip by a living cell in Atomic Force Microscopy (AFM) experiments permits the direct quantification of cell–substrate and cell–cell adhesion forces. This single-cell probe force measurement technique, when complemented by microscopy, allows controlled manipulation of the cell with defined location at the area of interest. In this work, a setup based on two glass half-slides, a non-fouling one with bacterial S-layer protein SbpA from L. sphaericus CMM 2177 and the second with a fibronectin layer, has been employed to measure the adhesion of MCF7 breast cancer cells to fibronectin films (using SbpA as control) and to other cells (symmetric vs. asymmetric systems). The measurements aimed to characterize and compare the adhesion capacities of parental cells and cells overexpressing the embryonic transcription factor Sox2, which have a higher capacity for invasion and are more resistant to endocrine therapy in vivo. Together with the use of fluorescence techniques (epifluorescence, Total Internal Fluorescence Microscopy (TIRF)), the visualization of vinculin and actin distribution in cells in contact with fibronectin surfaces is enabled, facilitating the monitoring and quantification of the formation of adhesion complexes. These findings demonstrate the strength of this combined approach to assess and compare the adhesion properties of cell lines and to illustrate the heterogeneity of adhesive strength found in breast cancer cells.

The Mechanical Properties, Secondary Structure, and Osteogenic Activity of Photopolymerized Fibroin.

Previously, we have described the preparation of a novel fibroin methacrylamide (FbMA), a polymer network with improved functionality, capable of photocrosslinking into Fb hydrogels with elevated stiffness. However, it was unclear how this new functionality affects the structure of the material and its beta-sheet-associated crystallinity. Here, we show that the proposed method of Fb methacrylation does not disturb the protein’s ability to self-aggregate into the stable beta-sheet-based crystalline domains. Fourier transform infrared spectroscopy (FTIR) shows that, although the precursor ethanol-untreated Fb films exhibited a slightly higher degree of beta-sheet content than the FbMA films (46.9% for Fb-F-aq and 41.5% for FbMA-F-aq), both materials could equally achieve the highest possible beta-sheet content after ethanol treatment (49.8% for Fb-F-et and 49.0% for FbMA-F-et). The elasticity modulus for the FbMA-F-et films was twofold higher than that of the Fb-F-et as measured by the uniaxial tension (130 ± 1 MPa vs. 64 ± 6 MPa), and 1.4 times higher (51 ± 11 MPa vs. 36 ± 4 MPa) as measured by atomic force microscopy. The culturing of human MG63 osteoblast-like cells on Fb-F-et, FbMA-F-et-w/oUV, and FbMA-F-et substrates revealed that the photocrosslinking-induced increment of stiffness increases the area covered by the cells, rearrangement of actin cytoskeleton, and vinculin distribution in focal contacts, altogether enhancing the osteoinductive activity of the substrate.

Dynamics and distribution of paxillin, vinculin, zyxin and VASP depend on focal adhesion location and orientation

Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial to many (patho-) physiological processes. We examined in two cell types from different species the binding dynamics of functionally related FA protein pairs: paxillin and vinculin versus zyxin and VASP. In photobleaching experiments ~40% of paxillin and vinculin remained stably associated with a FA for over half an hour. Zyxin and VASP predominantly displayed more transient interactions. We show protein binding dynamics are influenced by FA location and orientation. In FAs located close to the edge of the adherent membrane paxillin, zyxin and VASP were more dynamic and had larger bound fractions. Zyxin and VASP were also more dynamic and had larger bound fractions at FAs perpendicular compared to parallel to this edge. Finally, we developed a photoconversion assay to specifically visualise stably bound proteins within subcellular structures and organelles. This revealed that while paxillin and vinculin are distributed evenly throughout FAs, their stably bound fractions form small clusters within the FA-complex. These clusters are more concentrated for paxillin than for vinculin and are mostly found at the proximal half of the FA where actin also enters.

Osteoblast adhesion and response mediated by terminal -SH group charge surface of SiOxCy nanowires.

Robust cell adhesion is known to be necessary to promote cell colonization of biomaterials and differentiation of progenitors. In this paper, we propose the functionalization of Silicon Oxycarbide (SiOxCy) nanowires (NWs) with 3-mercaptopropyltrimethoxysilane (MPTMS), a molecule containing a terminal -SH group. The aim of this functionalization was to develop a surface capable to adsorb proteins and promote cell adhesion, proliferation and a better deposition of extracellular matrix. This functionalization can be used to anchor other structures such as nanoparticles, proteins or aptamers. It was observed that surface functionalization markedly affected the pattern of protein adsorption, as well as the in vitro proliferation of murine osteoblastic cells MC3T3-E1, which was increased on functionalized nanowires (MPTMS-NWs) compared to bare NWs (control) (p < 0.0001) after 48 h. The cells showed a better adhesion on MPTMS-NWs than on bare NWs, as confirmed by immunofluorescence studies on the cytoskeleton, which showed a more homogeneous vinculin distribution. Gene expression analysis showed higher expression levels for alkaline phosphatase and collagen I, putative markers of the osteoblast initial differentiation stage. These results suggest that functionalization of SiOxCy nanowires with MPTMS enhances cell growth and the expression of an osteoblastic phenotype, providing a promising strategy to improve the biocompatibility of SiOxCy nanowires for biomedical applications.

Interaction of osteoblast -TiO2 nanotubes in vitro: The combinatorial effect of surface topography and other physico-chemical factors governs the cell fate

In the context of surface modification of implants by TiO2 nanotubes, the topography of the nanotubes has been considered as a sole modulator of cellular response of adherent cells. However, a recent report has indicated about the existence of other contributing factors. Keeping this contradiction in mind, here we have performed a comprehensive study to account the contribution of the various factors such as morphological aspects, physico chemical aspects and the topographical features of TiO2 nanotubes on the cellular response. Here, the TiO2 nanotubes of varied topography were synthesized on the Ti6Al4V surface through anodic oxidation. SEM analysis showed that there was a variation in tube diameter, cross-sectional length, tube density and wall thickness along with the variation in oxidation time and voltage. An attempt to co-relate the cellular response of osteoblast cells (MG-63) cultured on the nanotubes has revealed that except cell proliferation; cell adhesion, spreading, vinculin distribution (focal adhesion) and differentiation (ALP activity) do not possess any direct relationship with the nanotube topography. Further, by comprehending the cellular responses with the variation in the extent of anatase phase (XRD study), surface roughness and wettability of nanotube surfaces, we established that the variation in cellular responses is a combinatorial effect of surface topography and other physico-chemical properties of TiO2 nanotubes. This study will help in developing bioactive implant surfaces modified with TiO2 nanotubes, for clinical application.

Symmetrical retrograde actin flow in the actin fusion structure is involved in osteoclast fusion.

ABSTRACTThe aim of this study was to elucidate the role of the zipper-like structure (ZLS), a podosome-related structure that transiently appears at the cell contact zone, in osteoclast fusion. Live-cell imaging of osteoclasts derived from RAW264.7 cells transfected with EGFP-actin revealed consistent symmetrical retrograde actin flow in the ZLS, but not in the podosome cluster, the podosome ring or the podosome belt. Confocal imaging showed that the distributions of F-actin, vinculin, paxillin and zyxin in the ZLS were different from those in the podosome belt. Thick actin filament bundles running outside the ZLS appeared to recruit non-muscle myosin IIA. The F-actin-rich domain of the ZLS contained actin-related protein 2/3 complex (Arp2/3). Inhibition of Arp2/3 activity disorganized the ZLS, disrupted actin flow, deteriorated cell-cell adhesion and inhibited osteoclast hypermultinucleation. In contrast, ML-7, an inhibitor of myosin light chain kinase, had little effect on the structure of ZLS and promoted osteoclast hypermultinucleation. These results reveal a link between actin flow in the ZLS and osteoclast fusion. Osteoclast fusion was promoted by branched actin elongation and negatively regulated by actomyosin contraction.

The distribution of vinculin to lipid rafts plays an important role in sensing stiffness of extracellular matrix.

Extracellular matrix (ECM) stiffness regulates cell differentiation, survival, and migration. Our previous study has shown that the interaction of the focal adhesion protein vinculin with vinexin α plays a critical role in sensing ECM stiffness and regulating stiffness-dependent cell migration. However, the mechanism how vinculin-vinexin α interaction affects stiffness-dependent cell migration is unclear. Lipid rafts are membrane microdomains that are known to affect ECM-induced signals and cell behaviors. Here, we show that vinculin and vinexin α can localize to lipid rafts. Cell-ECM adhesion, intracellular tension, and a rigid ECM promote vinculin distribution to lipid rafts. The disruption of lipid rafts with Methyl-β-cyclodextrin impaired the ECM stiffness-mediated regulation of vinculin behavior and rapid cell migration on rigid ECM. These results indicate that lipid rafts play an important role in ECM-stiffness regulation of cell migration via vinculin.

Impaired cytoskeletal arrangements and failure of ventral body wall closure in chick embryos treated with rock inhibitor (Y-27632).

Rho-associated kinase (ROCK) signaling regulates numerous fundamental developmental processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell contractility. ROCK knockout mice exhibit a ventral body wall defect (VBWD) phenotype due to disorganization of actin filaments at the umbilical ring. However, the exact molecular mechanisms leading to VBWD still remain unclear. Improper somitogenesis has been hypothesized to contribute to failure of VBW closure. We designed this study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) disrupts cytoskeletal arrangements in morphology during early chick embryogenesis, which may contribute to the development of VBWD. At 60h incubation, chick embryos were explanted into shell-less culture and treated with 50µL of vehicle for controls (n=33) or 50µL of 500µM of Y-27632 for the experimental group (Y-27, n=56). At 8h post-treatment, RT-PCR was performed to evaluate mRNA levels of N-cadherin, E-cadherin and connexin43. Immunofluorescence confocal microscopy was performed to analyze the expression and distribution of actin, vinculin and microtubules in the neural tube and somites. A further cohort of embryos was treated in ovo by dropping 50µL of vehicle or 50µL of different concentrations of Y-27632 onto the embryo and allowing development to 12 and 14days for further assessment. Gene expression levels of N-cadherin, E-cadherin and connexin43 were significantly decreased in treated embryos compared with controls (p<0.05). Thickened actin filament bundles were recorded in the neural tube of Y-27 embryos. In somites, cells were dissociated with reduced actin distribution in affected embryos. Clumping of vinculin expression was found in the neural tube and somites, whereas reduced expression of microtubules was observed in Y-27 embryos compared with controls. At 12 and 14days of development, affected embryos presented with an enlarged umbilical ring and herniation of abdominal contents through the defect. ROCK inhibition alters cytoskeletal arrangement during early chick embryogenesis, which may contribute to failure of anterior body wall closure causing VBWD at later stages of development.

Extracellular Substrate Stiffness Alters Macrophage Morphology and Function

Macrophages participate in immunity, tissue repair, and tissue homeostasis, and also mediate pathologic conditions including atherosclerosis and cancer. Depending on microenvironmental cues, macrophages may adopt a pro‐inflammatory or anti‐inflammatory phenotype. Tissue stiffness influences the behavior and differentiation of cells, yet little is known about how the physical extracellular environment influences macrophage function. To study the role of substrate stiffness in determining macrophage phenotype, we cultured murine macrophages on tunable polyacrylamide gels that approximate different tissue stiffnesses. Macrophages on rigid surfaces had larger surface area and reduced circularity compared to macrophages on compliant gels. On rigid surfaces, phagocytosis was increased, but lipid uptake was decreased. Cytokine secretion differed in macrophages grown on compliant versus rigid surfaces. Differences in response to the toll‐like receptor ligands CpG oligonucleotide and lipopolysaccharide were also noted. The distribution of vinculin, a component of mature focal adhesions that mediate mechanosensing, was punctate in macrophages grown on glass and diffuse in macrophages grown on compliant gels, suggesting a fundamental difference in the quantity and/or character of the focal adhesions. Together, our studies demonstrate that the mechanical properties of the extracellular environment have a direct impact on macrophage behavior and function. Furthermore, these findings have important implications for a variety of pathologic conditions that involve both macrophages and altered tissue stiffness such as fibrosis, atherosclerosis, and neoplasia.The studies were supported by NIH T32 Comparative Medicine Training Grant.

The molecular dynamics of crawling migration in microtubule-disrupted keratocytes.

Cell-crawling migration plays an essential role in complex biological phenomena. It is now generally believed that many processes essential to such migration are regulated by microtubules in many cells, including fibroblasts and neurons. However, keratocytes treated with nocodazole, which is an inhibitor of microtubule polymerization – and even keratocyte fragments that contain no microtubules – migrate at the same velocity and with the same directionality as normal keratocytes. In this study, we discovered that not only these migration properties, but also the molecular dynamics that regulate such properties, such as the retrograde flow rate of actin filaments, distributions of vinculin and myosin II, and traction forces, are also the same in nocodazole-treated keratocytes as those in untreated keratocytes. These results suggest that microtubules are not in fact required for crawling migration of keratocytes, either in terms of migrating properties or of intracellular molecular dynamics.

Palladin Plays an Important Role in Pulmonary Cellular Infiltration in Differentiation Syndrome

BackgroundAcute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation, which fuses the promyelocytic leukemia (PML) gene on chromosome 15 to the retinoic acid receptor-(RARα ) gene on chromosome 17. The introduction of all-trans retinoic acid (ATRA) into the treatment strategy of APL fundamentally changed the management and outcome of this disease. Treatment with ATRA relieves this blockage, resulting in terminal differentiation of the APL blasts.AimsTo investigate the molecular mechanisms of ATRA induced differentiation, we have described a series of novel genes up-regulated by ATRA. One of them, Rig-K (Retinoic-acid-induced gene-K), was the human homolog of palladin. This study is mainly focus on the function of palladin gene during the process of differentiation.Methods1. Lentivirus shRNA expression system was used to generate stable palladin konckdown NB4 cell lines. Cells morphological and the expression of differentiation markers were observed；2. Use flow cytometry and immunofluorescence to compare the difference of cell cycle, cell proliferation, apoptosis, cell migration and granulocyte related functions such as adhesion, phagocytosis between control and palladin knockdown group;3. Real-time PCR and chemokines antibody array were used to detect the effect of palladin reduction on the levels of chemokines secreted by NB4 cells；4. Use microgravity rotary cell culture system to study the infiltration capacity of differentiated NB4 cells to lung tissues. Observe whether palladin knockdown play a role in this processes.Results1. Palladin knockdown has minor effect on NB4 differentiation based on cell morphology and granulocytic differentiation-related antigens. Palladin knockdown can partly relieved the proliferation inhibition effect during differentiation. Cycle arrest can partly relieved by palladin knock down manifest as reduced G0/G1 arrest. Palladin knock down can also remarkable reduce apoptosis rate compared with control group at 96 hours time point;2. The phagocytosis level was about 50% dropped when the palladin was knocked down, suggest palladin play important roles in phagocytosis progress. Besides, we also observed the localization of palladin in the phagocytic cup, and palladin is colocalizated with F-actin in phagocytic cup. The migration level was also about 50% dropped when the palladin was knocked down in differentiated NB4 cells. Palladin colocalization with F-actin and vinculin, knockdown of palladin alter the distribution of F-actin and vinculin, suggest palladin take part in the formation of actin related structures;3. Differentiation induction of APL cells is associated with increased expression of specific adhesion molecules and inflammatory cytokines, palladin knockdown decrease the upregulation of many chemokines;4. Palladin knockdown also reduced infiltration ability of differentiated NB4 cells into lung tissues.ConclusionPalladin knockdown significantly decreased the granulocyte related function of differentiated APL cells, especially cell motile ability and secretion of chemokines. In vitro model shows that palladin knockdown can inhibit the invasion ability of differentiated APL cells into lung tissues, suggest palladin may play important roles in retinoic acid syndrome. DisclosuresNo relevant conflicts of interest to declare.

Physiologically induced restructuring of focal adhesions causes mobilization of vinculin by a vesicular endocytic recycling pathway

In epithelial cells, vinculin is enriched in cell adhesion structures but is in equilibrium with a large cytosolic pool. It is accepted that when cells adhere to the extracellular matrix, a part of the soluble cytosolic pool of vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FAs) by binding to plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We have previously shown that bradykinin (BK) induces both a reversible dissipation of vinculin from FAs, by the phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P2, and the concomitant internalization of vinculin. Here, by using an immunomagnetic method, we isolated vinculin-containing vesicles induced by BK stimulation. By analyzing the presence of proteins involved in vesicle traffic, we suggest that vinculin can be delivered in the site of FA reassembly by a vesicular endocytic recycling pathway. We also observed the formation of vesicle-like structures containing vinculin in the cytosol of cells treated with lipid membrane-affecting agents, which caused dissipation of FAs due to their deleterious effect on membrane microdomains where FAs are inserted. However, these vesicles did not contain markers of the recycling endosomal compartment. Vinculin localization in vesicles has not been reported before, and this finding challenges the prevailing model of vinculin distribution in the cytosol. We conclude that the endocytic recycling pathway of vinculin could represent a physiological mechanism to reuse the internalized vinculin to reassembly new FAs, which occurs after long time of BK stimulation, but not after treatment with membrane-affecting agents.

TIMP-1 modulates chemotaxis of human neural stem cells through CD63 and integrin signalling

We recently reported that hNSCs (human neural stem cells) have the interesting characteristic of migration towards an intracranial glioma. However, the molecules and mechanisms responsible for tumour tropism are unclear. In the present study, we used microarray and proteomics analyses to identify a novel chemoattractant molecule, TIMP-1 (tissue inhibitor of metalloproteinase-1), secreted from human brain tumour tissues. We demonstrate that TIMP-1 significantly enhances hNSC adhesion and migration in a cell culture system. These effects were critically dependent on CD63, as shRNA-mediated ablation of CD63 expression attenuated the response. TIMP-1 significantly increased the number of FAs (focal adhesions) and cytoskeletal reorganization for cell migration in hNSCs, whereas knockdown of CD63 resulted in decreased hNSC spreading, FAs and migration, even after TIMP-1 treatment. In addition, TIMP-1 binding to CD63 activated β1 integrin-mediated signalling through Akt and FAK phosphorylation, leading to pattern changes in distribution of vinculin and F-actin (filamentous actin). Furthermore, inactivation of β1 integrin by use of a blocking antibody or inhibition of PI3K (phosphoinositide 3-kinase) signalling impaired the migration of hNSCs towards TIMP-1. Collectively, our results underline TIMP-1 as a novel and effective key regulator of CD63 and β1 integrin-mediated signalling, which regulates hNSC adhesion and migration.

Rho-associated protein kinase modulates neurite extension by regulating microtubule remodeling and vinculin distribution.

Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distribution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulating Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite outgrowth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased membrane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vinculin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.

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.

The effect of surface topography on cell shape and early ERK1/2 signaling in macrophages; linkage with FAK and Src

Implant surface topography can modulate macrophage behavior during wound healing by the production of proinflammatory cytokines. This study investigated the activation of FAK, Src, and ERK1/2 signaling intermediates of the proinflammatory ERK1/2 pathway in RAW 264.7 macrophages in response to polished (P), coarse-grit-blasted (B), acid etched (E), and grit-blasted and etched (SLA) surface topographies. In addition, the effects of these topographies on cell spreading, vinculin organization, and viability were determined. Macrophages on the SLA surface changed from predominantly well-spread cells to ones with a more spherical morphology over time. In contrast, macrophages on the P surface changed from being predominantly spherical cells to well spread. The morphological changes were associated with changes in the distribution of vinculin. The overall patterns of the pFAK, pSrc, pERK1/2 levels as well as pERK1/2 nuclear translocation associated with cell shape with greater activation being seen with a more spread morphology. These results suggest that surface topography differentially activates signaling pathways that affect cell function and raise the possibility that topographies can be designed to optimize desired cell responses.

Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells

AimsFormation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells. Main methodsColon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization. Key findingsIncreased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential. SignificanceOur experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.

Integrin-mediated signaling contributes to gadolinium-containing-particle-promoted cell survival and G1 to S phase cell cycle transition by enhancing focal adhesion formation

We previously reported that Gd-containing particles formed under physiological conditions act as active entities to enhance cell survival and promote S phase entry via activation of both mitogen-activated protein kinase/extracellular-signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways. However, how they transduce the extracellular signal inside the cell remains unclear. The present study demonstrates that Gd-containing particles can alleviate serum-deprivation-induced cell death and promote G₁ to S phase cell cycle progression by enhancing cell adhesion to the extracellular matrix. As an indicator of adhesion, the vinculin distribution was detected by confocal laser scanning microscopy. The control cells exhibited fewer and less typical focal adhesions. After treatment with Gd-containing particles, a large number of vinculin-containing focal adhesions were maintained. In the presence of integrin antagonists, the percentage of S phase entry induced by Gd-containing particles was decreased and the enhancement of cell viability was also attenuated, along with a decrease in both cyclin D expression and ERK phosphorylation. In summary, the present results suggest that the integrin-mediated signaling pathway plays an important role in cell survival and G₁ to S phase transition promoted by Gd-containing particles by enhancing focal adhesion formation. The results presented here provide novel evidence to advance knowledge leading to further understanding of the mechanisms of both cell proliferation and cell survival promoted by Gd and may be helpful for developing effective measures to prevent or treat nephrogenic systemic fibrosis.

The difference of fibroblast behavior on titanium substrata with different surface characteristics

Connective tissue, one of the main components of peri-implant soft tissue, is key to the formation of the peri-implant mucosal seal and helping to prevent epithelial ingrowth. Rough surfaces (Rs), machined surfaces (Ms) or microgrooved surface (MG) are used in the neck area of commercially available titanium implants. In this paper, we aimed to evaluate the influence of surface topography of titanium substratum on connective tissue fibroblasts to gain a better understanding of this effect. Fibroblasts were cultured on titanium plates with Rs, Ms and MG. Adhesion cell number at day 3 was compared and protein distribution of both F-actin and vinculin was determined to observe cellular structure and adhesion. Cell adhesion strength was compared on each surface. At day 3, the number of fibroblasts attached on each substratum was in the order of MG ≈ Ms > Rs. Fibroblasts strongly expressed vinculin in the peripheral area on Ms and MG, and showed strong F-actin architecture. Decreased expression of vinculin and weaker continuity of F-actin were observed on Rs. Fibroblasts on MG were aligned along the grooves, with a significantly higher cell density, whereas cells on Ms and Rs had no clear orientation. The cell adhesion strength was significantly lower on Rs, and no significant difference was seen between MG and Ms. Both MG and Ms showed greater adhesion cell numbers and adhesion strength of fibroblasts when compared with Rs at day 3. The cell density on MG was greater than those on other substrata.