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Integrin receptors connect the extracellular matrix to the cell cytoskeleton to provide essential forces and signals. To examine the contributions of the beta 1 integrin cytoplasmic tail to adhesive forces, we generated cell lines...
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Integrin receptors connect the extracellular matrix to the cell cytoskeleton to provide essential forces and signals. To examine the contributions of the beta 1 integrin cytoplasmic tail to adhesive forces, we generated cell lines expressing wild-type and tail mutant beta 1 integrins in beta 1-null fibroblasts. Deletion of beta 1 significantly reduced cell spreading, focal adhesion assembly, and adhesive forces, and expression of human beta 1 (h beta 1) integrin in these cells restored adhesive functions. Cells expressing a truncated tail mutant had impaired spreading, fewer and smaller focal adhesions, reduced integrin binding to fibronectin, and lower adhesion strength and traction forces compared to h beta 1-expressing cells. All these metrics were equivalent to those for beta 1-null cells, demonstrating that the beta 1 tail is essential to these adhesive functions. Expression of the constitutively-active D759A h beta 1 mutant restored many of these adhesive functions in beta 1-null cells, although with important differences when compared to wild-type beta 1. Even though there were no differences in integrin-fibronectin binding and adhesion strength between h beta 1- and h beta 1-D759A-expressing cells, h beta 1-D759A-expressing cells assembled more but smaller adhesions than h beta 1-expressing cells. Importantly, h beta 1-D759A-expressing cells generated lower traction forces compared to h beta 1-expressing cells. These differences between h beta 1- and h beta 1-D759A-expressing cells suggest that regulation of integrin activation is important for fine-tuning cell spreading, focal adhesion assembly, and traction force generation. (C) 2014 Elsevier Inc. All rights reserved.
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Viral transmission from an infected cell to a target cell has been long appreciated to be more efficient than infection with a cell-free virus. New work using high-resolution, live-cell microscopy techniques provides important ins...
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Viral transmission from an infected cell to a target cell has been long appreciated to be more efficient than infection with a cell-free virus. New work using high-resolution, live-cell microscopy techniques provides important insights into the mechanisms underlying the efficiency of retrovirus transmission between cells.
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Focal brain ischemia is best studied in neocortex and striatum. Both show highly vulnerable neurons and high susceptibility to spreading depolarization (SD). Therefore, it has been hypothesized that these two variables generally c...
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Focal brain ischemia is best studied in neocortex and striatum. Both show highly vulnerable neurons and high susceptibility to spreading depolarization (SD). Therefore, it has been hypothesized that these two variables generally correlate. However, this hypothesis is contradicted by findings in cerebellar cortex, which contains highly vulnerable neurons to ischemia, the Purkinje cells, but is said to be less susceptible to SD. Here, we found in the rat cerebellar cortex that elevated K+ induced a long-lasting depolarizing event superimposed with SDs. Cerebellar SDs resembled those in neocortex, but negative direct current (DC) shifts and regional blood flow responses were usually smaller. The K+ threshold for SD was higher in cerebellum than in previous studies in neocortex. We then topically applied endothelin-1 (ET-1) to the cerebellum, which is assumed to cause SD via vasoconstriction-induced focal ischemia. Although the blood flow decrease was similar to that in previous studies in neocortex, the ET-1 threshold for SD was higher. Quantitative cell counting found that the proportion of necrotic Purkinje cells was significantly higher in ET-1-treated rats than sham controls even if ET-1 had not caused SDs. Our results suggest that ischemic death of Purkinje cells does not require the occurrence of SD.
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The paper presents recent results of the study of spreading and adhesion of human osteosarcoma cells on soft polydimethylsiloxane (PDMS) materials. Cell/surface interactions are studied on smooth and micro-grooved PDMS surfaces. T...
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The paper presents recent results of the study of spreading and adhesion of human osteosarcoma cells on soft polydimethylsiloxane (PDMS) materials. Cell/surface interactions are studied on smooth and micro-grooved PDMS surfaces. The viscoelastic spreading behavior of the human osteosarcoma cells (during the spreading stages on smooth PDMS surfaces) was studied using wetting theory. The HOS cell spreading behavior was also investigated in the micro-grooved PDMS surfaces in an effort to study the contact guidance formation process. The results show that the initial stages of HOS cell spreading can be modeled as a complete wetting process. The results also show that the cell cytoplasm contributes more to the spreading process than the nucleus. In the case of cell spreading on micro-grooved surfaces, the cell tractions resulted in significant deformation of the microgrooves. The tractions were also calculated, and found to be in good agreement with the results from other studies. The results suggest that the cell spreading-induced soft substrate deformation needs to be considered in the design of implantable bioMEMS structures.
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The spread of viral infection within a host can be restricted by bottlenecks that limit the size and diversity of the viral population. An essential process for alphaherpesvirus infection is spread from axons of peripheral nervous...
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The spread of viral infection within a host can be restricted by bottlenecks that limit the size and diversity of the viral population. An essential process for alphaherpesvirus infection is spread from axons of peripheral nervous system neurons to cells in peripheral epithelia (anterograde-directed spread, ADS). ADS is necessary for the formation of vesicular lesions characteristic of reactivated her-pesvirus infections; however, the number of virions transmitted is unknown. We have developed two methods to quantitate ADS events using a compartmentalized neuronal culture system. The first method uses HSV-1 and pseudorabies virus recombinants that express one of three different fluorescent proteins. The fluorescence profiles of cells infected with the virus mixtures are used to quantify the number of expressed viral genomes. Strikingly, although epithelial or neuronal cells express 3-10 viral genomes after infection by free virions, epithelial cells infected by HSV-1 or pseudorabies virus following ADS express fewer than two viral genomes. The second method uses live-cell fluorescence microscopy to track individual capsids involved in ADS. We observed that most ADS events involve a single capsid infecting a target epithelial cell. Together, these complementary analyses reveal that ADS events are restricted to small numbers of viral particles, most often a single virion, resulting in a single viral genome initiating infection.
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Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by beta 1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which t...
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Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by beta 1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate beta 1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of beta 1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase C epsilon bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of beta 1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase C epsilon is an essential regulatory step for the trafficking and activation of beta 1 integrins and cell spreading on collagen.
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Porous calcium polyphosphate (CPP) structures represent promising resorbable implant systems that can promote anchorage to connective tissues. Previous studies focused on chondrocyte interactions with CPP, but there are limited da...
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Porous calcium polyphosphate (CPP) structures represent promising resorbable implant systems that can promote anchorage to connective tissues. Previous studies focused on chondrocyte interactions with CPP, but there are limited data on interactions of soft connective tissue cells with these materials. We studied attachment, spreading, and matrix formation by human gingival fibroblasts when cultured on amorphous and crystalline CPP. Comparison with porous Ti6Al4V substrates of similar volume percent, porosity, and pore size distribution provided evaluations of fibroblast interactions with rapid, moderate, and nonbiodegrad-able systems, respectively. Cells were incubated on substrates in medium containing ascorbic acid and evaluated at 3, 24, 48, 72, and 96 h after plating. Attached cell counts, cytoplasmic actin filament area, and immunostained extra-cellular type 1 collagen or fibronectin were quantified by morphometric analyses using epifluorescence microscopy. Cell morphology and substrate interactions were evaluated by scanning electron microscopy. Spreading, attachment, and matrix production were similar for both CPP substrates. In contrast, titanium alloy substrates exhibited threefold more attachment and twofold more spreading than CPP substrates. The area per cell of immunostained extracellular collagen and fibronectin was similar for the three different substrates. The results indicate that the crystallinity and, hence, degradation rate of CPP substrates does not substantially affect the interactions of fibroblasts with CPP materials but that compared with titanium alloy substrates, spreading and attachment are inhibited.
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Osteoblasts are susceptible to the surface characteristics of bioceramics and stimulation from outside the cells. The purpose of this study was to evaluate the effects of electrical polarization on surface characteristics and oste...
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Osteoblasts are susceptible to the surface characteristics of bioceramics and stimulation from outside the cells. The purpose of this study was to evaluate the effects of electrical polarization on surface characteristics and osteoblastic adhesion. The surface characteristics revealed that electrical polarization had no effect on the surface roughness, crystallinity, and constituent elements. According to contact-angle measurements, electrically polarized hydroxyapatite (HA), which provides two kinds of surfaces, negatively charged HA (N-HA) and positively charged HA (P-HA). was even more hydrophilic than that of normal HA (O-HA). Morphological observations and quantitative analyses revealed that the typical adhered cells had a round shape on O-HA but had a spindle or fanlike spreading configuration on N-HA and P-HA I h after seeding. After 3 h of cultivation, the rate of the number of spread cells and the size of the focal adhesions on O-HA increased and approached that of N-HA and P-HA. However, the cell areas positively stained for actin, which indicates the degree of cell spreading, were distinctly larger on N-HA and P-HA than that on O-HA. The number of focal adhesions per cell was also less than that on N-HA and P-HA.
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The proline-rich tyrosine kinase 2, Pyk2, is a focal adhesion related kinase expressed in T cells that is tyrosine phosphorylated and activated by integrin, chemokine or T cell receptor stimulation. Ligation of the cell adhesion m...
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The proline-rich tyrosine kinase 2, Pyk2, is a focal adhesion related kinase expressed in T cells that is tyrosine phosphorylated and activated by integrin, chemokine or T cell receptor stimulation. Ligation of the cell adhesion molecule CD44 also induces Pyk2 phosphorylation and T cell spreading, and this is negatively regulated by the protein tyrosine phosphatase CD45. Here, we identify the activation requirements for Pyk2 and demonstrate its requirement for CD44-mediated elongated T cell spreading. Upon CD44-mediated cell spreading, Pyk2 was recruited to CD44 clusters in both CD45+ and CD45- T cells, yet was more strongly phosphorylated in T cells lacking CD45. In these cells, Pyk2 phosphorylation was dependent on Src family kinase activity and required actin polymerisation, phosphatidylinositol-3 kinase and phospholipase C activity as well as extracellular calcium. Inhibition of any of these events prevented Pyk2 phosphorylation and T cell spreading. Transfection of a truncated form of Pyk2 lacking the kinase domain, PRNK, inhibited CD44-mediated cell spreading, demonstrating an important role for Pyk2. However, inhibition of microtubule turnover by Taxol prevented elongated T cell spreading but did not affect Pyk2 phosphorylation, indicating that microtubule reorganisation is downstream, or independent, of Pyk2 phosphorylation. Together this demonstrates that multiple factors are required for CD44-induced Pyk2 activation, which plays a critical role in CD44-mediated elongated T cell spreading.
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In this report, we studied the interactions between biological cells and vertically aligned silicon nanowire (SiNW) arrays and focused on how SiNW arrays affected cellular behaviors such as cell adhesion and spreading. We observed...
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In this report, we studied the interactions between biological cells and vertically aligned silicon nanowire (SiNW) arrays and focused on how SiNW arrays affected cellular behaviors such as cell adhesion and spreading. We observed that SiNW arrays could support cell adhesion and growth and guide cell adhesion and spreading behaviors. The results also showed that SiNW arrays could not only enhance the cell-substrate adhesion force but also restrict cell spreading. Combining the results from scanning electron microscopy images of cell morphology and the expression analysis of genes and proteins related to cell adhesion and spreading process, we proposed a mechanism on how cell adhesion and spreading was controlled by arrayed SiNWs. The effects of SiNW arrays in guiding cell adhesion and spreading behavior might be useful in the development of cell microarrays, tissue engineering scaffolds, and molecule delivery vehicles in which strong cell-substrate adhesion and reduced cell-cell communication were beneficial.
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