Early molecular events in the interaction of enveloped viruses with cells

The fluorescence depolarization of 1,6-diphenyl-hexatriene was used to study the dynamic properties of the hydrophobic regions of the lipid envelopes of ortho- and paramyxoviruses as well as of the Rous sarcoma virus and of the membrane lipids of susceptible and nonsusceptible cells.The systems investigated where active and inactive influenza viruses, and NDV virus acting on chick embryo fibroblasts and Rous sarcoma virus acting on susceptible (C/E) and nonsusceptible (C/B) chicken-cell.Polarization degrees and mean rotational correlation times of DPH embedded in viral lipids were significantly higher than those of DPH in the cell membranes, due to a higher rigidity of the virus envelopes. When suspensions of labelled viruses and unlabelled cells or unlabelled viruses and labelled cells were mixed, a characteristic change of the fluorescence polarization degrees with time was observed. This behaviour was ascribed to a label transfer from virus to cell membranes or vice versa.While the rate constants of label transfer from virus to cells and cells to virus were about the same for the penetrating viruses the rate constants of label release from inactive virus to cells were much larger than for the migration in the opposite direction.     

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.     

The urokinase receptor: Focused cell surface proteolysis, cell adhesion and signaling

Plasma membrane urokinase-type plasminogen activator (uPA)-receptor (uPAR) is a GPI-anchored protein that binds with high-affinity and activates the serine protease uPA, thus regulating proteolytic activity at the cell surface. In addition, uPAR is a signaling receptor that often does not require its protease ligand or its proteolytic function.uPAR is highly expressed during tissue reorganization, inflammation, and in virtually all human cancers. Since its discovery, in vitro and in vivo models, as well as retrospective clinical studies have shown that over-expression of components of the uPA/uPAR-system correlates with increased proliferation, migration, and invasion affecting the malignant phenotype of cancer. uPAR regulates the cells-extracellular matrix interactions promoting its degradation and turnover through the plasminogen activation cascade.     

蛋白激酶PRKX对人肝癌细胞粘附和迁移能力的影响

目的观察蛋白激酶PRKX对人肝癌细胞SMMC-7721粘附和迁移能力的影响。方法采用脂质体转染的方法,将PRKX表达质粒转染到SMMC-7721细胞中,蛋白印迹方法鉴定转染前后PRKX蛋白的表达。细胞-基质粘附实验测定对照组和PRKX转染组SMMC-7721细胞的粘附能力。细胞迁移实验测定对照组和PRKX转染组SMMC-7721细胞的迁移能力。结果 SMMC-7721细胞转染组PRKX蛋白的表达增加,SMMC-7721细胞转染组的粘附能力和迁移能力均较对照组增加。结论 PRKX可增加人肝癌细胞SMMC-7721的粘附和迁移能力。     

TIMP-2 promotes cell spreading and adhesion via upregulation of Rap1 signaling

We previously demonstrated that TIMP-2 treatment of human microvascular endothelial cells (hMVECs) activates Rap1 via the pathway of paxillin-Crk-C3G. Here, we show that TIMP-2 overexpression in hMVECs by adenoviral infection enhances Rap1 expression, leading to further increase in Rap1-GTP. TIMP-2 expression, previously reported to inhibit cell migration, also leads to cell spreading accompanied with increased cell adhesion. HMVECs stably expressing Rap1 display a similar phenotype as hMVECs-TIMP-2, whereas the expression of inactive Rap1 mutant, Rap1(38N), leads to elongated appearance with greatly reduced cell adhesion. Furthermore, the phenotype of hMVECs-Rap1(38N) was not reversed by TIMP-2 overexpression. TIMP-2 greatly promotes the association of Rap1 with actin. Therefore, these findings suggest that TIMP-2 mediated alteration in cell morphology requires Rap1, TIMP-2 may recruit Rap1 to sites of actin cytoskeleton remodeling necessary for cell spreading, and enhanced cell adhesion by TIMP-2 expression may hinder cell migration.     

Silencing of VAMP3 inhibits cell migration and integrin-mediated adhesion

Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of β1 integrin at the cell surface but had no effect on total cellular β1 integrin, indicating that VAMP3 is required for trafficking of β1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.     

The effect of tributyltin on human eosinophylic leukemia EoL-1 cells

Organotin compounds are chemicals that are widely used in industry and agriculture as plastic stabilizers, catalysts and biocides. Many of them, including tributyltin (TBT), have been detected in human food and, as a consequence, detectable levels have been found in human blood. As organotin compounds were shown to possess immunotoxic activity, we focused our attention on the effect of TBT on the basic determinants of the function of eosinophils, i.e. cell adhesiveness and motility. We used human eosinophylic leukemia EoL-1 cells, a common in vitro cellular model of human eosinophils. Here, we demonstrate that TBT causes a dose-dependent decrease in the viability of EoL-1 cells. When administered at sub-lethal concentrations, TBT significantly decreases the adhesion of EoL-1 cells to human fibroblasts (HSFs) and inhibits their migration on fibroblast surfaces. Since the basic function of eosinophils is to invade inflamed tissues, our results indicate that TBT, and possibly other organotin compounds, may affect major cellular properties involved in the determination of in vivo eosinophil function. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication costs were covered by the organisers of this meeting.     

TEM8 expression stimulates endothelial cell adhesion and migration by regulating cell-matrix interactions on collagen

The TEM8 gene is selectively expressed in tumor versus normal blood vessels, though its function in endothelial cell biology is not known. Towards the goal of clarifying this function, we tested whether TEM8 overexpression, or blocking TEM8's function with a dominant negative protein, would modulate endothelial cell activities. We found that TEM8-expressing endothelial cells migrated at a rate 3-fold greater than control cells in a monolayer denudation assay. Also, the addition of recombinant TEM8 extracellular domain (TEM8-ED) specifically inhibited both chemokinetic and chemotactic migration on collagen in the denudation and Boyden chamber assays, respectively. The TEM8-ED binds preferentially to collagen, and TEM8 expression enhanced endothelial adhesion to collagen 3-fold; the latter response was antagonized by the TEM8-ED. Consistent with the TEM8-ED acting as a dominant negative inhibitor of endogenously expressed protein were data showing that the TEM8-ED had no effect on the activation of beta1 integrin. TEM8 protein is present in human umbilical vein in situ and is expressed in low passage HUVEC in vitro. TEM8 protein expression in HUVEC was increased 5-fold by the initiation of tube formation, correlating expression of TEM8 with the angiogenic response. Taken together, these results indicate that TEM8 plays a positive role in endothelial cell activities related to angiogenesis.     

Analysis of directional cell migration on defined FN gradients: role of intracellular signaling molecules

Directional cell migration on extracellular matrix (ECM) plays important roles in embryonic development and adult organisms. To study the mechanisms and signaling pathways involved in the regulation of directional cell migration, we created defined fibronectin (FN) gradients by using microfluidic systems. We found that fibroblasts exhibited haptotaxis towards higher FN concentration on the gradient. Furthermore, the net movements in the direction of FN gradients correlated with the increase in the slope of the gradient although the overall rate of migration was not correlated. Consistent with previous observations on the uniformly coated surface, local higher FN concentration led to reduced migration rate due to increased spreading. Upon transfection of N-WASP or activated Cdc42, but not FAK or Grb7, the cells showed increased directional migration. However, transfection of FAK, but not the other signaling molecules, led to an increase in the persistence of directional cell migration, which is dependent on the slope of the gradient as well as FAK interaction with PI3K. Together, these studies reveal some novel properties of directional cell migration on defined FN gradient and suggested a role for FAK signaling and N-WASP and Cdc42 in the differential regulation of the persistence and rate of directional cell migration.     

Laminin-5-deficient human keratinocytes: defective adhesion results in a saltatory and inefficient mode of migration

Laminin-5 is a major adhesion protein of the skin basement membrane and crucially involved in integrin-mediated cell substrate attachment of keratinocytes, which is important for hemidesmosomal anchorage as well as for keratinocyte migration during epidermal wound healing. To investigate its role in keratinocyte migration, we analyzed laminin-5-deficient cells of patients with a lethal variant of junctional epidermolysis bullosa. Normal migrating keratinocytes adopted monopolar morphology with a distinct front lamella and employed a continuous mode of translocation. In contrast, laminin-5-deficient cells assumed a stretched bipolar shape with two lamella regions and migrated in a discontinuous, saltatory manner characterized by significantly decreased directional persistence and reduced migration velocity. The distinct morphology as well as the migratory phenotype apparently resulted from a defect in the formation of cell substrate adhesions that were completely missing in the cell body and less stable in the lamella regions. Accordingly in normal keratinocytes, a bipolar shape and a saltatory migration mode were inducible by blocking laminin-5-mediated substrate adhesion. Our findings clearly point to an essential role of laminin-5 in forming dynamic cell substrate adhesion during migration of epidermal keratinocytes and provide an explanation for the cellular mechanisms that underlie the lethal form of junctional epidermolysis bullosa.     

Is there a pilot in a pseudopod?

The concept of pilot pseudopodia is reconsidered 30 years after its inauguration (Gerisch, G., Hülser, D., Malchow, D., Wick, U., 1975. Cell communication by periodic cyclic-AMP pulses. Phil. Trans. R. Soc. Lond. B 272, 181-192). The original hypothesis stated that protruding pseudopodia serve as dynamic sensory organelles that aid a cell in perceiving variations of chemoattractant concentration and, consequently, in navigation during chemotaxis. This influential idea is reevaluated in the light of recent findings about the mechanisms governing chemotactic cell motility, morphology and dynamics of pseudopodia, and about molecular constituents and regulators of pseudopod extension and retraction. It is proposed that stimulation by a chemoattractant modulates speed of pseudopod protrusion and thereby increases cell elongation. Elongation further enhances chemotactic sensitivity of the cell to shallow chemoattractant gradients, reinforces cell polarization, and finally leads to suppression of lateral pseudopodia and continuation of cell migration in the gradient direction.     

Type II phosphatidylinositol 4-kinase β is an integral signaling component of early T cell activation mechanisms

The early signaling events in T cell activation through CD3 receptor include a rapid change in intra cellular free calcium concentration and reorganization of actin cytoskeleton. Phosphatidylinositol 4-kinases (PtdIns 4-kinases) are implicated as key components in these early signaling events. The role of type II PtdIns 4-kinase β in CD3 receptor signaling was investigated with the help of short hairpin RNA sequences. Cross-linking of CD3 receptors on Jurkat T Cells with monoclonal antibodies showed an early increase in type II PtdIns 4-kinase activity and co-localization of type II PtdIns 4-kinase β with CD3 ζ. Transfection of Jurkat T Cells with shRNAs inhibited CD3 receptor mediated type II PtdIns 4-kinase activation with a concomitant reduction in intra cellular calcium release, suggesting a role for type II PtdIns 4-kinase β in CD3 receptor signal transduction. Knock-down of type II PtdIns 4-kinase β with shRNAs also correlated with a decrease in PtdIns 4-kinase activity in cytoskeleton fractions and reduced adhesion to matrigel surfaces. These results indicate that type II PtdIns 4-kinase β is a key component in early T cell activation signaling cascades.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

Roles and modes of action of nectins in cell–cell adhesion

Nectins are Ca²⁺-independent immunoglobulin (Ig)-like cell–cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell–cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell–cell junctions and cell–cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell–cell adhesion and recruit E-cadherin to the nectin-based cell–cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.