Integrin switching modulates adhesion dynamics and cell migration

When cells are stimulated to move, for instance during development, wound healing or angiogenesis, they undergo changes in the turnover of their cell-matrix adhesions. This is often accompanied by alterations in the expression profile of integrins—the extracellular matrix receptors that mediate anchorage within these adhesions. Here, we discuss how a shift in expression between two different types of integrins that bind fibronectin can have dramatic consequences for cell-matrix adhesion dynamics and cell motility.Key words: integrin, fibronectin, migration, cytoskeleton, dynamicsCells attach to the extracellular matrix (ECM) that surrounds them in specialized structures termed “cell-matrix adhesions.” These come in different flavors including “focal complexes” (small adhesions found in membrane protrusions of spreading and migrating cells), “focal adhesions” (larger adhesions connected by F-actin stress fibers that are derived from focal complexes in response to tension), “fibrillar adhesions” (elongated adhesions associated with fibronectin matrix assembly), and proteolytically active adhesions termed “podosomes” or “invadopodia” found in osteoclasts, macrophages and certain cancer cells. Common to all these structures is the local connection between ECM proteins outside- and the actin cytoskeleton within the cell through integrin transmembrane receptors. The intracellular linkage to filamentous actin is indirect through proteins that concentrate in cell-matrix adhesions such as talin, vinculin, tensin, parvins and others.¹Cell migration is essential for embryonic development and a number of processes in the adult, including immune cell homing, wound healing, angiogenesis and cancer metastasis. In moving cells, cell-matrix adhesion turnover is spatiotemporally controlled.² New adhesions are made in the front and disassembled in the rear of cells that move along a gradient of motogenic factors or ECM proteins. This balance between formation and breakdown of cell-matrix adhesions is important for optimal cell migration. Several mechanisms regulate the turnover of cell-matrix adhesions. Proteolytic cleavage of talin has been identified as an important step in cell-matrix adhesion disassembly³ and FAK and Src family kinases are required for cell-matrix adhesion turnover and efficient cell migration.^4,5 Besides regulating phospho-tyrosine-mediated protein-protein interactions within cell-matrix adhesions, the FAK/Src complex mediates signaling downstream of integrins to Rho GTPases, thus controlling cytoskeletal organization.^6,7 The transition from a stationary to a motile state could involve (local) activation of such mechanisms.Interestingly, conditions of increased cell migration (development, wound healing, angiogenesis, cancer metastasis) are accompanied by shifts in integrin expression with certain integrins being lost and others gained. Most ECM proteins can be recognized by various different integrins. For instance, the ECM protein, fibronectin (Fn) can be recognized by nine different types of integrins and most of these bind to the Arg-Gly-Asp (RGD) motif in the central cell-binding domain. Thus, cell-matrix adhesions formed on Fn contain a mixture of different integrins and shifts in expression from one class of Fn-binding integrins to another will alter the receptor composition of such adhesions. This may provide an alternative means to shift from stationary to motile.Indeed, we have found that the type of integrins used for binding to Fn strongly affects cell migration. We made use of cells deficient in certain Fn-binding integrins and either restored their expression or compensated for their absence by overexpression of alternative Fn-binding integrins. This allowed us to compare in a single cellular background cell-matrix adhesions containing α5β1 to those containing αvβ3. Despite the fact that these integrins support similar levels of adhesion to Fn, only α5β1 was found to promote a contractile, fibroblastic morphology with centripetal orientation of cell-matrix adhesions⁸ (Fig. 1). Moreover, RhoA activity is high in the presence of α5β1 and these cells move in a random fashion with a speed of around 25 mm/h. By contrast, in cells using αvβ3 instead, adhesions distribute across the ventral surface, RhoA activity is low, and these cells move with similar speed but in a highly persistent fashion.^8,9 Finally, photobleaching experiments using GFP-vinculin and GFP-paxillin demonstrated that cell-matrix adhesions containing α5β1 are highly dynamic whereas adhesions containing αvβ3 are more static.⁹Open in a separate windowFigure 1Immunofluorescence images. GE11 cells, epithelial β1 knockout cells derived from mouse embryos chimeric for the integrin β1 subunit endogenously express various av integrins, including low levels of αvβ3 and αvβ5. Ectopic expression of β1 leads to expression of α5β1 and induced α5β1-mediated adhesion to Fn (left image) whereas ectopic expression of β3 (in the β1 null background) leads to strong expression of αvβ3 and induced αvβ3-mediated adhesion to Fn (right image). Adhesions containing either α5β1 or αvβ3 show distinct distribution and dynamics (paxillin; green) and cause different F-actin organization (phalloidin; red). Cartoons: Differences in cell-matrix adhesion dynamics may be explained by differential binding of soluble Fn molecules (blue) or different molecular determinants of the interaction with immobilized Fn (red). See text for details.It has been observed that α5β1 and αvβ3 use different recycling routes. Interfering with Rab4-mediated recycling of αvβ3 causes increased Rab11-mediated recycling of α5β1 to the cell surface. In agreement with our findings, the shift to α5β1 leads to increased Rho-ROCK activity and reduced persistence of migration.¹⁰ One possible explanation for the different types of migration promoted by these two Fn-binding integrins might involve different signaling and/or adaptor proteins interacting with specific amino acids in their cytoplasmic tails. However, this appears not to be the case: α5β1 in which the cytoplasmic tails of α5 or β1 are replaced by those of αv or β3, respectively, behaves identical to wild type α5β1: it promotes a fibroblast-like morphology with centripetal orientation of cell-matrix adhesions and it drives a non-persistent mode of migration.^8,11 Together, these findings point to differences between α5β1 and αvβ3 integrins in the mechanics of their interaction with Fn, which apparently modulates intracellular signaling pathways in control of cell-matrix adhesion dynamics and cell migration.How might this work? It turns out that although α5β1 and αvβ3 similarly support cell adhesion to immobilized (stretched) Fn, only α5β1 efficiently binds soluble, folded (“inactive”) Fn.¹¹ We have proposed that such interactions with soluble Fn molecules (possibly secreted by the cell itself) may weaken the interaction with the immobilized ligand thereby causing enhanced cell-matrix adhesion dynamics in the presence of α5β1,¹¹ (Fig. 1). Preferential binding of soluble Fn by α5β1 could be explained by differences in accessibility of the RGD binding pocket between α5β1 (more exposed) and αvβ3 (more hidden) as suggested by others.¹² If this is the case, immobilization (“stretching”) of Fn apparently leads to reorientation of the RGD motif in such a way that it is easily accessed by both integrins.The issue is considerably complicated by the fact that other recognition motifs are present in the Fn central cell-binding domain. In addition to the RGD sequence in the tenth Fn type 3 repeat (IIIFn10), binding of α5β1, but not αvβ3, also depends on the PHSRN “synergy” sequence in IIIFn9.^13–15 The relative contribution of these motifs is controversial and there is structural data pointing either towards a model in which IIIFn9 interacts with α5β1 or towards a model in which IIIFn9 exerts long-range electrostatic steering resulting in a higher affinity interaction without contacting the integrin.^16,17 Cell adhesion studies have suggested that an interaction of α5β1 with the synergy region stabilizes the binding to RGD.^14,18 Such a two-step interaction may facilitate binding to full length, folded Fn for instance by altering the tilt angle between IIIFn9 and IIIFn10 leading to optimal exposure of the RGD loop, perhaps explaining why αvβ3 (which may not interact with the synergy site) poorly binds soluble Fn.Others have shown that the RGD motif alone is sufficient for mechanical coupling of αvβ3 to Fn whereas the synergy region is required to provide mechanical strength to the α5β1-Fn bond.¹⁹ It appears that the interaction of α5β1 with Fn is particularly dynamic with various conformations of α5β1 interacting with different Fn binding surfaces, including the RGD and synergy sequences as well as other regions in IIIFn9. Thus, besides the above model based on differential binding to soluble Fn molecules, differences in the complexity and dynamics of interactions with immobilized Fn that determine functional binding strength could also underlie the different dynamics of cell-matrix adhesions containing either α5β1 or αvβ3 (Fig. 1).Precisely how mechanical differences in receptor-ligand interactions result in such remarkably distinct cellular responses is poorly understood. In addition to effects on cell-matrix adhesion dynamics and cytoskeletal organization it is also associated with different activities of Rho GTPases, indicating that mechanical differences between these two integrins must translate into differential activation of intracellular signaling pathways.^8,9,11 Possibly, different adhesion dynamics due to distinct mechanisms of receptor-ligand interaction result in different patterns of F-actin organization, which, in turn, affects the formation of signaling platforms. It is also possible that differences in the extent of integrin clustering have an impact on the conformation of one or more cytoplasmic components of the cell-matrix adhesions containing either α5β1 or αvβ3. This could lead to hiding or exposing binding sites for signaling molecules (e.g., upstream regulators of Rho GTPases) or substrates. Whatever the mechanism involved, altering the integrin composition of cell-matrix adhesions through shifts in integrin expression as observed during development, angiogenesis, wound healing and cancer progression may be a driving force in the enhanced cell migration that characterizes those processes.     

Bacillus subtilis MreB paralogues have different filament architectures and lead to shape remodelling of a heterologous cell system

Like many bacteria, Bacillus subtilis cells contain three actin-like MreB proteins. We show that the three paralogues, MreB, Mbl and MreBH, have different filament architectures in a heterologous cell system, and form straight filaments, helices or ring structures, different from the regular helical arrangement in B. subtilis cells. However, when coexpressed, they colocalize into a single filamentous helical structure, showing that the paralogues influence each other's filament architecture. Ring-like MreBH structures can be converted into MreB-like helical filaments by a single point mutation affecting subunit contacts, showing that MreB paralogues feature flexible filament arrangements. Time-lapse and FRAP experiments show that filaments can extend as well as shrink at both ends, and also show internal rearrangement, suggesting that filaments consist of overlapping bundles of shorter filaments that continuously turn over. Upon induction in Escherichia coli cells, B. subtilis MreB (BsMreB) filaments push the cells into strikingly altered cell morphology, showing that MreB filaments can change cell shape. E. coli cells with a weakened cell wall were ruptured upon induction of BsMreB filaments, suggesting that the bacterial actin orthologue may exert force against the cell membrane and envelope, and thus possibly plays an additional mechanical role in bacteria.     

The efficiency of designs for fine-mapping of quantitative trait loci using combined linkage disequilibrium and linkage

In a simulation study, different designs were compared for efficiency of fine-mapping of QTL. The variance component method for fine-mapping of QTL was used to estimate QTL position and variance components. The design of many families with small size gave a higher mapping resolution than a design with few families of large size. However, the difference is small in half sib designs. The proportion of replicates with the QTL positioned within 3 cM of the true position is 0.71 in the best design, and 0.68 in the worst design applied to 128 animals with a phenotypic record and a QTL explaining 25% of the phenotypic variance. The design of two half sib families each of size 64 was further investigated for a hypothetical population with effective size of 1000 simulated for 6000 generations with a marker density of 0.25 cM and with marker mutation rate 4 × 10^-4 per generation. In mapping using bi-allelic markers, 42~55% of replicated simulations could position QTL within 0.75 cM of the true position whereas this was higher for multi allelic markers (48~76%). The accuracy was lowest (48%) when mutation age was 100 generations and increased to 68% and 76% for mutation ages of 200 and 500 generations, respectively, after which it was about 70% for mutation ages of 1000 generations and older. When effective size was linearly decreasing in the last 50 generations, the accuracy was decreased (56 to 70%). We show that half sib designs that have often been used for linkage mapping can have sufficient information for fine-mapping of QTL. It is suggested that the same design with the same animals for linkage mapping should be used for fine-mapping so gene mapping can be cost effective in livestock populations.     

Dynamic movement of actin-like proteins within bacterial cells

Actin proteins are present in pro- and eukaryotes, and have been shown to perform motor-like functions in eukaryotic cells in a variety of processes. Bacterial actin homologues are essential for cell viability and have been implicated in the formation of rod cell shape, as well as in segregation of plasmids and whole chromosomes. We have generated functional green fluorescent protein fusions of all three Bacillus subtilis actin-like proteins (MreB, Mbl and MreBH), and show that all three proteins form helical filaments underneath the cell membrane, the pattern of which is distinct for each protein. Time-lapse microscopy showed that the filaments are highly dynamic structures. A number of separate filaments of MreB and Mbl continuously move through the cell along helical tracks underneath the cell membrane. The speed of extension of the growing end of filaments is within the range of known actin polymerization (0.1 microm/s), generating a potential poleward or centreward pushing velocity at 0.24 microm/min for MreB or Mbl, respectively. During nutritional downshift and a block in topoisomerase IV activity, the filaments rapidly disintegrated, showing that movement occurs only in growing cells. Contrary to Mbl and MreBH filaments, MreB filaments were generally absent in cells lacking DNA, providing a further distinction between the three orthologues.     

Anti-alpha-fodrin antibodies do not add much to the diagnosis of Sjögren's syndrome

The presence of anti-alpha-fodrin autoantibodies has been reported to be a highly specific and sensitive test for the diagnosis of Sj?gren's syndrome (SjS). We looked (in Nijmegen) for anti-alpha-fodrin, anti-Ro60, and anti-La autoantibodies in a cohort of 51 patients with rheumatic diseases (primary SjS [21], secondary SjS 6, rheumatoid arthritis [RA] 12, systemic lupus erythematosus [SLE] 6, and scleroderma 6) and in 28 healthy subjects, using ELISA, immunoblotting, and immunoprecipitation. The same samples were analyzed with an alternative anti-alpha-fodrin ELISA in Hanover. The Nijmegen ELISA of the sera from primary SjS showed sensitivities of 43% and 48% for IgA- and IgG-type anti-alpha-fodrin antibodies, respectively. The Hanover ELISA showed sensitivities of 38% and 10% for IgA- and IgG-type anti-alpha-fodrin antibodies, respectively. The ELISAs for alpha-fodrin showed six (Nijmegen) and four (Hanover) anti-alpha-fodrin-positive RA sera. IgA and IgG anti-fodrin antibodies were also present in four patients with secondary SjS. The sensitivities of Ro60 and La-antibodies in the Nijmegen ELISA were 67% and 62%, respectively. Unlike anti-alpha-fodrin antibodies, all anti-Ro60 and anti-La positive sera could be confirmed by immunoblotting or RNA immunoprecipitation. Thus, anti-Ro and anti-La autoantibodies were more sensitive than anti-alpha-fodrin autoantibodies in ELISA and were more frequently confirmed by other techniques. Anti-La antibodies appear to be more disease-specific than anti-alpha-fodrin antibodies, which are also found in RA sera. Therefore, the measurement of anti-alpha-fodrin autoantibodies does not add much to the diagnosis of Sj?gren's syndrome.     

Clinical and angiographic results with the ACS MULTI-LINK DUET trade mark Coronary Stent System - the DUET Study

BACKGROUND: The DUET Study is a multicenter prospective efficacy and safety evaluation of the ACS MULTI-LINK DUET coronary stainless steel balloon-expandable stent. AIMS: The primary objective was to determine the one-month incidence of MACE (major adverse cardiac events). The secondary objectives were the acute success rate, the restenosis and reocclusion rates (assessed by quantitative coronary angiography (QCA)) at six months and the occurrence of MACE in hospital and at six months. METHODS: Two hundred and ten patients were enrolled between February and June 1998 in 18 European centers. Successful stent placement was achieved in 209 patients. All patients were treated with ticlopidine 500 mg/day for one month and with aspirin >/=100 mg/day. To allow the investigators to gain familiarity with the stent system, the first one to three patients per center formed a separate lead-in population leaving an intention-to-treat population of 157 patients. The majority of the intention-to-treat population were male (79%); 28% had unstable angina, 69% had stable angina, 44% had had a previous myocardial infarction, 15% had had a previous percutaneous transluminal coronary angioplasty, and 3% had a history of stroke. The target vessel was 38.5% left anterior descending artery, 20.5% left circumflex artery and 41.0% right coronary artery. RESULTS: All but one of the intention-to-treat patients were effectively stented (17 required multiple stents). Six-month angiographic follow-up was available in 90% of the intention-to-treat population. Minimal lumen diameter (MLD) postprocedure was 2.61 +/- 0.33 mm, with a residual diameter stenosis of 16%. Six-month follow-up data showed an MLD of 1.87 +/- 0.56 mm with a residual diameter stenosis of 36%. The binary restenosis rate (>/=50% residual stenosis) was 15.6%. Up to one month following the procedure 94.9% of the population was MACE-free, with two subacute occlusions. At six months all patients were alive, of whom 82.8% were MACE-free, and 73% were free of anginal complaints. CONCLUSION: The results observed in the current DUET registry are comparable to data of other balloon-expandable-stent trials, with a low incidence of clinical events at follow-up.     

The variability of the hepatitis B virus genome: statistical analysis and biological implications

A statistical analysis of the nucleotide sequence variability in 14 published hepatitis B virus (HBV) genomes was carried out using parametric and nonparametric methods. A parametric statistical model revealed that the different regions of the genome differed significantly in their variability. The conclusion was supported by a nonparametric kernel-density model of the HBV genome. Genes S, C, and P, region X, the precore region, and the pre-S2/pre-S1 regions were ranked in order of increasing variability. In many instances, conserved regions of the genome identified with sequences of known function in HBV biology. However, other characterized regions (such as pre-S) showed much variability despite the involvement of their encoded peptides in specific functions. Point mutations that may result in the formation of stop codons and amino acid changes may affect the clinical picture of HBV infection and may be reflected in atypical serological patterns.      

Definitive Evidence for the existence of tight junctions in invertebrates

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.     

Virtual reality 3D echocardiography in the assessment of tricuspid valve function after surgical closure of ventricular septal defect

Background

Chronic right ventricular apical pacing may have detrimental effect on left ventricular function and may promote to heart failure in adult patients with left ventricular dysfunction.

Methods

A group of 99 pediatric patients with previously implanted pacemaker was studied retrospectively. Forty-three patients (21 males) had isolated congenital complete or advanced atrioventricular block. The remaining 56 patients (34 males) had pacing indication in the presence of structural heart disease. Thirty-two of them (21 males) had isolated structural heart disease and the remaining 24 (13 males) had complex congenital heart disease. Patients were followed up for an average of 53 ± 41.4 months with 12-lead electrocardiogram and transthoracic echocardiography. Left ventricular shortening fraction was used as a marker of ventricular function. QRS duration was assessed using leads V₅ or II on standard 12-lead electrocardiogram.

Results

Left ventricular shortening fraction did not change significantly after pacemaker implantation compared to preimplant values overall and in subgroups. In patients with complex congenital heart malformations shortening fraction decreased significantly during the follow up period. (0.45 ± 0.07 vs 0.35 ± 0.06, p = 0.015). The correlation between the change in left ventricular shortening fraction and the mean increase of paced QRS duration was not significant. Six patients developed dilated cardiomyopathy, which was diagnosed 2 months to 9 years after pacemaker implantation.

Conclusion

Chronic right ventricular pacing in pediatric patients with or without structural heart disease does not necessarily result in decline of left ventricular function. In patients with complex congenital heart malformations left ventricular shortening fraction shows significant decrease.     

Optimization of a crossing system using mate selection

A simple model based on one single identified quantitative trait locus (QTL) in a two-way crossing system was used to demonstrate the power of mate selection algorithms as a natural means of opportunistic line development for optimization of crossbreeding programs over multiple generations. Mate selection automatically invokes divergent selection in two parental lines for an over-dominant QTL and increased frequency of the favorable allele toward fixation in the sire-line for a fully-dominant QTL. It was concluded that an optimal strategy of line development could be found by mate selection algorithms for a given set of parameters such as genetic model of QTL, breeding objective and initial frequency of the favorable allele in the base populations, etc. The same framework could be used in other scenarios, such as programs involving crossing to exploit breed effects and heterosis. In contrast to classical index selection, this approach to mate selection can optimize long-term responses.