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.     

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.     

T cell activation by mycobacterial antigens in inflammatory synovitis

To define which mycobacterial antigens were responsible for the activation of synovial fluid T lymphocytes, acetone-precipitated Mycobacterium tuberculosis (AP-MT) antigens were separated into five fractions following polyacrylamide gel electrophoresis and added to the mononuclear cell cultures of patients with inflammatory synovitis. Fractions 2 (50 to 70 kDa) and 5 (less than 28 kDa) resulted in significantly more proliferation than that of fractions 1, 3, and 4. The response to a purified mycobacterial 65-kDa heat shock protein (hsp), which migrated in fraction 2, was highly correlated (r = 0.89, P less than 0.001) with the response to the crude AP-MT. The proliferative response to a different hsp. the Escherichia coli DnaK, by synovial fluid lymphocytes was marginal. Analysis of the synovial fluid T cell response to mycobacterial culture filtrates by T cell Western blotting revealed dominant responses to antigen(s) in the range of 31 to 21 kDa in each responding patient, although no other consistent pattern of T cell activation was noted. Three lines of evidence suggested that the response to the low molecular weight fractions was directed against degradation fragments of the 65-kDa protein. These observations suggest that the activation of T lymphocytes obtained from inflammatory synovial fluids by crude mycobacterial antigens was due in large part to recognition of the 65-kDa mycobacterial hsp.     

Differential regulation of C-C chemokines during fibroblast-monocyte interactions: adhesion vs. inflammatory cytokine pathways.

The cell-to-cell interactions during chronic inflammatory diseases likely contribute to leukocyte accumulation leading to increased pathology and organ dysfunction. In particular, there is a paucity of information relating to the maintenance of chronic fibrotic diseases. Using a lung fibroblast line and enriched monocyte populations, we have investigated the activational events which contribute to the production of two C-C chemokines, macrophage inflammatory protein-1 alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1), during fibroblast-monocyte interactions. Neither the fibroblast cell line (16lu) nor isolated monocytes alone produced significant levels of MIP-1alpha or MCP-1. However, when isolated monocytes were layered onto 16 lu fibroblast monolayers a significant increase in MIP-1alpha and MCP-1 production was observed. The use of fixed cell populations indicated that the MIP-1alpha was derived from monocytes and MCP-1 from both cell populations. To examine the molecules which were required for chemokine production during the interaction, specific antibodies were used in the co-cultures. Blocking beta3-integrin interactions significantly inhibited MIP-1alpha production. In contrast, beta-integrin interactions had no effect on the MCP-1 production, while, neutralization of TNF significantly decreased MCP-1 production during the co-culture. These data indicate that fibroblast-monocyte interactions induce chemokine production through different mechanisms and a combination of these responses may contribute to the maintenance of the mononuclear cell accumulation during disease progression.     

Integrin alpha6 cleavage: a novel modification to modulate cell migration

Integrins play a major role in cell adhesion and migration. Previous work reported that a cleaved form of integrin alpha6 (alpha6p) was detected in invasive human prostate cancer tissue, absent in normal prostate tissue and was produced by urokinase-type Plasminogen Activator (uPA) in a plasmin-independent manner. Using site-directed mutagenesis we identified amino acid residues R594 and R595, located in the "stalk" region of integrin alpha6, as essential for cleavage. The cleavage site is located on the extracellular region of the protein between the beta-barrel domain and the thigh domain. Prostate cancer cells (PC3N) were stably transfected to overexpress the cleavable, wild-type (PC3N-alpha6-WT) or the non-cleavable form of integrin alpha6 (PC3N-alpha6-RR). The number of cells invading laminin 111- and laminin 332-coated filters by PC3N-alpha6-WT cells increased by threefold as compared to PC3N-alpha6-RR cells. Plasminogen activator inhibitor-1 (PAI-1) reduced the invasion of PC3N-alpha6-WT cells by approximately 42% through laminin 332-coated filters and plasmin inhibitor aprotinin had no significant effect. Linear cell migration increased production of integrin alpha6p in the PC3N-alpha6-WT cells and not in the PC3N-alpha6-RR cells and 32% of the PC3N-alpha6-WT cells migrated on laminin 111 in the linear migration assay as compared to the 5% PC3N-alpha6-RR cells. These data taken together suggest that the uPA-mediated cell surface cleavage of the alpha6 integrin extracellular domain is involved in tumor cell invasion and migration on laminin.     

Human T lymphocyte adhesion to endothelial cells and transendothelial migration. Alteration of receptor use relates to the activation status of both the T cell and the endothelial cell

An in vitro model of T cell adhesion to human umbilical vein endothelial cells (HUVEC) and transendothelial migration was used to determine whether the activation state of the T cell or cytokine exposure of the HUVEC altered T cell-HUVEC interactions or receptor utilization. Stimulation of T cells with the activator of protein kinase C, phorbol dibutyrate (PDB) alone or in combination with the calcium ionophore, ionomycin increased their binding to HUVEC. Much of the binding of control and activated T cells to HUVEC was mediated by leukocyte function-associated Ag-1 (LFA-1) (CD11a/CD18), because mAb to either chain of this molecule inhibited binding substantially, but not completely. Activation of HUVEC with IL-1 also increased binding of T cells. Binding of control T cells to IL-1-stimulated HUVEC, however, was found to be LFA-1 independent, because mAb to CD11a/CD18 failed to block the interaction. In contrast, binding of activated T cells to IL-1-stimulated HUVEC was partially inhibited by mAb to LFA-1. Binding of activated T cells to IL-1-stimulated HUVEC also involved CD44 because this interaction was partially blocked by mAb to this determinant. When T cell migration was analyzed, it was found that the migration of PDB-activated T cells was three to four-fold more than that of control T cells. Migration through HUVEC and random migration were both enhanced by PDB stimulation. However, when the T cells were costimulated with PDB and ionomycin, migration was not increased above that of control T cells. PDB-activated T cells appeared to use LFA-1 for migration regardless of the activation status of the HUVEC, because mAb to CD11a/CD18 partially blocked their migration after binding to HUVEC. There was also a modest inhibition of PDB-activated T cell migration by mAb to CD44. In contrast, migration of control T cells involved neither LFA-1 nor CD44. Finally, binding of control T cells to high endothelial venules of peripheral lymphoid tissue was found to be CD11a/CD18 and CD44 independent, and completely inhibited by activation with either PDB or the combination of PDB and ionomycin. These results demonstrate that T cells use LFA-1 and CD44 as well as other as yet unidentified adhesion receptors for interactions with HUVEC, and that use of these adhesion receptors is mutable and related to the activation state of the T cell and cytokine stimulation of the HUVEC.     

The dendritic cell cytoskeleton promotes T cell adhesion and activation by constraining ICAM-1 mobility

Integrity of the dendritic cell (DC) actin cytoskeleton is essential for T cell priming, but the underlying mechanisms are poorly understood. We show that the DC F-actin network regulates the lateral mobility of intracellular cell adhesion molecule 1 (ICAM-1), but not MHCII. ICAM-1 mobility and clustering are regulated by maturation-induced changes in the expression and activation of moesin and α-actinin-1, which associate with actin filaments and the ICAM-1 cytoplasmic domain. Constrained ICAM-1 mobility is important for DC function, as DCs expressing a high-mobility ICAM-1 mutant lacking the cytoplasmic domain exhibit diminished antigen-dependent conjugate formation and T cell priming. These defects are associated with inefficient induction of leukocyte functional antigen 1 (LFA-1) affinity maturation, which is consistent with a model in which constrained ICAM-1 mobility opposes forces on LFA-1 exerted by the T cell cytoskeleton, whereas ICAM-1 clustering enhances valency and further promotes ligand-dependent LFA-1 activation. Our results reveal an important new mechanism through which the DC cytoskeleton regulates receptor activation at the immunological synapse.     

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.     

Mechanisms for vascular cell adhesion molecule-1 activation of ERK1/2 during leukocyte transendothelial migration

Background

During inflammation, adhesion molecules regulate recruitment of leukocytes to inflamed tissues. It is reported that vascular cell adhesion molecule-1 (VCAM-1) activates extracellular regulated kinases 1 and 2 (ERK1/2), but the mechanism for this activation is not known. Pharmacological inhibitors of ERK1/2 partially inhibit leukocyte transendothelial migration in a multi-receptor system but it is not known whether VCAM-1 activation of ERK1/2 is required for leukocyte transendothelial migration (TEM) on VCAM-1.

Methodology/Principal Findings

In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells. VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2. Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling. Furthermore, VCAM-1-specific leukocyte migration under physiological laminar flow of 2 dynes/cm² was blocked by pretreatment of endothelial cells with dominant-negative ERK2 K52R or the MEK/ERK inhibitors, PD98059 and U0126, indicating for the first time that ERK regulates VCAM-1-dependent leukocyte transendothelial migration.

Conclusions/Significance

VCAM-1 activation of endothelial cell NADPH oxidase/PKCα/PTP1B induces transient ERK1/2 activation that is necessary for VCAM-1-dependent leukocyte TEM.     

cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edge dynamics

Epithelial cell migration is a complex process crucial for embryonic development, wound healing and tumor metastasis. It depends on alterations in cell–cell adhesion and integrin–extracellular matrix interactions and on actomyosin-driven, polarized leading edge protrusion. The small GTPase Rap is a known regulator of integrins and cadherins that has also been implicated in the regulation of actin and myosin, but a direct role in cell migration has not been investigated. Here, we report that activation of endogenous Rap by cAMP results in an inhibition of HGF- and TGFβ-induced epithelial cell migration in several model systems, irrespective of the presence of E-cadherin adhesion. We show that Rap activation slows the dynamics of focal adhesions and inhibits polarized membrane protrusion. Importantly, forced integrin activation by antibodies does not mimic these effects of Rap on cell motility, even though it does mimic Rap effects in short-term cell adhesion assays. From these results, we conclude that Rap inhibits epithelial cell migration, by modulating focal adhesion dynamics and leading edge activity. This extends beyond the effect of integrin affinity modulation and argues for an additional function of Rap in controlling the migration machinery of epithelial cells.     

Integrin organization: linking adhesion ligand nanopatterns with altered cell responses

This paper presents a modelling framework in which the mechanochemical properties of smooth muscle cells may be studied. The activation of smooth muscles is considered in a three-dimensional continuum model which is key to realistically capture the function of hollow organs such as blood vessels. On the basis of a general thermodynamical framework the mechanical and chemical phases are specialized in order to quantify the coupled mechanochemical process. A free-energy function is proposed as the sum of a mechanical energy stored in the passive tissue, a coupling between the mechanical and chemical kinetics and an energy related purely to the chemical kinetics and the calcium ion concentration. For the chemical phase it is shown that the cross-bridge model of Hai and Murphy [1988. Am. J. Physiol. Cell Physiol. 254, C99-C106] is included in the developed evolution law as a special case. In order to show the specific features and the potential of the proposed continuum model a uniaxial extension test of a tissue strip is analysed in detail and the related kinematics and stress-stretch relations are derived. Parameter studies point to coupling phenomena; in particular the tissue response is analysed in terms of the calcium ion level. The model for smooth muscle contraction may significantly contribute to current modelling efforts of smooth muscle tissue responses.     

Actin,microtubules and focal adhesion dynamics during cell migration

Cell migration is a complex cellular behavior that results from the coordinated changes in the actin cytoskeleton and the controlled formation and dispersal of cell-substrate adhesion sites. While the actin cytoskeleton provides the driving force at the cell front, the microtubule network assumes a regulatory function in coordinating rear retraction. The polarity within migrating cells is further highlighted by the stationary behavior of focal adhesions in the front and their sliding in trailing ends. We discuss here the cross-talk of the actin cytoskeleton with the microtubule network and the potential mechanisms that control the differential behavior of focal adhesions sites during cell migration.     

Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell

Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, or platelets result in increased cell adhesion to immobilized integrin ligands. However, the mechanism by which this occurs is not clear, and it is possible that anti-PECAM-1 mAbs elicit this effect by simply sequestering PECAM-1, via antibody-induced patching and capping, away from stimulatory receptors that it normally regulates. To determine whether dimerization or oligomerization of PECAM-1 directly initiates signal transduction pathways that affect integrin function in an antibody-independent manner, stable human embryonic kidney-293 cell lines were produced that expressed chimeric PECAM-1 cDNAs containing one or two FK506-binding protein (FKBP) domains at their COOH terminus. Controlled dimerization initiated by addition of the bivalent, membrane-permeable FKBP dimerizer, AP1510, nearly doubled homophilic binding capacity, whereas AP1510-induced oligomers favored cis PECAM-1/PECAM-1 associations within the plane of the plasma membrane at the expense of trans homophilic adhesion. Importantly, AP1510-induced oligomerization resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2-transfected cells on immobilized fibronectin, a reaction that was mediated by the integrin alpha(5)beta(1). These data demonstrate that signals required for integrin activation can be elicited by clustering of PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosine-based inhibitory motif family of regulatory receptors.     

Integrating adhesion, protrusion, and contraction during cell migration

Cell migration is fastest when the strength of the adhesion between the cell and the substrate is neither too strong nor too weak. In this issue of Cell, reveal how adhesion and cytoskeletal dynamics are integrated to optimize migration speed.     

Progesterone enhances vascular endothelial cell migration via activation of focal adhesion kinase

The mechanisms of progesterone on endothelial cell motility are poorly investigated. Previously we showed that progesterone stimulated endothelial cell migration via the activation of actin-binding protein moesin, leading to actin cytoskeleton remodelling and the formation of cell membrane structures required for cell movement. In this study, we investigated the effects of progesterone on the formation of focal adhesion complexes, which provide anchoring sites for cell movement. In cultured human umbilical endothelial cells, progesterone enhanced focal adhesion kinase (FAK) phosphorylation at Tyr(397) in a dose- and time-dependent manner. Several signalling inhibitors interfered with progesterone-induced FAK activation, including progesterone receptor (PR) antagonist ORG 31710, specific c-Src kinase inhibitor PP2, phosphatidylinosital-3 kinase (PI3K) inhibitor wortmannin as well as ρ-associated kinase (ROCK-2) inhibitor Y27632. It suggested that PR, c-Src, PI3K and ROCK-2 are implicated in this action. In line with this, we found that progesterone rapidly promoted c-Src/PI3K/Akt activity, which activated the small GTPase RhoA/ρ-associated kinase (ROCK-2) complex, resulting in FAK phosphorylation. In the presence of progesterone, endothelial cells displayed enhanced horizontal migration, which was reversed by small interfering RNAs abrogating FAK expression. In conclusion, progesterone promotes endothelial cell movement via the rapid regulation of FAK. These findings provide new information on the biological actions of progesterone on human endothelial cells that are relevant for vascular function.     

Soluble CD8 during T cell activation

The CD8 Ag has long been used as a surface marker for the identification of cytotoxic and suppressor cells. Recently CD8-positive cells have been shown to release a soluble form of the CD8 Ag. We have devised a sandwich monoclonal enzyme immunoassay for the quantitation of this released CD8. Soluble CD8 was released in response to lymphocyte activation. In vitro, PHA or anti-CD3 mAb-mediated T cell activation led to release of CD8 into the culture supernatant. In vivo, serum from patients with EBV-induced infectious mononucleosis (IM), a disease associated with intense CD8+ T cell activation, demonstrated elevations in soluble CD8 (7939 U/ml, day 0) compared to serum from normal controls (289 U/ml). Levels of soluble CD8 correlated (r = 0.82, p less than 0.001) with the increased percentage of CD8+/HLA-DR+ (activated CD8+ T cells) observed in acute IM. Sequential analysis of serum during the course of IM shows that soluble CD8 levels parallel the decline in CD8+/HLA-DR+ cells that occurs with the resolution of the disease. These data suggest that released CD8 may be of value in monitoring the involvement of CD8+ T cells in response to a pathologic event. The functional role of the released CD8 molecule will require further investigation.     

Cytoskeletal rearrangement during migration and activation of T lymphocytes.

T lymphocytes have an inherent ability to migrate along a chemotactic gradient, which enables them to exit the bloodstream and reach different tissues. Motile T cells display a polarized morphology with two distinct cell compartments: the leading edge and the uropod. During cell polarization, chemoattractant receptors, cell-adhesion molecules and cytoskeletal proteins are redistributed within these cellular compartments. The polarity of T lymphocytes changes during the establishment of antigen-specific cell-cell interactions, and this involves rearrangement of cytoskeletal proteins. This article discusses the regulation of these cytoskeletal rearrangements, and their role in the activation, migration and effector function of T cells.     

Microscope-based techniques to study cell adhesion and migration

Modern light microscopy has evolved to provide a variety of quantitative imaging techniques and also the capability to perturb structure-function relationships in living cells. The advances have been especially useful in the study of cell adhesion and migration. This review will focus on how such microscopy-based techniques can be useful in situ to study the molecular interactions and dynamics, to locally perturb actin-based structures and to measure the traction forces exerted by motile cells.