The Spontaneously Adhesive Leukocyte Function-associated Antigen-1 (LFA-1) Integrin in Effector T Cells Mediates Rapid Actin- and Calmodulin-dependent Adhesion Strengthening to Ligand under Shear Flow

Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5–20-s contact time). The maximum unbinding force for this interaction was ∼140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.     

Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation

Yeast Abp1p is a cortical actin cytoskeleton protein implicated in cytoskeletal regulation, endocytosis, and cAMP-signaling. We have identified a gene encoding a mouse homologue of Abp1p, and it is identical to SH3P7, a protein shown recently to be a target of Src tyrosine kinases. Yeast and mouse Abp1p display the same domain structure including an N-terminal actin-depolymerizing factor homology domain and a C-terminal Src homology 3 domain. Using two independent actin-binding domains, mAbp1 binds to actin filaments with a 1:5 saturation stoichiometry. In stationary cells, mAbp1 colocalizes with cortical F-actin in fibroblast protrusions that represent sites of cellular growth. mAbp1 appears at the actin-rich leading edge of migrating cells. Growth factors cause mAbp1 to rapidly accumulate in lamellipodia. This response can be mimicked by expression of dominant-positive Rac1. mAbp1 recruitment appears to be dependent on de novo actin polymerization and occurs specifically at sites enriched for the Arp2/3 complex. mAbp1 is a newly identified cytoskeletal protein in mice and may serve as a signal-responsive link between the dynamic cortical actin cytoskeleton and regions of membrane dynamics.     

The mammalian actin-binding protein 1 is critical for spreading and intraluminal crawling of neutrophils under flow conditions

Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the β(2) integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbp1 severely compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that mAbp1 was required for β(2) integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon β(2) integrin-mediated adhesion and chemotactic migration of human neutrophil-like differentiated HL-60 cells, mAbp1 was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence microscopy revealed that mAbp1 formed propagating waves toward the front of the lamellipodium, which are characteristic for dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbp1 to actin was increased upon β(2) integrin-mediated adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaffected in the absence of mAbp1. In contrast, the downregulation of mAbp1 by RNA interference technique in neutrophil-like differentiated HL-60 cells or the genetic ablation of mAbp1 in leukocytes led to defective migration under flow conditions in vitro and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbp1 is of fundamental importance for spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during inflammation.     

The actin-depolymerizing factor homology and charged/helical domains of drebrin and mAbp1 direct membrane binding and localization via distinct interactions with actin

Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with Cdc42, regulates actin polymerization and molecular motor-based motility. Drebrin and mammalian Abp1 (mAbp1) are actin-binding proteins found previously to bind to Golgi membranes in an ARF1-dependent manner in vitro. Despite sharing homology through two shared actin binding domains, drebrin and mAbp1 have different subcellular localization and bind to distinct actin structures on the Golgi apparatus. We find that the actin-depolymerizing factor homology (ADFH) and charged/helical actin binding domains of drebrin and mAbp1 are sufficient for regulated binding to Golgi membranes and subcellular localization. We have used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. We find that a linker region between the ADFH and charged/helical domains confers Golgi binding properties to mAbp1. mAbp1 binds to a specific actin pool through its ADFH/linker domain that is not bound by drebrin. Drebrin localization to the cell surface was found to involve motifs within the charged/helical domain. Our results indicate that targeting of these proteins is directed through multiple distinct interactions with the actin cytoskeleton. The mechanisms for selective recruitment of mAbp1 and drebrin to Golgi membranes indicate how actin-based structures are able to select specific actin-binding proteins and, thus, carry out multiple different functions within cells.     

Actin-binding Protein-1 Interacts with WASp-interacting Protein to Regulate Growth Factor-induced Dorsal Ruffle Formation

Growth factor stimulation induces the formation of dynamic actin structures known as dorsal ruffles. Mammalian actin-binding protein-1 (mAbp1) is an actin-binding protein that has been implicated in regulating clathrin-mediated endocytosis; however, a role for mAbp1 in regulating the dynamics of growth factor–induced actin-based structures has not been defined. Here we show that mAbp1 localizes to dorsal ruffles and is necessary for platelet-derived growth factor (PDGF)-mediated dorsal ruffle formation. Despite their structural similarity, we find that mAbp1 and cortactin have nonredundant functions in the regulation of dorsal ruffle formation. mAbp1, like cortactin, is a calpain 2 substrate and the preferred cleavage site occurs between the actin-binding domain and the proline-rich region, generating a C-terminal mAbp1 fragment that inhibits dorsal ruffle formation. Furthermore, mAbp1 directly interacts with the actin regulatory protein WASp-interacting protein (WIP) through its SH3 domain. Finally, we demonstrate that the interaction between mAbp1 and WIP is important in regulating dorsal ruffle formation and that WIP-mediated effects on dorsal ruffle formation require mAbp1. Taken together, these findings identify a novel role for mAbp1 in growth factor–induced dorsal ruffle formation through its interaction with WIP.     

Anchorage dependence of mitogen-induced G1 to S transition in primary T lymphocytes

Anchorage dependence defines the cellular requirement for integrin-mediated adhesion to substrate to initiate DNA replication in response to growth factors. In this study we investigated whether normal T cells, which spend extended periods in a nonadherent state, show similar requirements for cell cycle progression in response to TCR stimulation. Resting primary T lymphocytes were induced to enter the cell cycle by TCR triggering, and leukocyte integrins were either engaged using purified ICAM-1 or inhibited with function-blocking mAbs. Our data indicate that leukocyte integrins complement TCR-driven mitogenic signals not as a result of their direct clustering but, rather, via integrin-dependent organization of the actin cytoskeleton. Leukocyte integrin-dependent reorganization of the actin cytoskeleton cooperates with the TCR to effect mitogen-activated protein kinase activation, but also represents a required late (4-8 h poststimulation) component in the mitogenic response of normal T cells. Prolonged leukocyte integrin-dependent spreading, in the context of intercellular contact, is a requisite for the production of the mitogenic cytokine IL-2, which, in turn, is involved in the induction of D3 cyclin and is primarily responsible for the decrease in the cyclin-dependent kinase inhibitor p27kip, resulting in retinoblastoma protein inactivation and S phase entry. Thus, T lymphocytes represent a peculiar case of anchorage dependence, in which signals conveyed by integrins act sequentially with the activating stimulus to effect a sustained production of the essential mitogenic cytokine.     

Rapid up-regulation of alpha4 integrin-mediated leukocyte adhesion by transforming growth factor-beta1

The alpha4 integrins (alpha4beta1 and alpha4beta7) are cell surface heterodimers expressed mostly on leukocytes that mediate cell-cell and cell-extracellular matrix adhesion. A characteristic feature of alpha4 integrins is that their adhesive activity can be subjected to rapid modulation during the process of cell migration. Herein, we show that transforming growth factor-beta1 (TGF-beta1) rapidly (0.5-5 min) and transiently up-regulated alpha4 integrin-dependent adhesion of different human leukocyte cell lines and human peripheral blood lymphocytes (PBLs) to their ligands vascular cell adhesion molecule-1 (VCAM-1) and connecting segment-1/fibronectin. In addition, TGF-beta1 enhanced the alpha4 integrin-mediated adhesion of PBLs to tumor necrosis factor-alpha-treated human umbilical vein endothelial cells, indicating the stimulation of alpha4beta1/VCAM-1 interaction. Although TGF-beta1 rapidly activated the small GTPase RhoA and the p38 mitogen-activated protein kinase, enhanced adhesion did not require activation of both signaling molecules. Instead, polymerization of actin cytoskeleton triggered by TGF-beta1 was necessary for alpha4 integrin-dependent up-regulated adhesion, and elevation of intracellular cAMP opposed this up-regulation. Moreover, TGF-beta1 further increased cell adhesion mediated by alpha4 integrins in response to the chemokine stromal cell-derived factor-1alpha. These data suggest that TGF-beta1 can potentially contribute to cell migration by dynamically regulating cell adhesion mediated by alpha4 integrins.     

Golgi vesicle proteins are linked to the assembly of an actin complex defined by mAbp1

Recent studies indicate that regulation of the actin cytoskeleton is important for protein trafficking, but its precise role is unclear. We have characterized the ARF1-dependent assembly of actin on the Golgi apparatus. Actin recruitment involves Cdc42/Rac and requires the activation of the Arp2/3 complex. Although the actin-binding proteins mAbp1 (SH3p7) and drebrin share sequence homology, they are differentially segregated into two distinct ARF-dependent actin complexes. The binding of Cdc42 and mAbp1, which localize to the Golgi apparatus, but not drebrin, is blocked by occupation of the p23 cargo-protein-binding site on coatomer. Exogenously expressed mAbp1 is mislocalized and inhibits Golgi transport in whole cells. The ability of ARF, vesicle-coat proteins, and cargo to direct the assembly of cytoskeletal structures helps explain how only a handful of vesicle types can mediate the numerous trafficking steps in the cell.     

Endothelial cell cortactin coordinates intercellular adhesion molecule-1 clustering and actin cytoskeleton remodeling during polymorphonuclear leukocyte adhesion and transmigration

Endothelial cell ICAM-1 interacts with leukocyte beta(2) integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.     

Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.     

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.     

Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle

The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). beta1 integrins are ECM receptors in skeletal muscle, and mutations that affect the alpha7beta1 integrin cause myopathy in humans. In mice, beta1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate beta1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.     

Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1

The immune cell specific protein Fyn-T binding protein (Fyb) has been identified as a target of the Yersinia antiphagocytic effector Yersinia outer protein H (YopH), but its role in macrophages is unknown. By using Fyb domains as bait to screen a mouse lymphoma cDNA library, we identified a novel interaction partner, mammalian actin binding protein 1 (mAbp1). We show that mAbp1 binds the Fyb N-terminal via its C-terminally located src homology 3 domain. The interaction between Fyb and mAbp1 is detected in macrophage lysates and the proteins co-localize with F-actin in the leading edge. Hence, mAbp1 is likely to constitute a downstream effector of Fyb involved in F-actin dynamics.     

α-Catenin Localization and Sarcomere Self-Organization on N-Cadherin Adhesive Patterns Are Myocyte Contractility Driven

The N-cadherin (N-cad) complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. α-catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that α-catenin localizes to regions of highest internal stress in myocytes. This localization suggests that α-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization.     

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.     

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.     

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In parallel to integrins, other adhesion systems mediate adhesion and cytoskeletal coupling to the extracellular matrix (ECM). These include multifunctional cell surface receptors (syndecans and CD44) and discoidin domain receptors, which together coordinate ligand binding with direct or indirect cytoskeletal coupling and intracellular signalling. We review the way that the different adhesion systems for ECM components impact cell migration in two- and three-dimensional migration models. We further discuss the hierarchy of these concurrent adhesion systems, their specific tasks in cell migration and their contribution to migration in three-dimensional multi-ligand tissue environments.     

Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1

Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes.     

Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle.

Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with "focal adhesion complexes" (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli.     

Adhesion Complexes Formed by OVCAR-4 Cells on Laminin 1 Differ from Those Observed on Fibronectin

Cell adhesion to laminin 1 or to fibronectin is mediated by distinct sets of integrins and is differentially regulated by protein kinase C (PKC). It suggests that upon integrin ligation to laminin 1 or to fibronectin different intracellular signaling pathways could be activated. We have therefore investigated the formation of signaling complexes induced during cell adhesion to laminin 1 or to fibronectin. Following cell adhesion to laminin 1 the re-arrangement of the cytoskeleton was slower than that observed on fibronectin and it was activated by treating the cells with H-7, an inhibitor of PKC. Conversely, treatment of laminin-adhering cells with a PKC activator resulted in a rapid disorganization of the actin cytoskeleton while a similar treatment had no effect on fibronectin-adhering cells. These results suggested that the structural organization of the adhesion complexes might be substrate-specific and might correspond to a different arrangement of cytoskeletal and/or cytoplasmic proteins. Reflection interference contrast microscopy (RICM) images revealed that cell-substratum contacts formed on laminin 1 were not well differentiated in contrast to those developed on fibronectin. However, immunofluorescence staining revealed a similar organisation of actin microfilaments, talin and phosphotyrosyl-containing proteins on both substrates. In contrast, differences were observed for vinculin distribution within cells spread on fibronectin or on laminin I. Following cell adhesion to fibronectin most of the vinculin appeared as thick patches at the tips of the actin stress fibers while in laminin-adhering cells vinculin was recruited into thin streaks localized at the end of only some actin stress fibers.