Cell-matrix Junctions Research Articles

A novel culture platform for fast proliferation of human annulus fibrosus cells.

Tissue engineering provides a promising approach to treat degenerative disc disease, which usually requires a large quantity of seed cells. A simple and reliable in vitro culture system to expand seed cells in a timely fashion is necessary to implement the application clinically. Here, we sought to establish a cost-effective culture system for expanding human annulus fibrosus cells using extracellular matrix (ECM) proteins as culture substrates. Cells were cultured onto a plastic surface coated with various types of ECMs, including fibronectin, vitronectin, collagen type I, gelatin and cell-free matrix deposited by human nucleus pulposus cells. AF cell morphology, growth, adhesion and phenotype (anabolic and catabolic markers) were assessed by microscopy, real-time RT-PCR, western blotting, zymography, immunofluorescence staining and biochemical assays. Fibronectin, collagen and gelatin promoted cell proliferation and adhesion in a dose-dependent manner. Fibronectin elevated mRNA expression of proteoglycan and enhanced glycosaminoglycan production. Both collagen and gelatin increased protein expression of type II collagen. Consistent with increased cell adhesion, collagen and fibronectin promoted formation of focal adhesion complexes in the cell-matrix junction, suggesting enhanced binding of the actin network with both ECM substrates. On the other hand, fibronectin, collagen and gelatin decreased expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 in media. Finally, a mixture of fibronectin (1.7μg/mL) and collagen (1.3μg/mL) was identified as the most promising in vitro culture substrate system in promoting proliferation and maintaining anabolic-catabolic balance. Our method provides a simple and cost-effective platform for tissue engineering applications in intervertebral disc research.

Crim1 regulates integrin signaling in murine lens development

The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1(glcr11), which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1(null) and Crim1(cko), we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active β1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.

Editorial: Podocyte Pathology and Nephropathy.

Editorial: Podocyte Pathology and Nephropathy.

57. A transmembrane mega highway for cryoprotective agent delivery via self-assembling organic nanopores with tunable function

Cryoprotective agents (CPAs) are critical additives that improve the post-thaw viability of cryopreserved biological systems by preventing ice crystal nucleation and growth. Membrane permeable CPAs also prevent osmotic shrinkage of the cells and reduce the volume of available water by penetrating and equilibrating across the cell membrane. All known CPAs exhibit various levels of cytotoxicity at their effective concentration which may be decreased by reducing the CPA loading temperature and exposure time. However, most CPAs become effectively impermeable at sub-zero temperatures. This is largely associated with the dysfunction of transmembrane protein channels at low temperature that serve as mass transport gates. An unbalanced gradient of CPA and salt across cell membrane leads to detrimental cell shrinkage as water efflux out of the cell. This could be detrimental to the fate of large, complex solid tissues/organs by damaging cell–cell and cell–matrix junctions. To facilitate the intracellular delivery and transmembrane equilibration of the CPA at sub-zero temperatures, we propose size and function tunable organic nanopores as a “mega highway” to deliver CPAs across cell membrane which allows a significant decrease in both the CPA exposure time and temperature during freezing. This method may be particularly effective when CPA loading in tissues using the “liquidus tracking” or step-wise methods where increasingly concentrated solutions of CPA are loaded in the tissue/organ at progressively decreasing temperatures. Recent work in Gong’s group has demonstrated the reliability of constructing well-defined nanotubular assemblies via the enforced stacking of shape-persistent macrocycles based on the interplay of multiple hydrogen-bonding, dipole-dipole, and aromatic π - π stacking interactions. The resultant nanotubes have modifiable surfaces and non-collapsible inner pores of a uniform diameter defined by the constituent macrocycles. The robust self-assembling nanopores have been found to act as transmembrane channels which can mediate highly selective transmembrane ion transport with high stability, unprecedented for synthetic ion channels, as well as exhibiting highly efficient transmembrane water permeability, which is comparable to aquaporin. Aquaporins have been found to efficiently assist transport of water and glycerol during freezing to prevent cell rupture under osmotic pressure. In this proposal, selected organic nanopores are delivered into a target system at physiological temperature (37 °C), followed by CPA loading at hypothermic temperature (e.g. 1 °C). A high influx rate of CPAs through shape-persistent nanopores can be maintained during cooling as a function of the concentration gradient across cell membrane, thereby reducing the required time to reach vitrification concentrations. Upon rewarming, these organic nanopores will facilitate rapid removal of CPAs to reduce exposure time and the consequent toxic side effects. The proposed incorporation of synthetic nanopores can significantly reduce toxicity and cell injury due to osmotic shrinkage caused by CPAs and salt during both the cooling and rewarming processes via (1) reducing CPA exposure time and (2) enabling rapid CPA loading and unloading at lower temperatures. Moreover, versatile functional organic nanotubes of diverse sizes and properties by modifying inner macrocyclic cavities allow selective CPA transport by while preventing ion exchange. Incorporation of functional supramolecular assemblies to enhance membrane permeability of CPAs could lead to a revolutionary solution to cryopreserve complex biological systems.

Targeting Abl kinases to regulate vascular leak during sepsis and acute respiratory distress syndrome.

The vascular endothelium separates circulating fluid and inflammatory cells from the surrounding tissues. Vascular leak occurs in response to wide-spread inflammatory processes, such as sepsis and acute respiratory distress syndrome, because of the formation of gaps between endothelial cells. Although these disorders are leading causes of mortality in the intensive care unit, no medical therapies exist to restore endothelial cell barrier function. Recent evidence highlights a key role for the Abl family of nonreceptor tyrosine kinases in regulating vascular barrier integrity. These kinases have well-described roles in cancer progression and neuronal morphogenesis, but their functions in the vasculature have remained enigmatic until recently. The Abl family kinases, c-Abl (Abl1) and Abl related gene (Arg, Abl2), phosphorylate several cytoskeletal effectors that mediate vascular permeability, including nonmuscle myosin light chain kinase, cortactin, vinculin, and β-catenin. They also regulate cell-cell and cell-matrix junction dynamics, and the formation of actin-based cellular protrusions in multiple cell types. In addition, both c-Abl and Arg are activated by hyperoxia and contribute to oxidant-induced endothelial cell injury. These numerous roles of Abl kinases in endothelial cells and the current clinical usage of imatinib and other Abl kinase inhibitors have spurred recent interest in repurposing these drugs for the treatment of vascular barrier dysfunction. This review will describe the structure and function of Abl kinases with an emphasis on their roles in mediating vascular barrier integrity. We will also provide a critical evaluation of the potential for exploiting Abl kinase inhibition as a novel therapy for inflammatory vascular leak syndromes.

Matrix metalloproteinases promote arterial remodeling in aging, hypertension, and atherosclerosis.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which are highly activated by inflammatory signaling, including that of angiotensin II (Ang II; Figure).1,2 Activated MMPs are able to degrade collagen, elastin, and other extracellular molecules,2 probably resulting in aging, hypertension, and atherosclerotic effects within the arterial wall (for reviews).1,2 The modified extracellular matrix and its context of vasoconstrictors and vasodilators via cleavage by MMPs creates a proinflammatory microenvironment that shifts the phenotypes of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs; for reviews).1,2 These phenotypic shifts in which cells become secretory, migratory, proliferative and senescent facilitate arterial remodeling such as intimal-medial thickening (IMT), fibrosis, calcification, and aneurysms that are associated with a decrease in endothelial-dependent vasodilation and an increase in stiffness (for reviews).1,2 Impressively, MMP inhibition effectively retards/alleviates arterial structural remodeling, decreases stiffness, and improves vascular endothelial function in animal models,3–8 providing a rationale for the translation of MMP actions to therapeutic approaches in aging humans to curb the epidemic of cardiovascular disease. Figure. Matrix metalloproteinase (MMP) activation plays a central role in arterial proinflammatory remodeling and stiffening, which is a structural and functional signature of aging, hypertension, and atherosclerosis. BM indicates basement membrane; EC, endothelial cell; ECM, extracellular matrix; IMT, intimal-medial thickening; and VSMC, vascular smooth muscle cell. MMP activation facilitates arterial remodeling during aging (Figure; Table). View this table: Table. MMP Activation is Involved in Arterial Remodeling and Functional Changes ### Endothelial Inflammation Increased MMP activity facilitates endothelial inflammation, such as EC senescence/apoptosis/necrosis, thrombosis, and dysfunction (Figure; Table). MMP-1 enhances EC senescence via p53 activation.9 MMP-2 cleaves the intercellular and cell–matrix junctions, including vascular endothelial–cadherin and β- and γ-catenin, which initiates EC apoptosis or necrosis mediated by activated caspase 3, contributing to increased …

Substrate Stiffness Affects Human Keratinocyte Colony Formation.

Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned along the periphery of an evolving cell colony.

Crim1 regulates integrin signaling in murine lens development.

The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1glcr11, which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1null and Crim1cko, we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active β1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.

The non-muscle functions of actinins: an update

α-Actinins are a major class of actin filament cross-linking proteins expressed in virtually all cells. In muscle, actinins cross-link thin filaments from adjacent sarcomeres. In non-muscle cells, different actinin isoforms play analogous roles in cross-linking actin filaments and anchoring them to structures such as cell-cell and cell-matrix junctions. Although actinins have long been known to play roles in cytokinesis, cell adhesion and cell migration, recent studies have provided further mechanistic insights into these functions. Roles for actinins in synaptic plasticity and membrane trafficking events have emerged more recently, as has a 'non-canonical' function for actinins in transcriptional regulation in the nucleus. In the present paper we review recent advances in our understanding of these diverse cell biological functions of actinins in non-muscle cells, as well as their roles in cancer and in genetic disorders affecting platelet and kidney physiology. We also make two proposals with regard to the actinin nomenclature. First, we argue that naming actinin isoforms according to their expression patterns is problematic and we suggest a more precise nomenclature system. Secondly, we suggest that the α in α-actinin is superfluous and can be omitted.

Talins and kindlins: partners in integrin-mediated adhesion

Integrin receptors provide a dynamic, tightly-regulated link between the extracellular matrix (or cellular counter-receptors) and intracellular cytoskeletal and signalling networks, enabling cells to sense and respond to their chemical and physical environment. Talins and kindlins, two families of FERM-domain proteins, bind the cytoplasmic tail of integrins, recruit cytoskeletal and signalling proteins involved in mechanotransduction and synergize to activate integrin binding to extracellular ligands. New data reveal the domain structure of full-length talin, provide insights into talin-mediated integrin activation and show that RIAM recruits talin to the plasma membrane, whereas vinculin stabilizes talin in cell-matrix junctions. How kindlins act is less well-defined, but disease-causing mutations show that kindlins are also essential for integrin activation, adhesion, cell spreading and signalling.

Cells behaviors and genotoxicity on topological surface

To investigate different cells behaviors and genotoxicity, which were driven by specific microenvironments, three patterned surfaces (pillars, wide grooves and narrow grooves) and one smooth surface were prepared by template-based technique. Vinculin is a membrane-cytoskeletal protein in focal adhesion plaques and associates with cell–cell and cell–matrix junctions, which can promote cell adhesion and spreading. The immunofluorescence staining of vinculin revealed that the narrow grooves patterned substrate was favorable for L929 cell adhesion. For cell multiplication, the narrow grooves surface was fitted for the proliferation of L929, L02 and MSC cells, the pillars surface was only in favor of L929 cells to proliferate during 7days of cell cultivation. Cell genetic toxicity was evaluated by cellular micronuclei test (MNT). The results indicated that topological surfaces were more suitable for L929 cells to proliferate and maintain the stability of genome. On the contrary, the narrow grooves surface induced higher micronuclei ratio of L02 and MSC cells than other surfaces. With the comprehensive results of cell multiplication and MNT, it was concluded that the wide grooves surface was best fitted for L02 cells to proliferate and have less DNA damages, and the smooth surface was optimum for the research of MSC cells in vitro.

Abstract 1915: The role of the E2F2 and ID3 transcription factors in colon tumour growth and cellular dissemination.

Abstract In order to identify molecular markers prognostic of initiation and/or progression of human colon cancer (CC), a genome-wide analysis was performed and highlighted a micro-deletion at the 1p36.11-12 region in 23% (n = 115) and 47% (n = 59) of adenomas and carcinomas, respectively. This region contains the E2F2 and ID3 genes, which are known to regulate the cell cycle and cell differentiation, respectively. In addition, E2F2, is described as either oncogenic or tumour suppressor, depending on the tissue or cell type. The micro-deletion incidence depends on tumour stages (60% in early stages whereas only 34% in metastatic stages and further clinical analysis showed that patients with deleted E2F2 had a lower rate of recurrence and a better overall survival. Also, RT-QPCR evidenced that E2F2 and ID3 transcript expression levels decrease in human CC. Therefore, to assess the role of the deletion in tumour growth and metastatic activity, we evaluated the functional consequences of the loss of these genes both in vitro in human colon cancer cells that were transiently depleted of either E2F2 or ID3 and in vivo in immunodeficient mice. Both gene transcript expressions were down-regulated by transient siRNA transfection in the human epithelial CC cell line Caco-2/TC7. Consequences were evaluated by immunocytochemistry for proteins involved in the cell architecture and in cell-cell and cell-matrix junctions, and at the expression level by RT-QPCR and Western Blot analyses. Functional analyses were assessed for the migratory potential with the wound healing assay, for proliferation with the MTS assay, and for adhesion on substrates such as laminin, collagen I and fibronectin. The in vivo metastastic potential was evaluated with E2F2- and ID3-silenced cells that were intra-splenically implanted. E2F2 down-regulation and that of ID3 at a lesser extend, reduced proliferation and induced severe morphological modifications, associated with relocalization of structural members of adherens junctions (beta-catenin, APC), tight junctions (Claudin-1, ZO-1) and cytoskeleton (F-Actin, Cytokeratin-19). The integrins alpha5, alphaV, alpha2 and beta-1, were downregulated and the adhesion properties on laminin-111, but not on collagen I or fibronectin were lost. Moreover, inhibition of E2F2 and ID3 expression leads to a decreased migratory potential. The intra-spenic injection in immunodeficient mice of HT29 cells stably transduced to underexpress E2F2 and ID3, leads to lower / slower development of liver metastases. In conclusion, for a clinical aspect, the micro-deletion of the 1p36.11-12 region is a good prognosis for the event-free survival, and from a more basic and functional aspect, the loss of E2F2 and ID3 expression regulates tumor growth and dissemination through functions involving migration and adhesion. Citation Format: Dominique Guenot, Manon Voegelin, Eric Guérin, Céline Nicolet, Marie-Pierre Gaub. The role of the E2F2 and ID3 transcription factors in colon tumour growth and cellular dissemination. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1915. doi:10.1158/1538-7445.AM2013-1915

Alpha-actinin interactions with syndecan-4 are integral to fibroblast–matrix adhesion and regulate cytoskeletal architecture

All cells of the musculoskeletal system possess transmembrane syndecan proteoglycans, notably syndecan-4. In fibroblasts it regulates integrin-mediated adhesion to the extracellular matrix. Syndecan-4 null mice have a complex wound repair phenotype while their fibroblasts have reduced focal adhesions and matrix contraction abilities. Signalling through syndecan-4 core protein to the actin cytoskeleton involves protein kinase Cα and Rho family G proteins but also direct interactions with α-actinin. The contribution of the latter interaction to cell–matrix adhesion is not defined but investigated here since manipulation of Rho GTPase and its downstream targets could not restore a wild type microfilament organisation to syndecan-4 null cells. Microarray and protein analysis revealed no significant alterations in mRNA or protein levels for actin- or α-actinin associated proteins when wild type and syndecan-4 knockout fibroblasts were compared. The binding site for syndecan-4 cytoplasmic domain was identified as spectrin repeat 4 of α-actinin while further experiments confirmed the importance of this interaction in stabilising cell–matrix junctions. However, α-actinin is also present in adherens junctions, these organelles not being disrupted in the absence of syndecan-4. Indeed, co-culture of wild type and knockout cells led to adherens junction-associated stress fibre formation in cells lacking syndecan-4, supporting the hypothesis that the proteoglycan regulates cell–matrix adhesion and its associated microfilament bundles at a post-translational level. These data provide an additional dimension to syndecan function related to tension at the cell–matrix interface, wound healing and potentially fibrosis.

Collagen XIII: A type II transmembrane protein with relevance to musculoskeletal tissues, microvessels and inflammation

Collagen XIII and the homologous collagens XXIII and XXV form a subgroup of type II transmembrane proteins within the collagen superfamily. Collagen XIII consists of a short cytosolic domain, a transmembrane domain and a large extracellular ectodomain, which may be shed into the pericellular matrix. It has been proposed that collagen XIII may function as an adhesion molecule, due to its cellular localization at focal contacts, numerous interactions with basement membrane (BM) and other extracellular matrix (ECM) proteins and expression at various cell–cell and cell–matrix junctions. Recent in vivo studies highlight its involvement in the development, differentiation and maturation of musculoskeletal tissues and vessels and in maintaining tissue integrity.

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin

The cytoskeletal proteins talin and vinculin are localized at cell-matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F-actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four- and five-helix bundles that harbor amphipathic α-helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four-helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α-helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four-helix bundle.

Effect of microtubule-targeting drugs on cell–cell and cell–matrix junctions in tumor epithelial cells

In this study, we investigated the effects of microtubule-targeting drugs, which either destabilize (the Vinca alkaloid vincristine) or stabilize (the taxane derivative docetaxel) microtubules, on the cell-cell and cell-matrix adhesive junctions of Caco-2 tumor epithelial cells, using fluorescence imaging and functional assays. We found that, in sub-confluent (but not confluent) cells, vincristine (but not docetaxel) affected cell-cell junction morphology. Furthermore, docetaxel (but not vincristine) attenuated the formation of the peri-junctional actomyosin ring and enhanced the internalization of junctional adhesion molecule-A. However, these effects of vincristine and docetaxel did not translate into appreciable functional changes during the opening and resealing of the cell-cell junctions. We also found that vincristine caused enlargement of focal adhesions (the major cell-matrix junctions) without affecting cell adhesion onto the matrix. Thus, we conclude that the microtubule-targeting drugs interfere to variable degrees with the morphology and/or function of the cell-cell and cell-matrix adhesive junctions. In addition, the results highlight the importance of considering the cellular context and dynamics (e.g. cell confluence and junction opening, respectively), when determining the final effects of microtubule manipulation on cell adhesiveness.

Dynamic Relationship of Focal Contacts and Hemidesmosome Protein Complexes in Live Cells

Epidermal cells adhere to the basement membrane zone through cell-matrix junctions termed hemidesmosomes. During wound healing, hemidesmosomes are disassembled to allow keratinocytes to move over wound sites. Such movement is mediated by both hemidesmosome protein complexes (HPCs) and focal contacts (FCs). In this study, we analyzed the interaction between HPCs and FCs in live HaCat cells expressing yellow fluorescent protein (YFP)-tagged beta4 integrin and cyan fluorescent protein (CFP)-tagged alpha-actinin as markers of HPCs and FCs, respectively. In HaCat cells migrating to repopulate wounds, FC proteins cluster rapidly in the direction of the wound. HPC assembly then follows and the newly formed HPCs occupy sites vacated by the disassembled FCs. HPC dynamics are dramatically reduced, and HaCat cells cease migration upon treatment with reagents that affect FC integrity/function. Upon treatment with reagents that destabilize HPCs, the dynamics of FCs in HaCat cells at the edges of wounds are enhanced, although FC assembly is irregular and the migration of the cells is aberrant. We also show that the complex interaction between hemidesmosomes and FCs in keratinocytes is myosin dependent and requires energy. In summary, we suggest that HPCs and FCs dynamics are tightly co-regulated in keratinocytes undergoing migration during wound healing.

Distribution of costameric proteins in normal human ventricular and atrial cardiac muscle.

In the mature heart, the intercalated disc and costameres provide the cell-cell and cell-matrix junctions respectively. Intercalated disc is situated at the bipolar ends of the cardiomyocytes and the myofibrils are anchored at this structure. The costameres mediate integration with the extracellular matrix that covers individual cardiomyocytes laterally. Costameres are considered as "proteic machinery" that appears to comprise two protein complexes: the dystrophin-glycoprotein complex (DGC) and the vinculin-talin-integrin system. There are structural differences between atrial and ventricular myocytes, but there have been relatively few studies that have analyzed costameres and focal adhesion function in cardiac cells. Our previous study carried out only on atrial myocytes, demonstrated that the DGC and talin-vinculin-integrin complexes had a costameric distribution that, unlike skeletal muscle, it localized only on the I band. We performed a further immunohistochemical analysis extending also the evaluation to the normal human cardiac muscle fibers obtained from ventricle and interventricular septum, in order to define the distribution and the spatial relationship between the proteins of the two complexes also in the other heart districts. Immunoconfocal microscopy of cardiac tissue revealed the costameric distribution of DGC and of vinculin-talin-integrin system, the association of all tested proteins in intercalated disks, in disagreement with other Authors, and in T-tubule with irregular spokelike extensions penetrating toward the center of the cell. Moreover, our data showed that all tested proteins colocalize between each other.

Altered Cytoskeletal Structure of Smooth Muscle Cells in Ureteropelvic Junction Obstruction

Purpose Ureteropelvic junction obstruction (UPJO) is the most common cause of congenital hydronephrosis. It was suggested an important role in smooth muscle cell (SMC) malfunction and apoptosis as primary anomaly in UPJO. SMC cytoskeleton in UPJO, however, has not been investigated. Vinculin-talin-integrin arrangement is a known systems of interaction between extracellular matrix and sarcolemma-associated cytoskeleton. Our purpose is to evaluate the cytoskeletal structure of SMC of infants affected by UPJO. Material and Methods Tissue specimens obtained from ten pyeloplasty were divided into 3 sections, renal pelvis above the obstruction, UPJ and ureter below the obstruction. For control group, eight unaffected UPJ of matched-aged infants were autopsied. Specimens, fixed in paraformaldeyde, were examinated using anti-β1-D, anti-β1-A, anti-α7-B and anti-α7-A integrins, anti-talin, anti-vinculin and anti-β-dystroglycan antibodies. Sections were observed using a confocal laser scanning microscopy. Results A significant loss of talin, vinculin, β-dystroglycan, β1-D and α7-D integrins were detected in obstructed UPJ, while a significant enhancement of β1-A and α7-A integrins was shown as compared to control UPJ. Conclusions In UPJO we observed an alternatively spliced isoform of both α and β subunits. Integrins play a crucial role in cell adhesion including cell-matrix and intracellular interactions, they are involved in cell migration, survival and differentiation and transmit signals by organizing the cytoskeleton.Expression of β1-A and α7-A integrins appears to be developmentally regulated and needs to meet the requirements of the different biochemical adhesion situations during SMC development. We observed a loss of talin, a flexible cystoskeletal protein important in the assembly of cell-matrix junctions, a lacking of vinculin, protein for control of apoptosis, and of β-dystroglycan, a transmembrane glycoprotein fundamental in connecting the intracellular cytoskeleton to extracellular matrix and able also to modulate the expression of α7-B integrin. Our data suggest that primary anomaly in UPJO might be attributed to a critical alteration of SMC cytoskeleton, promoting SMC abnormal function and apoptosis.

Structural Determinants of Integrin Binding to the Talin Rod

The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin β-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, “IBS2-A” (1974-2139) and “IBS2-B” (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.