The interaction between urokinase receptor and vitronectin in cell adhesion and signalling

The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells present in all tissue and organs. Cell-matrix interactions play fundamental roles during embryonic development, morphogenesis, tissue homoeostasis, wound healing, and tumourigenesis. Cell-matrix communication is kept in balance by physical contact and by transmembrane integrin receptors providing the dynamic link between the extracellular and intracellular environments through bi-directional signalling. The urokinase-type plasminogen activator receptor (uPAR) is a plasma membrane receptor overexpressed during inflammation and in almost all human cancers. One of its functions is to endorse ECM remodelling through the activation of plasminogen and downstream proteases, including matrix-metalloproteases (MMPs). Beside its role in ECM degradation, uPAR modulates cell-matrix contact through a direct engagement with the ECM component, vitronectin (Vn), and by regulating the activity state of integrins thus promoting or inhibiting integrin signalling and integrin-mediated cell adhesion to other ECM components, like fibronectin and collagen. In this review we have centred our attention on the non-proteolytic function of uPAR as a mediator of cell adhesion and downstream signalling.     

Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices

Summary Interactions between epithelial cells and their environment are critical for normal function. Mammary epithelial cells require hormonal and extracellular matrix (ECM) signalling for the expression of tissue specific characteristics. With regard to ECM, cultured mammary epithelial cells synthesize and secrete milk proteins on stromal collagen I matrices. The onset of function coincides both with morphogenesis of a polarized epithelium and with deposition of basement membrane ECM basal to the cell layer. Mammary specific morphogenesis and biochemical differentiation is induced if mammary cells are cultured directly on exogenous basement membrane (EHS). Thus ECM may effect function by the concerted effect of permissivity for cell shape changes and the direct biochemical signalling of basement membrane molecules.A model is discussed where initial ECM control of mammary epithelial cell function originates in the interstitial matrix of stroma and subsequently transfers to the basement membrane when the epithelial cells have accumulated and deposited an organized basement membrane matrix.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

WAKs; cell wall associated kinases.

Students of metazoan biology have traditionally viewed the extracellular matrix (ECM) as a substrate with which cells interact to participate in developmental pattern formation and define a specific location. In contrast, the plant cell wall has been viewed as a cage that limits and thus directs plant cell morphology, and perhaps for this reason many have shied away from calling the plant cell wall the ECM. The recent discovery of a variety of receptor molecules and their ligands on the surface of plant cells and the intimate role cell walls play in development should direct our thinking toward a more dynamic view of the plant cell wall. A recent example, is the discovery of wall associated kinases (WAKs), which may well signal between the ECM and the cell and are required for cell expansion.     

Structure and function of the receptor-like protein kinases of higher plants

Cell surface receptors located in the plasma membrane have a prominent role in the initiation of cellular signalling. Recent evidence strongly suggests that plant cells carry cell surface receptors with intrinsic protein kinase activity. The plant receptor-like protein kinases (RLKs) are structurally related to the polypeptide growth factor receptors of animals which consist of a large extracytoplasmic domain, a single membrane spanning segment and a cytoplasmic domain of the protein kinase gene family. Most of the animal growth factor receptor protein kinases are tyrosine kinases; however, the plant RLKs all appear to be serine/threonine protein kinases. Based on structural similarities in their extracellular domains the RLKs fall into three categories: the S-domain class, related to the self-incompatibility locus glycoproteins of Brassica; the leucine-rich repeat class, containing a tandemly repeated motif that has been found in numerous proteins from a variety of eukaryotes; and a third class that has epidermal growth factor-like repeats. Distinct members of these putative receptors have been found in both monocytyledonous plants such as maize and in members of the dicotyledonous Brassicaceae. The diversity among plant RLKs, reflected in their structural and functional properties, has opened up a broad new area of investigation into cellular signalling in plants with far-reaching implications for the mechanisms by which plant cells perceive and respond to extracellular signals.     

Actin dynamics at sites of extracellular matrix degradation

The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.     

Extracellular matrix molecules, their receptors, and secreted proteases in synaptic plasticity

Neural cells secrete diverse molecules, which accumulate in the extracellular space and form the extracellular matrix (ECM). Interactions between cells and the ECM are well recognized to play the crucial role in cell migration and guidance of growing axons, whereas formation of mature neural ECM in the form of perineuronal nets is believed to restrict certain forms of developmental plasticity. On the other hand, major components of perineuronal nets and other ECM molecules support induction of functional plasticity, the most studied form of which is long-term potentiation. Here, we review the underlying mechanisms by which ECM molecules, their receptors and remodeling proteases regulate the induction and maintenance of synaptic modifications. In particular, we highlight that activity-dependent secretion and activation of proteases leads to a local cleavage of the ECM and release of signaling proteolytic fragments. These molecules regulate transmitter receptor trafficking, actin cytoskeleton, growth of dendritic spines, and formation of dendritic filopodia.     

Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon

The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated "fibripositors") that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.     

The interaction between uPAR and vitronectin triggers ligand‐independent adhesion signalling by integrins

The urokinase‐type plasminogen activator receptor (uPAR) is a non‐integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure–function analyses of uPAR, VN and integrins, we document that uPAR‐mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin–matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non‐canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non‐integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.     

Receptor kinases with leucine-rich repeats are enriched in Triton X-100 insoluble plasma membrane microdomains from plants

In mammals, signalling components at the cell surface are clustered in Triton X-100 insoluble plasma membrane microdomains. We isolated plasma membrane microdomains from Arabidopsis and mustard cotyledons and determined their protein composition by mass spectrometry. Although the protein composition of the plant vesicles differ from the composition of the animal vesicles, they are also enriched in signalling components. We identified at least seven receptor kinases with leucine-rich repeats, 10 other kinases, the β subunit of heterotrimeric G-proteins and five small GTP-binding proteins. Thus, specific signalling components are highly enriched in plant plasma membrane microdomains while others are excluded.     

Semiotic Tools For Multilevel Cell Communication

Cell communication plays a key role in multicellular organisms. In developing embryos as in adult organisms, cells communicate by coordinating their differentiation through the establishment and/or renewal of a variety of cell communication channels. Under both these conditions, cells interact by either receptor signalling, surface recognition of specific cell adhesion molecules or transfer of cytoplasmic components through junctional coupling. In recent years, it has become apparent that cells may also communicate through the extracellular release of microvesicles. They may originate as either exosomes from the endosomal compartment upon fusion of multivesicular bodies with the plasma membrane or be shed directly from the plasma membrane via extensions of the cell surface. Microvesicles may disperse over long distances through body fluids and deliver their molecular cargo onto a variety of target cells. As a general rule, the metabolic fate of these cells is determined by the molecular nature of the vesicular cargo, while targeting itself depends on the affinity of the molecules expressed on the enclosing membrane. In this paper, we will be arguing that intercellular vesicular transfer is substantially different from other types of cell communication, allowing cells and molecules to interact on varying levels. Cells interacting via ligand signalling owe their specificity to the steric coupling with cognate receptor molecules. As such, it is a pure molecular process that affects target cells only upon integration into their responding repertoire. In this relationship, coupled cells are reciprocally adapted to each other through the selection of their respective signalling capacities, following exploration of their receptor specificity. Interaction by intercellular vesicles realizes a substantially different type of cell communication. Vesicular traffic allows donor cells to carry out a horizontal type of gene transfer and target this information over long distances via independently controlled mechanisms. Because of this independence, cells interacting via vesicular traffic are not expected to adapt their signalling correspondences, but to control instead the efficiency of their cargo delivery irrespective of the receptor repertoire expressed by the target tissue. In this paper, the multifaceted functions of the intercellular vesicular traffic will be discussed in a multilevel biosemiotic perspective with the aim of unravelling the cellular mechanisms devised by nature to accomplish communication.     

Perturbing plasma membrane hemichannels attenuates calcium signalling in cardiac cells and HeLa cells expressing connexins

Many cell signalling pathways are driven by changes in cytosolic calcium. We studied the effects of a range of inhibitors of connexin channels on calcium signalling in cardiac cells and HeLa cells expressing connexins. Gap 26 and 27, peptides that mimic short sequences in each of the extracellular loops of connexin 43, and anti-peptide antibodies generated to extracellular loop sequences of connexins, inhibited calcium oscillations in neonatal cardiac myocytes, as well as calcium transients induced by ATP in HL-1 cells originating from cardiac atrium and HeLa cells expressing connexin 43 or 26. Comparison of single with confluent cells showed that intracellular calcium responses were suppressed by interaction of connexin mimetic peptides and antibodies with hemichannels present on unapposed regions of the plasma membrane. To investigate how inhibition of hemichannels in the plasma membrane by the applied reagents was communicated to calcium store operation in the endoplasmic reticulum, we studied the effect of Gap 26 on calcium entry into cells and on intracellular IP3 release; both were inhibited by Gap 26. Calcium transients in both connexin 43- and connexin 26-expressing HeLa cells were inhibited by the peptides suggesting that the extended cytoplasmic carboxyl tail domain of larger connexins and their interactions with intracellular scaffolding/auxiliary proteins were unlikely to feature in transmitting peptide-induced perturbations at hemichannels in the plasma membrane to IP3 receptor channel central to calcium signalling. The results suggest that calcium levels in a microenvironment functionally connecting plasma membrane connexin hemichannels to downstream IP3-dependent calcium release channels in the endoplasmic reticulum were disrupted by the connexin mimetic peptide, although implication of other candidate hemichannels cannot be entirely discounted. Since calcium signalling is fundamental to the maintenance of cellular homeostasis, connexin hemichannels emerge as therapeutic targets open to manipulation by reagents interacting with external regions of these channels.     

closca, a new gene required for both Torso RTK activation and vitelline membrane integrity. Germline proteins contribute to Drosophila eggshell composition

The Drosophila eggshell is a specialised extracellular matrix (ECM) that surrounds and protects the oocyte and the embryo until its eclosion. In addition, the vitelline membrane, the innermost layer of the eggshell, holds the local determinant required to activate the Torso RTK pathway, which establishes the embryonic terminal regions. Here we report the identification and characterisation of closca, a gene encoding a new member of a group of proteins that act non-redundantly in vitelline membrane biogenesis and in Torso signalling. We also show that the Nasrat protein, another member of this group, is incorporated into the vitelline membrane, thereby indicating that the eggshell is a shared ECM that receives contributions from both follicle cells and the germline. This observation also provides a new scenario that accounts for the long known contribution of germline products to vitelline membrane biogenesis and to the follicle cell-dependent activation of the Torso receptor.     

Angiotensin-converting enzyme signalling in human preadipocytes and adipocytes

Angiotensin-converting enzyme (ACE, kininase II) is a plasma membrane zinc metallopeptidase that acts as a key enzyme for the extracellular conversion of vasoactive peptides. Recently, ACE outside-in signalling in endothelial cells has been described. The present study tested the hypothesis that ACE signalling is not restricted to endothelial cells and may act as an additional peptide receptor on human preadipocytes and adipocytes. ACE protein levels were not changed during adipose conversion of human primary preadipocytes. The enzyme was primarily localized to the non-detergent-resistant fraction of the membrane and phosphorylated in non-dividing cells. Antibody arrays of whole cell lysate detected putative ACE-interacting proteins, which all share important roles in cell cycle control and/or apoptosis. These findings suggest that ACE is a versatile molecule, involved both in the regulation of extracellular peptide concentrations and direct intracellular signalling. In human adipose cells ACE may potentially influence exit from the cell cycle, differentiation, and programmed cell death signalling.     

Extracellular ATP: a potential regulator of plant cell death

Adenosine 5′‐triphosphate (ATP) has been regarded as an intracellular energy currency molecule for many years. In recent decades, it has been determined that ATP is released into the extracellular milieu by animal, plant and microbial cells. In animal cells, this extracellular ATP (eATP) functions as a signalling compound to mediate many cellular processes through its interaction with membrane‐associated receptor proteins. It has also been reported that eATP is a signalling molecule required for the regulation of plant growth, development and responses to environmental stimuli. Recently, the first plant receptor for eATP was identified in Arabidopsis thaliana. Interestingly, some studies have shown that eATP is of particular importance in the control of plant cell death. In this review article, we summarize and discuss the theoretical and experimental advances that have been made with regard to the roles and mechanisms of eATP in plant cell death. We also make an attempt to address some speculative aspects to help develop and expand future research in this area.     

Ultrastructural evidence for contacts between migrating L5222 rat leukemia cells and extracellular matrix components of the rat mesentery

The nature of interactions between cells migrating through tissues and their structural surroundings are largely unknown. We have therefore examined the ultrastructural relationship between L5222 rat leukemia cells, moving through the loose connective tissue of the mesentery, and components of the extracellular matrix (ECM). Ultrathin tissue sections, fixed in the presence of ruthenium hexammine trichloride (RHT), revealed the following: Constitutents of fibrillar and nonfibrillar elements of the ECM are in contact with the plasma membrane of L5222 cells. Linear nonfibrillar ECM elements contact the plasma membrane at point-like sites, often associated with root-like structures present within the submembraneous microfilament mesh. Aggregates of ECM material are connected to patch-like cell membrane sites, associated with a condensed, plate-like part of the microfilament mesh. Point-like and patch-like contacts are more numerous at the anterior part of polarized migrating L5222 cells than on the posterior end. In round resting leukemia cells they are evenly distributed around the cell periphery. We suggest that the ECM-cell membrane contacts represent tissue adhesion sites. We therefore hypothesize that in migrating cells a coordinate interaction occurs between the contact sites and the continuous microfilament meshwork which results in a simultaneous backward movement of ECM-membrane contacts on the cell body and in a net forward movement of the whole cell. Since Dembo et al. (1981) present a similar mechanism for in vitro locomotion of granulocytes, we assume that blood cell locomotion in vivo and in vitro depends on similar molecular mechanisms: force generation by the cell, transmembraneous linkage between cytoskeletal and ECM elements, and membrane fluidity. The major difference in blood cell locomotion through a three-dimensional tissue or on a plane substratum would then be given by the distribution of contact sites, occurring around the cell periphery or limited to the ventral cell surface, respectively.     

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.     

Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.     

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.