The extracellular matrix (mesoglea) of hydrozoan jellyfish and its ability to support cell adhesion and spreading

The outer mesoglea (extracellular matrix; ECM) of hydrozoan jellyfish was found to contain a species-specific meshwork of striated fibers of different diameters. In the mesoglea, molecules were identified which exhibit several features of well known vertebrate ECM: a laminin-like molecule which appears cross-shaped on electronmicrographs, a fibronectin-like molecule (both detectable by their immunoreactivity at the exumbrella side) and a species-specific collagen consisting of 3 different -chains of which at least 2 can be decorated with con A. The -chains are linked by disulfide bridges. Acetic acid extraction of the mesoglea and subsequent salt precipitation yields fibrils which appear banded in the electron microscope and support species-specific adhesion and spreading of isolated tissue. These precipitated fibrils are mainly composed of the disulfide-linked collagen.     

Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.     

Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9′10), (ii) an Arg to Ala synergy site mutation (FnIII9^R→^A10), (iii) a two-Gly (FnIII9^2G10) insertion, and (iv) a four-Gly (FNIII9^4G10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9′10 and FnIII9^2G10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9′10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9′10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.     

Integrin Signaling: Cytoskeletal Complexes,MAP Kinase Activation,and Regulation of Gene Expression

Members of the integrin family of adhesion receptors mediate interactions of cells with the extracellular matrix. Besides their role in tissue morphogenesis by anchorage of cells to basement membranes and migration along extracellular matrix proteins, integrins are thought to play a key role in mediating the control of gene expression by the extracellular matrix. Studies over the past 10 years have shown that integrin-mediated cell adhesion can trigger signal transduction cascades involving translocation of proteins and protein tyrosine phosphorylation events. In this review, we discuss approaches used in our lab to study early events in integrin signalling as well as further downstream changes.     

Temporal analysis of vascular smooth muscle cell elasticity and adhesion reveals oscillation waveforms that differ with aging

A spectral analysis approach was developed for detailed study of time‐resolved, dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion to identify differences in VSMC from young and aged monkeys. Atomic force microscopy (AFM) was used to measure Young’s modulus of elasticity and adhesion as assessed by fibronectin (FN) or anti‐beta 1 integrin interaction with the VSMC surface. Measurements demonstrated that VSMC cells from old vs. young monkeys had increased elasticity (21.6 kPa vs. 3.5 kPa or a 612% increase in elastic modulus) and adhesion (86 pN vs. 43 pN or a 200% increase in unbinding force). Spectral analysis identified three major frequency components in the temporal oscillation patterns for elasticity (ranging from 1.7 × 10^?3 to 1.9 × 10^?2 Hz in old and 8.4 × 10^?4 to 1.5 × 10^?2 Hz in young) and showed that the amplitude of oscillation was larger (P < 0.05) in old than in young at all frequencies. It was also observed that patterns of oscillation in the adhesion data were similar to the elasticity waveforms. Cell stiffness was reduced and the oscillations were inhibited by treatment with cytochalasin D, ML7 or blebbistatin indicating the involvement of actin–myosin‐driven processes. In conclusion, these data demonstrate the efficacy of time‐resolved analysis of AFM cell elasticity and adhesion measurements and that it provides a uniquely sensitive method to detect real‐time functional differences in biomechanical and adhesive properties of cells. The oscillatory behavior suggests that mechanisms governing elasticity and adhesion are coupled and affected differentially during aging, which may link these events to changes in vascular stiffness.     

Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix

Fibronectin and laminin production by human keratinocytes cultured in serum-free, low-calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7-day keratinocyte cultures was determined by 18 hr 35S-methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.     

The beta-amyloid peptide of Alzheimer's disease decreases adhesion of vascular smooth muscle cells to the basement membrane

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Abeta) builds up. In this study, the possibility that Abeta disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Abeta in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Abeta was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Abeta that was fluorescently labelled at the N-terminus (fluo-Abeta) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Abeta. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Abeta1-40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Abeta treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Abeta on cell adhesion. The studies suggest that Abeta can decrease VSMC viability by disrupting VSMC-extracellular matrix (ECM) adhesion.     

Integrin α3β1 Engagement Disrupts Intercellular Adhesion

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of β1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell–cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell–cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand—collagen type I, fibronectin, or laminin 1—MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell–cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional β1 integrin and specifically α3β1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial–mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin–ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.     

Integrins stimulate phosphorylation of neurofilament NF-M subunit KSP repeats through activation of extracellular regulated-kinases (Erk1/Erk2) in cultured motoneurons and transfected NIH 3T3 cells

Integrin-mediated interactions of cells with components of the extracellular matrix (ECM) regulate cell survival, cell proliferation, cell differentiation and cell migration through activation of multiple intracellular signal transduction pathways. In this study, we have demonstrated that integrin-matrix interactions promote KSP tail-domain phosphorylation of neurofilament medium molecular weight subunits (NF-M) in cultured rat spinal cord motoneurons and NF-M transfected NIH 3T3 cells. We found that laminin and fibronectin induce NF-M tail-domain phosphorylation in motoneurons and NIH 3T3 cells transfected with NF-M, respectively. This phosphorylation was selectively inhibited by PD98059, a specific MEK1 inhibitor. This suggests that laminin and fibronectin-induced MEK1 activation and the downstream targets Erk1 and Erk2 are involved in NF-M KSP tail-domain phosphorylation. This pathway appears to represent one of the mechanisms whereby integrin-extracellular matrix interactions are involved in phosphorylation of the NF-M KSP tail domain.     

Aspects of Mineral Structure in Normally Calcifying Avian Tendon

Structural characteristics of normally calcifying leg tendons of the domestic turkey Meleagris gallopavo have been observed for the first time by tapping mode atomic force microscopy (TMAFM), and phase as well as corresponding topographic images were acquired to gain insight into the features of mineralizing collagen fibrils and fibers. Analysis of different regions of the tendon has yielded new information concerning the structural interrelationships in vivo between collagen fibrils and fibers and mineral crystals appearing in the form of plates and plate aggregates. TMAFM images show numerous mineralized collagen structures exhibiting characteristic periodicity (54-70 nm), organized with their respective long axes parallel to each other. In some instances, mineral plates (30-40 nm thick) are found interspersed between and in intimate contact with the mineralized collagen. The edges of such plates lie parallel to the neighboring collagen. Many of these plates appear to be aligned to form larger aggregates (475-600 nm long x 75-90 nm thick) that also retain collagen periodicity along their exposed edges. Intrinsic structural properties of the mineralizing avian tendon have not previously been described on the scale reported in this study. These data provide the first visual evidence supporting the concept that larger plates form from parallel association of smaller ones, and the data fill a gap in knowledge between macromolecular- and anatomic-scale studies of the mineralization of avian tendon and connective tissues in general. The observed organization of mineralized collagen, plates, and plate aggregates maintaining a consistently parallel nature demonstrates the means by which increasing structural complexity may be achieved in a calcified tissue over greater levels of hierarchical order.     

Deposition of Basement Membrane Proteins in Attachment and Neurite Formation of Cultured Murine C-1300 Neuroblastoma Cells

The deposition of the basement membrane glycoproteins, laminin, fibronectin, and type IV procollagen was studied by indirect immunofluorescence microscopy during the attachment and differentiation of murine C-1300 neuroblastoma cells. A typical cytoplasmic perinuclear staining for the basement membrane antigens was seen both in undifferentiated and differentiated cells. Freshly seeded suspended cells lacked surface fluorescence but in two hours after plating, distinct punctate laminin deposits became discernible on the ventral surface of the cells. Notably, in sparsely seeded undifferentiated cultures, the cell-associated extracellular laminin deposits could only be detected under the primary attaching cells, whereas daughter cells in clonal cell colonies lacked such fluorescence. In cultures induced to neurite formation with dibutyryl cyclic AMP, laminin deposition was also detected in association with the growing cytoplasmic extensions. No distinct differences were found between the secreted proteins of cultures of differentiated and nondifferentiated neuroblastoma cells, but the patterns of fucosylation of high-molecular weight proteins in the two cultures were markedly different. We conclude that cultured neuroblastoma cells both synthesize, secrete and deposit laminin. The distribution of laminin during neuroblastoma cell attachment and neurite extension suggests that this glycoprotein may be involved in cell–to–substratum interactions in C-1300 cell cultures.     

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.