Type VIII collagen: heterotrimeric chain association

Two chains, alpha1(VIII) and alpha2(VIII), have been described for type VIII collagen. Early work suggested that these chains were present in a 2:1 ratio, although recent work has shown that homotrimers can form and predominate in some tissues. In order to address the question of whether the alpha1(VIII) and alpha2(VIII) chains could co-polymerise we made a shortened alpha1(VIII) chain and expressed this with full length alpha2(VIII) chain in an in vitro translation system supplemented with semi-permeabilised cells. Heterotrimers containing either two or one alpha2(VIII) were evident. Interestingly, a point mutation in the NC1 domain of the alpha1(VIII) chain abrogated trimer formation. In addition we were able to demonstrate chain association of the alpha1(X) chain of type X collagen with the shortened alpha1(VIII) chain. Variations in chain association were seen when altered ratios of message were used. These results demonstrate the importance of the NC1 domain in chain association and suggest that gene expression regulates the composition and function of type VIII collagen by varying chain composition.     

Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels

The hepatic vitamin A-storing Ito cell has been implicated as a causative cell in hepatic fibrogenesis. Using a modification of a recent method (Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8681-8685), rat Ito cells were isolated and passaged in vitro on collagen-coated plastic dishes through cell generation 40-50. The collagen synthetic phenotype for Ito cells grown on various extracellular matrices was demonstrated by immunofluorescence and quantitated by competition enzyme-linked immunosorbent assays. When grown on a type I collagen matrix, Ito cells produced type IV greater than type III greater than type I collagen. When grown on a type IV collagen matrix, the cells produced relatively equal amounts of types I and III collagen. The absolute amounts of type I collagen produced were greater when cells were grown on type IV versus type I matrix. When 10(-5) M retinol was added to cell cultures, there was a uniform increase in type III collagen regardless of matrix type but a decrease in type I collagen when cells were grown on a type IV matrix and a large increase in type I collagen when cells were grown on a type I collagen matrix. The levels of cellular retinol binding protein, a key cytosolic retinol transport protein, were quantitated by high performance liquid chromatography and compared for cells grown on type I versus type IV collagen matrices. It was found that cells on a type I matrix contain 4.96 +/- 2.8 times more cellular retinol binding protein than do cells grown on a type IV matrix. In conclusion, Ito cell collagen synthesis may be altered by underlying extracellular matrix and exogenous retinol. This in vitro culture system should allow the study of regulatory factors and possible therapeutic anti-fibrogenic mediators.     

Type VIII collagen from bovine Descemet's membrane: structural characterization of a triple-helical domain

Bovine corneal Descemet's membrane (DM) was subjected to limited pepsin digestion. Soluble native collagens were fractionated by differential salt precipitation, and a mixture of type V collagen and collagenous fragments with a chain Mr of 50,000 (50K) was obtained at a concentration of 1.5 M NaCl. Further purification of the 50K collagen by molecular sieve and high-performance liquid chromatography resulted in the isolation of two-non-disulfide-bonded polypeptides, 50K-A and 50K-B, which were susceptible to several neutral proteases, including bacterial collagenase. By the criteria of peptide mapping, amino acid composition, and N-terminal sequence analysis, 50K-A and 50K-B were structurally dissimilar, although both chains contained Gly-X-Y repeats. 50K-A and 50K-B were immunologically and structurally distinct from collagen type I, III, IV, V, and VI. Immunohistochemical studies of bovine ocular tissue showed preferential distribution of the collagen containing the 50K fragment in the DM, with a more disperse arrangement of apparently interconnecting fibrils in the corneal stroma. Type VIII collagen isolated from the culture medium of metabolically radiolabeled bovine corneal endothelial (BCE) cells and its pepsin-resistant Mr 50 000 domain(s) both cross-reacted with antisera to 50K polypeptides from the corneal DM. Additionally, the CNBr peptide maps of pepsin-resistant Mr 50 000 polypeptides of type VIII collagen isolated from BCE cells and bovine corneal DM were highly similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.     

Liquid crystallinity in condensed type I collagen solutions. A clue to the packing of collagen in extracellular matrices.

We recently described a new type of assembly of collagen molecules, forming typical liquid crystalline phases in highly concentrated solutions after sonication. The present work shows that intact 300 nm long collagen molecules also form cholesteric liquid crystalline domains, but the time required is much longer, several weeks instead of several days. Differential calorimetry and X-ray diffraction show that sonication does not alter the triple-helical structure of the collagen fragments. In the viscous solutions, observed between crossed polars in optical microscopy, the textures vary as a function of the concentration. Molecules first align near the air interface at the coverslip edge, then as the concentration increases by slow evaporation of the solvent, the birefringence extends inwards and liquid crystalline domains progressively appear. For concentrations estimated to be above 100 mg/ml, typical textures and defects of cholesteric phases are obtained, at lower concentrations zig-zag extinction patterns and banded patterns are observed; all these textures are described and interpreted. The cholesteric packing of collagen fibrils in various extracellular matrices is known, and the relationship that can be made between the ordered phases obtained with collagen molecules in vitro and the related geometrical structures observed between fibrils in vivo is thoroughly discussed.     

Native collagen has a two-bonded structure

An intermediate in the assembly of bacteriophage φX174 virions that sediments at 12 S and contains equimolar amounts of the capsid proteins coded by genes F and G was characterized. A functional B gene product seems necessary for the synthesis of this aggregate, that results from the joining of one 6 S particle (containing five molecules of G protein) to one 9 S particle (a pentamer of F protein). B protein is not found in the 12 S structure.     

Apoptosis is physiologically restricted to a specialized cytoplasmic compartment in rat spermatids

Cytoplasmic caudal tags of maturing spermatids condense and are detached from the spermatidal cells just before the spermatids are released as spermatozoa. The detached cytoplasmic masses are termed "residual bodies." Features of residual bodies seem to be compatible with those of apoptosis and, just as occurs with apoptotic bodies, residual bodies are phagocytosed by Sertoli cells. Since in vitro studies have demonstrated that nucleus and cytoplasm apoptosis events can be independent phenomena, we reasoned that apoptosis pathways might be restricted to the caudal tag of the maturing spermatids in order to originate residual bodies. Consistent with this idea, here we showed that annexin V specifically bound the membranes of isolated residual bodies and that expression levels of caspase-1, c-jun, p53, and p21 were specifically increased in these cytoplasmic compartments. Electron microscopy of cytoplasmic lobes and residual bodies confirmed that their ultrastructural features were those of apoptosis. These data indicate that the mechanism responsible for the formation of residual bodies is similar to that for apoptotic bodies; and the study presents evidence, for the first time, that apoptotic signaling molecules can be restricted to a cytoplasmic compartment and proceed in the presence of a healthy nucleus.     

Identification of a specialized extracellular matrix component in Drosophila imaginal discs

We have generated a monoclonal antibody that recognizes a major component of a specialized extracellular matrix in Drosophila imaginal discs. In mature larvae, antibody binding is observed almost exclusively on imaginal discs. On the basal surface of the thoracic discs, the antigen is localized to particular regions of the epithelium, and ultrastructural studies indicate that the antigen is found in a fibrous network secreted between the cells and the basal lamina. The localized expression indicates that the matrix is not simply related to disc differentiation, as all regions of the columnar disc epithelium are determined to secrete adult cuticle. A correlation of the antigen distribution with known developmental events leads us to propose that the antigen-containing network provides an extensible matrix for the rapid elongation of the disc epithelium during evagination; consistent with this, the antigen is a component of the matrix between the dorsal and ventral surfaces of the evaginated wing pouch. The antigen is very large (greater than 5 X 10(5) Da), can be labeled metabolically with methionine and sulfate, and is digested by chondroitinase ABC; these biochemical characteristics indicate that the antigen is a proteoglycan.     

A specialized nucleosome has a "point" to make

Three recent papers, including Mizuguchi et al. (2007) in this issue, show that the nonhistone protein Scm3 is required for the recruitment of the histone H3 variant Cse4 to centromeres in budding yeast. Scm3 forms a chromatin component with Cse4:histone H4 tetramers that appear to lack H2A/H2B histones. These studies provide key insights into the pathway that recruits Cse4 to centromeres and have important implications for other functions of chromatin.     

Cell transport in model extracellular matrices

The rapid transport of cells has been shown to occur by ordered countercurrent convection. This convection can be created by mixtures of macromolecules which make up the extracellular matrix and by the degradation and aggregation products of these macromolecules. The ordered countercurrent convection is manifested in the form of structured flows and arises in isothermal systems with small concentration gradients of solutes. The flows are gravity driven but may rapidly move at angles close to the horizontal axis if they are mechanically constrained to do so. These flows have been shown to rapidly transport cells at rates ranging from 1 to 100 mm h-1, depending on the conditions of the experiment. The transport of cells is nonspecific in that various cell types (chondrocytes, fibroblasts, endothelial cells, and red blood cells) as well as inert particles of similar size (latex beads 6-microns diam) are transported at similar rates. Latex bead transport by structured flow has also been demonstrated to occur in confined spaces in the form of Teflon tubing down to 200 microns in diameter and at angles in the range of 45-90 degrees to the horizontal axis. The flows may also occur over relatively long distances for a prolonged period of time. The conditions for flow formation are simple and widespread. It is suggested that it may contribute to the forces involved in the movement of cells in the extracellular matrix in vivo especially during remodeling and embryogenesis.     

Dendritic fibroblasts in three-dimensional collagen matrices

Cell motility determines form and function of multicellular organisms. Most studies on fibroblast motility have been carried out using cells on the surfaces of culture dishes. In situ, however, the environment for fibroblasts is the three-dimensional extracellular matrix. In the current research, we studied the morphology and motility of human fibroblasts embedded in floating collagen matrices at a cell density below that required for global matrix remodeling (i.e., contraction). Under these conditions, cells were observed to project and retract a dendritic network of extensions. These extensions contained microtubule cores with actin concentrated at the tips resembling growth cones. Platelet-derived growth factor promoted formation of the network; lysophosphatidic acid stimulated its retraction in a Rho and Rho kinase-dependent manner. The dendritic network also supported metabolic coupling between cells. We suggest that the dendritic network provides a mechanism by which fibroblasts explore and become interconnected to each other in three-dimensional space.     

Fibroblast biology in three-dimensional collagen matrices

Research on fibroblast biology in three-dimensional collagen matrices offers new opportunities to understand the reciprocal and adaptive interactions that occur between cells and surrounding matrix in a tissue-like environment. Such interactions are integral to the regulation of connective tissue morphogenesis and dynamics that characterizes tissue homeostasis and wound repair. During fibroblast-collagen matrix remodeling, mechanical signals from the remodeled matrix feed back to modulate cell behavior in an iterative process. As mechanical loading (tension) within the matrix increases, the mechanisms used by cells to remodel the matrix change. Fibroblasts in matrices that are under tension or relaxed respond differently to growth factor stimulation, and switching between mechanically loaded and unloaded conditions influences whether cells acquire proliferative/biosynthetic active or quiescent/resting phenotypes.     

HMGB1, an architectural chromatin protein and extracellular signalling factor,has a spatially and temporally restricted expression pattern in mouse brain

HMGB1 is an abundant chromatin component, so far considered ubiquitous. HMGB1 also has an extracellular signalling role: when passively released by necrotic cells, it triggers inflammation; moreover, it can be actively secreted by myeloid cells, neurons and neuronal cancer cells. We show here that HMGB1 protein is undetectable in most cells in adult mouse brain, and is present in a subset of brain cells during development, with a very complex temporal, spatial and subcellular expression pattern. HMGB1 is expressed in the cortical plate of E14.5 embryos, predominantly in the nucleus, although roughly 1% of cells show a cytoplasmic localization as well. In E16 embryos, HMGB1 is nuclearly expressed in scattered cells apparently moving from the ventricular zone to the cortical plate. HMGB1 expression is strongly down-regulated at later developmental stages; in adult mice significant expression is maintained only in areas of continuing neurogenesis. Finally, HMGB1 subcellular localization changes during retinoic acid induced differentiation of P19 neuroblastoma cells.     

Imaging cardiac extracellular matrices: a blueprint for regeneration

Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated.     

Neurofascin assembles a specialized extracellular matrix at the axon initial segment

Action potential initiation and propagation requires clustered Na(+) (voltage-gated Na(+) [Nav]) channels at axon initial segments (AIS) and nodes of Ranvier. In addition to ion channels, these domains are characterized by cell adhesion molecules (CAMs; neurofascin-186 [NF-186] and neuron glia-related CAM [NrCAM]), cytoskeletal proteins (ankyrinG and betaIV spectrin), and the extracellular chondroitin-sulfate proteoglycan brevican. Schwann cells initiate peripheral nervous system node formation by clustering NF-186, which then recruits ankyrinG and Nav channels. However, AIS assembly of this protein complex does not require glial contact. To determine the AIS assembly mechanism, we silenced expression of AIS proteins by RNA interference. AnkyrinG knockdown prevented AIS localization of all other AIS proteins. Loss of NF-186, NrCAM, Nav channels, or betaIV spectrin did not affect other neuronal AIS proteins. However, loss of NF-186 blocked assembly of the brevican-based AIS extracellular matrix, and NF-186 overexpression caused somatodendritic brevican clustering. Thus, NF-186 assembles and links the specialized brevican-containing AIS extracellular matrix to the intracellular cytoskeleton.     

Immunolocalization of type VIII collagen in vascular tissue

Type VIII collagen was first detected in the culture medium of aortic endothelial cells. Subsequently its synthesis by a number of other cell lines was demonstrated. Its presence in vascular tissue is reported here. It is a component of sheep aorta and carotid artery but could not be demonstrated in the jugular vein. It is principally localized in the subendothelial region but this can only be demonstrated after pretreatment of the tissue with proteases. Thus type VIII collagen is a constituent of blood vessels.     

Mechanical model for a collagen fibril pair in extracellular matrix

In this paper, we model the mechanics of a collagen pair in the connective tissue extracellular matrix that exists in abundance throughout animals, including the human body. This connective tissue comprises repeated units of two main structures, namely collagens as well as axial, parallel and regular anionic glycosaminoglycan between collagens. The collagen fibril can be modeled by Hooke’s law whereas anionic glycosaminoglycan behaves more like a rubber-band rod and as such can be better modeled by the worm-like chain model. While both computer simulations and continuum mechanics models have been investigated for the behavior of this connective tissue typically, authors either assume a simple form of the molecular potential energy or entirely ignore the microscopic structure of the connective tissue. Here, we apply basic physical methodologies and simple applied mathematical modeling techniques to describe the collagen pair quantitatively. We found that the growth of fibrils was intimately related to the maximum length of the anionic glycosaminoglycan and the relative displacement of two adjacent fibrils, which in return was closely related to the effectiveness of anionic glycosaminoglycan in transmitting forces between fibrils. These reveal the importance of the anionic glycosaminoglycan in maintaining the structural shape of the connective tissue extracellular matrix and eventually the shape modulus of human tissues. We also found that some macroscopic properties, like the maximum molecular energy and the breaking fraction of the collagen, were also related to the microscopic characteristics of the anionic glycosaminoglycan.     

The radial decline of nerve impulses in a restricted cylindrical extracellular space

In order to increase the potentials recorded extracellularly from nerve fibres, peripheral nerves are often placed in restricted space with cylindrical geometry. Equations are derived for computing the potentials expected at the surface of the cylinder, based on the potentials at the external surface of a small nerve fibre located on the long axis of the cylinder. These equations are evaluated numerically, using two formulae for a nerve impulse given in the literature. In both cases there is little attenuation for cylinders with radii less than 0.5 mm, but the potential declines approximately as a power of radius b for 1<b<10 mm. Various factors which might affect these results under different experimental conditions are discussed.