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This chapter will outline a number of assays in use in the authors′ laboratory for the quantification of apoptosis in cultured mammary epithelial cells. These assays are each directed at particular stages of apoptosis, which are outlined below. There are a number of commercial assays available, utilizing such reagents as fluorescent caspase substrates, which determine an average level of apoptosis throughout a population of cells. However, we find a considerable use for assays that identify individual apoptotic cells, and it is chiefly these that will be described here. [ABSTRACT FROM AUTHOR]

Understanding the function of ECM and cell-matrix interactions in mammalian development has reached new levels of sophistication with the introduction of gene knockout technology. Indeed, two of the chapters in this volume provide detailed methods for producing mice with deletions in specific ECM genes (seeChapters 13 and 14). However, in some knockout mice, animals die during late embryogenesis or shortly after birth. In such cases, it is possible to analyze embryonic developmental phenotypes, but it is less easy to determine the in vivo role of cell-matrix interactions in adult tissues. [ABSTRACT FROM AUTHOR]

The basis for cell movement and the maintenance of organized epithelial structures formed by "stationary" cells is a phenomenon known as cell adhesion, i.e., the establishment of the firm anchorage of a cell to an underlying substratum. Both cell adhesion and the subsequent process of cell locomotion occur via the interaction of cell-adhesion molecules, integrins, cell-surface proteoglycans, nonintegrin receptors, and selections with each other or with extracellular ligands such as various ECM constituents. The number of extracellular matrix (ECM) components discovered to be implicated in cell adhesion and migration phenomena is rapidly growing. This fact, in conjunction with the progressively unveiled diversity in the mode by which these ECM molecules affect cell behavior, has raised the demands on the in vitro assays aimed at the analysis of cell adhesion. Probably, the requirement for a higher accuracy and versatility; for an increased capability to most closely reproduce the in vivo situations; and for the uncompromised possibility to apply the assays to the analysis of ex vivo cells; represent the primary demands that have forced investigators to ameliorate the currently available cell-adhesion protocols, as well as device novel alternative ones. [ABSTRACT FROM AUTHOR]

The basement membrane that separates an epithelium or other parenchymal tissues from the connective tissue has been postulated for a long time to be of epithelial origin. Because of difficulties in interpreting results obtained mostly by autoradiographic labeling at the light microscopy level, new analytical tools and models were important to develop. In particular, Lipton (1) using an in vitro coculture system was probably the first to provide evidence for a dual origin of the basement membrane: indeed a distinct basement membrane structure developed in myoblast cultures only after the addition of muscle fibroblasts. [ABSTRACT FROM AUTHOR]

Solid-phase assays provide a simple, rapid and robust method for the analysis of protein-protein interactions; i.e., does protein A interact with protein B? In this assay, protein A (here termed "receptor") is adsorbed to the wells of an enzyme-linked immunosorbent assay (ELISA) plate (solid phase). The plate is then blocked using bovine serum albumin (BSA), and biotin-labeled protein B (here termed 'ligand') is added. After washing the wells to remove unbound ligand, bound ligand is detected by addition of an avidin-peroxidase conjugate followed by a colorimetric detection step. [ABSTRACT FROM AUTHOR]

Cartilage is a dense connective tissue that functions to withstand and distribute load (1). Articular cartilage lines the ends of long bones and distributes loads across the joints. It consists of a dense collagenous matrix (primarily collagen type II, with smaller amounts of other collagens, including types I, V, VI, IX, and XI), embedded in a high concentration of aggregating proteoglycan, aggrecan. The collagen provides tensile properties, and the proteoglycans confer compressive properties and resiliency. There is a sparse population of a single cell type, the chondrocyte, distributed throughout the tissue. These cells synthesize and maintain the cartilaginous matrix in a regulated fashion that involves breakdown of matrix components, release of proteolytic products from the tissue, and synthesis and incorporation of new components into the matrix. [ABSTRACT FROM AUTHOR]

Tissue engineering seeks to produce living,three-dimensional cellular constructs that can be used as clinical replacements of damaged tissues and organs as well as research tools to study cell and matrix interactions that occur in higher-order systems.To organize the cells into a three-dimensional structure in vitro,a provisional extracellular matrix support is required.The two main methods to achieve this are a)to culture the stromal cells on a three-dimensional synthetic meshwork,or else b)embed the cells within a three-dimensional type I collagen lattice.The contracted collagen lattice can be used for a variety of practical applications including the support of epithe- lial growth and differentiation in order to produce a skin replacement (1-5). However,this model system can also be exploited for experiments to study cell-matrix interactions such as the influence of tension on cell phenotype (6). [ABSTRACT FROM AUTHOR]

This chapter will outline in detail the two standard assays used in the author' s laboratory for quantitating the adhesion of cells to an immobilized substrate. The attachment assay, which employs a colorimetric detection of bound cells, is based on Kueng et al. (1), and the spreading assay, which employs phase contrast microscopy to measure the flattening of adherent cells, is based on the method of Yamada and Kennedy (2). [ABSTRACT FROM AUTHOR]

In the developing central nervous system (CNS), oligodendrocyte precursor cells (OPCs) originate in discreet regions, which may be at a significant distance from their final position. These cells must, therefore, undergo extensive migration in order to reach this final destination (1). The regulation of OPC migration remains to be fully elucidated, with the role of external factors such as extracellular matrix proteins, and growth factors, as well as cell-cell interactions with other neural cells currently being investigated. In order to assess the possible regulation of OPC migration, we have refined two separate assays that present the OPCs with putative regulatory molecules in different forms. [ABSTRACT FROM AUTHOR]

Many important processes in the development of the vertebrate central nervous system (CNS) rely on signals induced by extracellular matrix (ECM) components (1,2). These molecules are known to contain multiple functional domains that deliver distinct signals to the cells with which they interact. We will focus here on a well-known ECM glycoprotein, laminin-1, which plays a variety of roles in the development of the CNS (2). [ABSTRACT FROM AUTHOR]

Dissociated cell culture has proved extremely valuable for studying the functional relationship between specific cell surface and extracellular matrix (ECM) molecules and a variety of cellular behaviors such as proliferation, differentiation, migration, and neuronal process outgrowth. When cells are cultured on substrata made from purified cell surface or ECM components, the direct actions of these molecules on cells can be examined easily. However, for some in vitro studies, it is desirable to examine the functions of such molecules in an environment that more closely approximates that which exists in vivo. Such studies might include, for example, investigations in which the endogenous distribution and heterogeneity of such molecules is important, screens for uncharacterized factors that effect a particular cell behavior, or studies examining the combinatorial effects of the many cell surface and ECM components that occur in intact tissue. [ABSTRACT FROM AUTHOR]

The insulin-like growth factor-binding proteins (IGFBPs) are carrier proteins that are present in all extracellular fluids (1). Similarly, insulin-like growth factor-I (IGF-I) and -II are synthesized by connective tissue cells that are present within the stroma of all tissues, and therefore their presence is ubiquitous in all organs (2). IGF receptors are present in all tissues, which accounts for balanced growth of organs, and deletion of the IGF-I gene results in a balanced decrease in organ growth, whereas overexpression of IGF-I results in a symmetric increase in growth (3). [ABSTRACT FROM AUTHOR]

Recent advances in developmental biology have highlighted the importance of the synergistic relationships between structural components of the extracellular matrix (ECM) and growth factors that play fundamental roles in tissue morphogenesis and repair. Increasing evidence suggests that the binding of growth factors to the ECM is a major mechanism for regulation of growth factor activity (for review seeref. (1). Association of growth factors with the ECM allows storage of large quantities of growth factors in a readily mobilized form, which could allow extracellular signaling to proceed rapidly in the absence of new protein synthesis. This may be particularly important in situations such as tissue repair following injury. Storage of growth factors in the ECM may also facilitate communication between cells that are widely separated in time, by allowing transmission of signals from one cell to a different cell that comes in contact with the same matrix later. Another intriguing possibility is that matrix-bound growth factors could act as a "memory," which provides information about the history of cellular activity of the tissue. Finally, it is possible that matrix-bound growth factors may actually signal differently than their soluble counterparts, for example by escaping internalization after binding to receptors, thus producing an extended signal. Thus matrix-bound growth factors may provide an important new dimension to cell-cell communication by greatly increasing the diversity of action of growth factors. [ABSTRACT FROM AUTHOR]

We present a method that uses microcontact printing of alkanethiols on gold to generate patterned substrates presenting "islands" of extracellular matrix (ECM) surrounded by nonadhesive regions such that single cells attach and spread only on the adhesive regions. We have used this micropatterning technology to demonstrate that mammalian cells can be switched between growth and apoptosis programs in the presence of saturating concentrations of growth factors by either promoting or preventing cell spreading (1). From the perspective of fundamental cell biology, these results suggested that the local differentials in growth and viability that are critical for the formation of complex tissue patterns may be generated by local changes in cell-ECM interactions. In the context of cell culture technologies, such as bioreactors and cellular engineering applications, the regulation of cell function by cell shape indicates that the adhesive microenvironment around cells can be carefully optimized by patterning a substrate in addition to using soluble factors (2). Micropatterning technology will play a central role both in our understanding how ECM and cell shape regulate cell physiology and in facilitating the development of cellular biosensor and tissue engineering applications (3-5). [ABSTRACT FROM AUTHOR]

The introduction of recombinant DNA has become a common tool for studying functional and structural properties of a wide variety of proteins. Functional analysis of protein can be studied by suppression of gene expression, thus introducing a plasmid which expresses an antisense RNA in mammalian cells. Several extracellular matrix proteins require an assembly of subunits to form functional heteromultimers with distinct features. Thus, expression of a specific mutant protein can interfere with the assembly of multimeric protein, resulting in a dominant-negative phenotype. In these studies, an efficient delivery of DNA into appropriate target cells represents a critical step. Although many procedures of transfection of the plasmid DNA into mammalian cells are available, viral infection represents a far superior mode of delivery because retroviral vectors were shown to transduce genes of interest into tissue-culture cells with success rates approaching 100%. The retrovirus inserts the viral genome into the chromosome of the infected cell permanently, usually without any measurable effect on the viability of the infected cells. The result is an efficient gene-transfer system in which most recipient cells will incorporate and express the transduced gene. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) plays a critical role in normal development, tissue repair, and is altered in several disease states. During these processes, expression of ECM genes is regulated temporally and spatially. Identification of regulatory elements that direct tissue- and stage-specific expression is important for understanding of the role of ECM genes in cellular differentiation and function. A significant number of ECM genes have been cloned and their regulatory elements have been identified. Reporter gene systems are widely used to study gene regulation and function (1-3). In this chapter, we describe methods for the identification of promoters and enhancers of cartilage collagen genes for the α1(II) and α2(XI) chains using transgenic mice and transient transfection in cell cultures (4-9). [ABSTRACT FROM AUTHOR]

Genetically engineered mutant strains of mice have become invaluable too to decipher complex biological processes, such as ECM assembly, and to dissect phenotypic overlaps, like the genodermatoses. Homologous gene targe ing in embryonic stem (ES) cells is increasingly favored over the transgen approach because it allows the study of mutations in homozygosity and without altering the remainder of the genome (1). Homologous gene targeting has been successfully employed to abrogate the expression or alter the structure of ECM-coding genes (2,3). In both cases, the experimental protocol consists of the following three steps: a. construction of the targeting vector; b. homologous recombination in ES cells; and c. generation of mutant mouse strains. [ABSTRACT FROM AUTHOR]

The analysis of phenotypes caused by null and mutant alleles is a very powerful means to understand gene function in vivo. Historically, this experimental approach has been widely and successfully used in invertebrate models. Now, thanks to the gene-targeting technology in ES cells, the genome of a mammalian organism such as the mouse can be artificially modified by precise alterations. The system exploits the ability of ES cells to be cultured and manipulated in vitro without losing their totipotency (1,2). Mutations in specific genes can be achieved by in vitro selection of ES cell clones in which the locus of interest has been targeted by homologous recombination (3,4). The peculiar property of being totipotent allows ES cells, once injected in the cavity of a blastocyst, to contribute to the formation of all cell types of a chimeric embryo. Whenever a chimeric mouse possesses ES-derived germ cells, the mutation can be propagated to its offspring. Heterozygous mice are then mated to generate the homozygous offspring needed for phenotypic analysis. [ABSTRACT FROM AUTHOR]

Genetic disorders of cartilage (chondrodysplasias) are a clinically and genetically heterogeneous group of diseases ranging in severity from relatively mild to severe and lethal forms (1-2). There are over 100 unique well-characterized chondrodysplasia phenotypes and remarkable progress has been made in the last few years identifying the underlying genetic basis of many of these disorders (3). In most cases, a molecular genetics approach was employed involving a combination of genetic linkage mapping, positional (candidate) cloning and DNA sequence analysis (4-9). By its nature this approach requires extensive mutation screening in any potential candidate gene, first to determine if it is the disease gene and then subsequently to identify a range of disease causing mutations. In chondrodysplasia phenotypes this approach has been hampered by a difficulty in obtaining appropriate pathological tissue, such as cartilage, for the isolation of mRNA. This problem is compounded by the complex genomic structure of many genes that encode cartilage structural ECM molecules. For most cartilage diseases, a combination of these difficulties has necessitated screening for mutations in a large numbers of exons using a variety of techniques such as single-stranded conformational polymorphism (SSCP) (10), conformational sensitive gel electrophoresis (CSGE) (11), heteroduplex (12) and chemical cleavage mismatch (CCM) analysis (13). [ABSTRACT FROM AUTHOR]

The examination of ECM macromolecules by conventional transmission electron microscopy (TEM) or scanning transmission EM (STEM) can provide important information on macromolecular organization and interactions. [ABSTRACT FROM AUTHOR]

Intermolecular binding forces are intrinsic property of cohesive structures and should be used as the main quantitative criteria for assessing and defining their functional contribution to the maintenance of the anatomical integrity of the adult and embryonal multicellular organism, to fertilization, to blood cell adhesiveness in normal and pathological conditions, to tumor cell adhesion, to parasite-host interactions, and to cellular associations in symbiotic organism. Recently, a novel technology of atomic force microscopy (AFM) measurements of binding strength between a single pair of cell adhesion molecules in various physiological solution was developed (1). Binding studies, calorimetric, and spectroscopic analyses do not provide direct information about binding forces and are thus complementary kinetic methods to AFM measurements. [ABSTRACT FROM AUTHOR]

Collagen fibrils in tissue are generally heterotypic with more than one type of collagen molecule incorporated into the fibril structure. Furthermore specific macromolecules are bound onto the fibril surface influencing both the assembly and the interaction of the fibril with the surrounding matrix. The electron cryomicroscopy procedures described here form part of a program of work to determine the structure and assembly of tissue fibrils containing surface-associated components. [ABSTRACT FROM AUTHOR]

Integrins are heterodimeric cell-surface receptors involved in a variety of functions such as binding to the ECM, regulation of the cellular organization, cell migration, proliferation, differentiation, and gene expression (1,2). Integrins in their native environment can diffuse in the plane of the membrane, and this mobility is required for processes like the assembly of integrins to focal contacts or cell migration (3,4). [ABSTRACT FROM AUTHOR]

Extracellular matrices (ECM) contain a mixture of fibrillar and nonfibrillar macromolecular components, which together form a composite structure (1-3). It is the ECM that defines the architecture, the form, and the biomechanical properties of different tissues (4,5). Among the nonfibrillar macromolecules, the highly charged proteoglycans and hyaluronan are major components that occur at high concentration and greatly influence the movement of solutes and water between the tissue and the circulation, and control the access to cells of nutrients, metabolites, growth factors, and chemokines (6-7). This local environmental regulation may have important consequences on cellular functions, especially in tissues with large dense ECMs, such as articular cartilage. [ABSTRACT FROM AUTHOR]

The collagens, proteoglycans, and glycoproteins of the extracellular matrix (ECM) are a diverse collection of macromolecules, heterogeneous in size, structure, and function. However, their primary structures have revealed that their polypeptide components are frequently comprised of a series of sequence repeats or "modules" (1). This mosaic nature of ECM macromolecules permits their properties to be analyzed by a "dissection" strategy (2), where protein fragments generated by proteolysis, chemical synthesis, or recombinant expression are employed in structural and functional investigations. [ABSTRACT FROM AUTHOR]

For recombinant expression of extracellular matrix (ECM) proteins or their individual domains, the use of transformed mammalian cells offers two major advantages. First, eukaryotic expression can be expected under optimum conditions to produce a large proportion of correctly folded molecules. ECM proteins are made from a group of 25 structurally known (1) and about 200 cDNA derived domains many of which regularly reappear in the different proteins. These have often a complex secondary structure, maintained by multiple disulfide bonds. Whereas by denaturing and then carefully renaturing, an approximation to the native structure may be obtained using prokaryotic expression systems, the best that may be expected is that a small percentage of the protein folds into such a conformation. Second, most ECM proteins are at least to some extent glycosylated and often heavily so, and the use of the mammalian system offers the best approximation to the sugar structures present in the native form of the molecule. [ABSTRACT FROM AUTHOR]

At least 19 proteins have now been defined as collagens (1,2), but many of those recently discovered are present in tissues in such small amounts that their isolation for characterization at the protein level has so far been impossible. Some of the fibril-forming collagens are now in medical use, in applications ranging from the use of type I collagen as a biomaterial and a delivery system for several drugs to trials for the potential of type II collagen as an oral tolerance-inducing agent for the treatment of rheumatoid arthritis (3,4). An efficient recombinant expression system for collagens can thus be expected to have numerous scientific and medical applications. The systems commonly used for expressing other proteins in lower organisms are not suitable as such for the production of recombinant collagens, however, as bacteria and yeast have no prolyl 4-hydroxylase activity and insect cells have insufficient levels of it. Prolyl 4-hydroxylase, an α2β2 tetramer in vertebrates, plays a central role in the synthesis of all collagens, as 4-hydroxyproline-deficient collagen polypeptide chains cannot form triple helices that are stable at 37°C (5,6). All attempts to assemble an active prolyl 4-hydroxylase tetramer from its subunits in vitro have been unsuccessful, but active recombinant human prolyl 4-hydroxylase has been produced in insect cells and yeast by coexpression of its α- and β-subunits (7,7). We have recently shown that recombinant human type III collagen with a stable triple helix can be efficiently produced in insect and yeast cells by simultaneous coexpression with the recombinant human prolyl 4-hydroxylase (8,9). This chapter describes detailed procedures for the production of stable recombinant human type III collagen in insect cells and in the yeast Pichia [ABSTRACT FROM AUTHOR]

Recent developments in the application of eukaryotic recombinant protein techniques have provided new tools with which to dissect and map functional activities in basement membrane glycoproteins. This has been particularly valuable in the case of laminins where the relationship between structure and function has been difficult to establish. Several characteristics of the laminins lie at the heart of the problem. First, the molecules are very large, each assembled from three multidomain subunits joined in a long-coiled coil Fig. 1). Second, laminins bear substantial disulfide and carbohydrate modifications that are crucial for proper conformation. Many, perhaps most, laminin activities present in native laminin are lost upon heat- or chaotropic-denaturation. Native activities are often not retained in short peptides or even in recombinant fragments generated in prokaryotic cells. Furthermore, there is evidence to suggest that synthetic laminin peptides can exhibit "cryptic" activities not found in native protein. [ABSTRACT FROM AUTHOR]

The primary functional role of collagen is as a supporting tissue and it is now well established that the aggregated forms of the collagen monomers are stabilized to provide mechanical strength by a series of intermolecular crosslinks. These links are formed by oxidative deamination of the ε-amino group of the single lysine in the amino and carboxy-telopeptides by lysyl oxidase. The aldehyde thus formed reacts with an ε-amino group of a lysine at a specific point in the triple helix because of the quarter-staggered end-overlap alignment of the molecules in the fibers. The chemistry of these crosslinks is dependent on both the nature and age of the collagenous tissue (1,2). Differences in the crosslinks are because of the degree of hydroxylation of both the telopeptide and the specific lysine in the triple helix. Thus, the amounts of intermediate crosslinks present in immature tissue, dehydro-hydroxylysinonorleucine (A-HLNL), and hydroxylysino-keto-norleucine (HLKNL) may vary considerably between tissues, e.g., rat tail tendon and skin contain A-HLNL whereas cartilage and bone contain predominantly HLKNL. [ABSTRACT FROM AUTHOR]

This chapter will describe the preparation and use of a semipermeabilized (SP) cell system that reconstitutes the initial stages in the assembly and modification of proteins entering the secretory pathway (1). The procedure involves treating cells grown in culture with the detergent digitonin and isolating the cells free from their cytosolic component (2). The expression of proteins in an SP cell system allows protein assembly to be studied in an environment that more closely resembles that of the intact cell. As this is an in vitro system, the individual components can be manipulated easily, providing a means by which cellular processes can be studied under a variety of conditions. In addition, membrane-permeable chemical crosslinking reagents can be added in order to facilitate the study of interaction between proteins within the endoplasmic reticulum (ER) lumen (1,3). Furthermore, as the ER remains morphologically intact, the spatial localization of folding and transport processes within the reticular network may also be examined. [ABSTRACT FROM AUTHOR]