SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.     

The Bi-Functional Organization of Human Basement Membranes

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.     

Molecular interactions in the retinal basement membrane system: A proteomic approach

Basement membranes (BMs) are physiologically insoluble extracellular matrix sheets present in all multicellular organisms. They play an important role in providing mechanical strength to tissues and regulating cell behavior. Proteomic analysis of BM proteins is challenged by their high molecular weights and extensive post-translational modifications. Here, we describe the direct analysis of an in vivo BM system using a mass spectrometry (MS) based proteomics approach. Retinal BMs were isolated from embryonic chick eyes. The BM macromolecules were deglycosylated and separated by low percentage gradient SDS PAGE, in-gel digested and analyzed by LC-MS/MS. This identified over 27 extracellular matrix proteins in the retinal BM. A semi-quantitative measure of protein abundance distinguished, nidogens-1 and -2, laminin subunits α1, α5, β2, and γ1, agrin, collagen XVIII, perlecan, FRAS1 and FREM2 as the most abundant BM protein components. Laminin subunits α3, β1, γ2, γ3 and collagen IV subunits α5 and α6 were minor constituents. To examine binding interactions that contribute to the stability of the retinal BM, we applied the LC-MS/MS based approach to detect potential BM complexes from the vitreous. Affinity-captured nidogen- and heparin-binding proteins from the vitreous contained > 10 and > 200 proteins respectively. Comparison of these protein lists with the retinal BM proteome reveals that glycosaminoglycan and nidogen binding interactions play a central role in the internal structure and formation of the retinal BM. In addition, we studied the biomechanical qualities of the retinal BM before and after deglycosylation using atomic force microscopy. These results show that the glycosaminoglycan side chains of the proteoglycans play a dominant role in regulating the thickness and elasticity of the BMs by binding water to the extracellular matrix. To our knowledge, this is the first large-scale investigation of an in vivo BM system using MS-based proteomics.     

Perlecan maintains the integrity of cartilage and some basement membranes

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.     

Laminin α1-Chain Shows a Restricted Distribution in Epithelial Basement Membranes of Fetal and Adult Human Tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) α1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln α1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16–19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln α1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln α1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln α1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

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.     

Basement membranes in development and disease

Basement membranes (BMs) are specializations of the extracellular matrix that act as key mediators of development and disease. Their sheet like protein matrices typically serve to separate epithelial or endothelial cell layers from underlying mesenchymal tissues, providing both a biophysical support to overlying tissue as well as a hub to promote and regulate cell–cell and cell–protein interactions. In the latter context, the BM is increasingly being recognized as a mediator of growth factor interactions during development. In this review, we discuss recent findings regarding the structure of the BM and its roles in mediating the normal development of the embryo, and we examine congenital diseases affecting the BM which impact embryonic development and health in later life. Birth Defects Research (Part C) 90:8–31, 2010. © 2010 Wiley‐Liss, Inc.     

The role of basement membranes in cardiac biology and disease

Basement membranes (BMs) are highly specialised extracellular matrix (ECM) structures that within the heart underlie endothelial cells (ECs) and surround cardiomyocytes and vascular smooth muscle cells. They generate a dynamic and structurally supportive environment throughout cardiac development and maturation by providing physical anchorage to the underlying interstitium, structural support to the tissue, and by influencing cell behaviour and signalling. While this provides a strong link between BM dysfunction and cardiac disease, the role of the BM in cardiac biology remains under-researched and our understanding regarding the mechanistic interplay between BM defects and their morphological and functional consequences remain important knowledge-gaps. In this review, we bring together emerging understanding of BM defects within the heart including in common cardiovascular pathologies such as contractile dysfunction and highlight some key questions that are now ready to be addressed.     

Segmental and restricted localization pattern of Fras1 in the developing meningeal basement membrane in mouse

The Fras1/Frem family of extracellular matrix proteins consists of Fras1 and its structurally related proteins, Frem1 (Fras1-related extracellular matrix protein 1), Frem2 and Frem3. These are co-localized in embryonic epithelial basement membranes (BMs), where they contribute to epithelial–mesenchymal adhesion. Although Fras1 localization pattern in epithelial BMs has been well defined, it has not yet been comprehensively studied in the central nervous system. Here, we demonstrate the immunohistochemical profile of Fras1 in the developing mouse brain and reveal an exclusively meningeal BM protein deposition. Interestingly, Fras1 displays a segmental localization pattern, which is restricted to certain regions of the meningeal BM. Frem2 protein displays a similar localization pattern, while Frem3 is rather uniformly distributed throughout the meningeal BM. Fras1 and Frem2 proteins are detected in regions of the BM that underlie organizing centers, such as the roof plate (RP) of diencephalon, midbrain and hindbrain, and the RP-derived structures of telencephalon (choroid plexus and hem). Organizing centers exert their activity via the production of bioactive molecules, which are potential Fras1 ligands. The restricted pattern of Fras1 and Frem2 proteins indicates a molecular compartmentalization of the meningeal BM that could reflect, yet unspecified, functional and structural differences.     

Human pathogens utilize host extracellular matrix proteins laminin and collagen for adhesion and invasion of the host

Laminin (Ln) and collagen are multifunctional glycoproteins that play an important role in cellular morphogenesis, cell signalling, tissue repair and cell migration. These proteins are ubiquitously present in tissues as a part of the basement membrane (BM), constitute a protective layer around blood capillaries and are included in the extracellular matrix (ECM). As a component of BMs, both Lns and collagen(s), thus function as major mechanical containment molecules that protect tissues from pathogens. Invasive pathogens breach the basal lamina and degrade ECM proteins of interstitial spaces and connective tissues using various ECM-degrading proteases or surface-bound plasminogen and matrix metalloproteinases recruited from the host. Most pathogens associated with the respiratory, gastrointestinal, or urogenital tracts, as well as with the central nervous system or the skin, have the capacity to bind and degrade Lns and collagen(s) in order to adhere to and invade host tissues. In this review, we focus on the adaptability of various pathogens to utilize these ECM proteins as enhancers for adhesion to host tissues or as a targets for degradation in order to breach the cellular barriers. The major pathogens discussed are Streptococcus, Staphylococcus, Pseudomonas, Salmonella, Yersinia, Treponema, Mycobacterium, Clostridium, Listeria, Porphyromonas and Haemophilus; Candida, Aspergillus, Pneumocystis, Cryptococcus and Coccidioides; Acanthamoeba, Trypanosoma and Trichomonas; retrovirus and papilloma virus.     

Clonorchis sinensis Infestation Promotes Three-Dimensional Aggregation and Invasion of Cholangiocarcinoma Cells

Numerous experimental and epidemiological studies have demonstrated a correlation between Clonorchis sinensis (C. sinensis) infestation and cholangiocarcinoma (CCA). However, the role of C. sinensis in the increased invasiveness and proliferation involved in the malignancy of CCA has not been addressed yet. Here, we investigated the possibility that C. sinensis infestation promotes expression of focal and cell-cell adhesion proteins in CCA cells and secretion of matrix metalloproteinases (MMPs). Adhesion proteins help maintain cell aggregates, and MMPs promote the three-dimensional invasion of cells into the neighboring extracellular matrix (ECM). Using a novel microfluidic assay, we quantitatively addressed the role of excretory-secretory products (ESPs) gradients from C. sinensis in promoting the invasion of cells into the neighboring ECM.     

Modified basement membrane composition during bronchopulmonary tumor progression.

During tumor progression, the extracellular matrix (ECM) and particularly the basement membrane (BM) appear to be dynamic structures that are not only degraded but also deposited around tumor clusters. In this study we examined by immunohistochemistry the localization of three chains of Type IV collagen (alpha1, alpha3 and alpha5), Type VII collagen, and laminin 5 at different stages of bronchopulmonary cancers. In normal tissues, alpha1(IV) chain was detected in all BMs (bronchial, vascular, alveolar, and glandular), alpha5(IV) chain was present only in vascular BM, and laminin 5 and Type VII collagen were co-localized in bronchial and glandular BMs, whereas alpha3(IV) immunolabeling was totally absent from normal bronchi. In well-differentiated carcinomas, alpha3(IV) chain staining was found in some neosynthetized BMs interfacing the tumor cell and the stromal compartment, contrasting with the total absence of labeling in normal tissues. alpha1(IV) chain showed strong reactivity in all BM. Laminin 5 and Type VII collagen were also detected in neosynthetized BM. In poorly differentiated invasive cancers, alpha3(IV) chain and Type VII collagen were not found, whereas laminin 5 and alpha1(IV) chain persisted. The most important modifications in BM composition during tumor progression therefore appear to be the appearance of the alpha3 (IV) chain in well-differentiated carcinomas and its subsequent disappearance in poorly differentiated carcinomas, together with the loss of type VII collagen. alpha5(IV) chain distribution was restricted in vascular BM of well- and poorly differentiated carcinomas. These results show that the composition of BM is modified during the progression of bronchopulmonary tumor, emphasizing that the BM represents a dynamic element in tumor progression and has an important role in tumor cell invasiveness.     

To form and function: on the role of basement membrane mechanics in tissue development,homeostasis and disease

The basement membrane (BM) is a special type of extracellular matrix that lines the basal side of epithelial and endothelial tissues. Functionally, the BM is important for providing physical and biochemical cues to the overlying cells, sculpting the tissue into its correct size and shape. In this review, we focus on recent studies that have unveiled the complex mechanical properties of the BM. We discuss how these properties can change during development, homeostasis and disease via different molecular mechanisms, and the subsequent impact on tissue form and function in a variety of organisms. We also explore how better characterization of BM mechanics can contribute to disease diagnosis and treatment, as well as development of better in silico and in vitro models that not only impact the fields of tissue engineering and regenerative medicine, but can also reduce the use of animals in research.     

Colonial development in Pandorina morum. II. Colony morphogenesis and formation of the extracellular matrix

The colonial green alga, Pandorina morum, resembles its unicellular relative Chlamydomonas in both intracellular architecture and the composition of the extracellular matrix. Despite these similarities, cell division in Pandorina leads to the formation of a colony instead of the 8 or 16 single cells produced by cell division in Chlamydomonas. To study colony formation, partially synchronized cultures of P. morum were sampled periodically and stained with ruthenium red for electron microscopy. The cells of the colony were found to be held together during development by medial and basal connections between cells; the basal connections include strands of chloroplast. Studies of cells removed from the parental matrix before division confirmed that the cytoplasmic connections are strong enough to maintain the colonial configuration. After the medial connections break, the cells of the plate of the developing colony swing outward and attain the nearly spherical colonial configuration; the basal connections are still present. After this inversion, the formation of the extracellular matrix begins, with the colonial boundary appearing first. Capsule and sheath then form on the outer and inner faces of the colonial boundary until the extracellular matrix is complete. The process is compared to previous observations of Volvox, and possible evolutionary implications are discussed.     

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.     

unc-44 Ankyrin and stn-2 gamma-syntrophin regulate sax-7 L1CAM function in maintaining neuronal positioning in Caenorhabditis elegans

The L1 family of single-pass transmembrane cell adhesion molecules (L1CAMs) is conserved from Caenorhabditis elegans and Drosophila to vertebrates and is required for axon guidance, neurite outgrowth, and maintenance of neuronal positions. The extracellular region of L1CAMs mediates cell adhesion via interactions with diverse cell-surface and extracellular matrix proteins. In contrast, less is known regarding the function of the intracellular domains in the L1CAM cytoplasmic tail. Previously, we identified a role of the C. elegans L1CAM homolog, SAX-7, in maintaining neuronal and axonal positioning. Here, we demonstrate that this function is dependent on three conserved motifs that reside in the SAX-7 cytoplasmic tail: (1) the FERM-binding motif, (2) the ankyrin-binding domain, and (3) the PDZ-binding motif. Furthermore, we provide molecular and genetic evidence that UNC-44 ankyrin and STN-2 γ-syntrophin bind SAX-7 via the respective ankyrin-binding and PDZ-binding motifs to regulate SAX-7 function in maintaining neuronal positioning.     

An ultrastructural analysis of isolated basement membranes in the acellular renal cortex: A comparative study of human and laboratory animals

Freshly harvested kidneys from New Zealand white rabbits, Sprague-Dawley white rats, rhesus monkeys, and transplant-quality human kidneys were used in this study. Minced renal cortical tissue blocks (<2 mm³) were treated with 1 mM EDTA, 3% Triton X-100, 0.025% DNAse, and 4% sodium deoxycholate in an effort to remove all cellular elements and leave the extracellular matrix (ECM) intact. These preparations showed remarkable structural preservation and all components of the ECM, including basement membranes (BMs), maintained their in vivo histoarchitectural relationships. By light microscopy, at least four major BM types were recognizable, including Bowman's capsular BM (BCBM), tubular BM (TBM), glomerular BM (GBM), and peritubular capillary BM (PTCBM). Scanning electron microscopy demonstrated that, despite the lack of supporting interstitium, GBMs in human, monkey, and rat (and rabbit to a lesser degree) exhibit intrinsic structural rigidity such that their convoluted spheroidal shapes are maintained following cell removal. Transmission electron microscopy showed that major BM types are morphologically heterogeneous and vary markedly within and between species. Randomized measurements showed that isolated BM thicknesses (lamina densa only) compared favorably with those reported in cellular preparations. Mean thicknesses of GBMs were within normal ranges in all species with or without power transformations to reduce right-sided skew of distribution curves. In all species, thickness of BCBM > TBM > GGBM > PTCBM. The striking morphologic heterogeneity of major BM types demonstrated in the acellular renal cortex is not surprising in view of recent biochemical analyses that show that BMs derived from different sources are compositionally disparate. We conclude that BMs should be evaluated and characterized individually and that morphologic definition of isolated BMs is necessary prior to further analysis.