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Methods in Cell Biology

Robert P Mecham
No abstract text is available yet for this article.
2018: Methods in Cell Biology
Greg M Harris, Irene Raitman, Jean E Schwarzbauer
The ability to create cell-derived decellularized matrices in a dish gives researchers the opportunity to possess a bioactive, biocompatible material made up of fibrillar proteins and other factors that recapitulates key features of the native structure and composition of in vivo microenvironments. By using cells in a culture system to provide a natural ECM, decellularization allows for a high degree of customization through the introduction of selected proteins and soluble factors. The culture system, culture medium, cell types, and physical environments can be varied to provide specialized ECMs for wide-ranging applications to study cell-ECM signaling, cell migration, cell differentiation, and tissue engineering purposes...
2018: Methods in Cell Biology
Annemarie K Leonard, Elizabeth A Loughran, Yuliya Klymenko, Yueying Liu, Oleg Kim, Marwa Asem, Kevin McAbee, Matthew J Ravosa, M Sharon Stack
This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography...
2018: Methods in Cell Biology
Chien-Cheng Shih, Dennis M Oakley, Matthew S Joens, Robyn A Roth, James A J Fitzpatrick
Over the last 2 decades, nonlinear imaging methods such as multiharmonic imaging microscopy (MHIM) have become powerful approaches for the label-free visualization of biological structures. Multiharmonic signals are generated when an intense electromagnetic field propagates through a sample that either has a specific molecular orientation or exhibits certain physical properties. It can provide complementary morphological information when integrated with other nonlinear optical imaging techniques such as two-photon excitation (TPE)...
2018: Methods in Cell Biology
Mats Paulsson, Raimund Wagener
Marilins mediate interactions between macromolecular components of the extracellular matrix, e.g., collagens and proteoglycans. They are composed of von Willebrand factor type A and epidermal growth factor-like domains and the subunits oligomerize via coiled-coil domains. Matrilin-1 and -3 are abundant in hyaline cartilage, whereas matrilin-2 and -4 are widespread but less abundant. Mutations in matrilin genes have been linked to chondrodysplasias and osteoarthritis and recently characterization of matrilin-deficient mice revealed novel functions in mechanotransduction, regeneration, or inflammation...
2018: Methods in Cell Biology
Donald A Fowler, Michael B Filla, Charles D Little, Brenda J Rongish, Hans C E Larsson
Understanding the morphogenesis and differentiation of tissues and organs from progenitor fields requires methods to visualize this process. Despite an ever-growing recognition that ECM plays an important role in tissue development, studies of ECM movement, and patterns in live tissue are scarce. Here, we describe a method in which a living limb bud is immunolabeled prior to fixation using fluorescent antibodies that recognize two ECM constituents, fibronectin and fibrillin 2. The results show that undifferentiated mesenchyme in quail embryos can be distinguished from prechondrogenic cellular condensations, in situ, via ECM antibodies-indicating the developmental transition from naïve mesenchyme to committed skeletal tissue...
2018: Methods in Cell Biology
Devadarssen Murdamoothoo, Anja Schwenzer, Jessica Kant, Tristan Rupp, Anna Marzeda, Kim Midwood, Gertraud Orend
The extracellular matrix molecule tenascin-C (TNC) has received a lot of attention since its discovery 30 years ago because of its multiple roles in tissue repair, and in pathologies such as chronic inflammation, fibrosis, and cancer. Mouse models with high or no TNC expression have enabled the validation of key roles for TNC in immunity and angiogenesis. In parallel, many approaches including primary cell or organ cultures have shed light on the cellular and molecular mechanisms by which TNC exerts its multiple actions in vivo...
2018: Methods in Cell Biology
Sean P Giblin, Devadarssen Murdamoothoo, Claire Deligne, Anja Schwenzer, Gertraud Orend, Kim S Midwood
The extracellular matrix molecule tenascin-C (TNC) was discovered over 30 years ago, and its tightly regulated pattern of expression since sparked keen interest in the scientific community. In adult tissues, TNC expression is restricted to specific niches and areas of active remodeling or high mechanical strain. However, while most healthy tissues contain little TNC, its transient expression upon cellular stress or tissue injury helps to mediate repair and restore homeostasis. Persistent expression of TNC is associated with chronic inflammation, fibrosis, and cancer, where methods for its detection are emerging as a reliable means to predict disease onset, prognosis, and response to treatment...
2018: Methods in Cell Biology
John M Sipes, Joanne E Murphy-Ullrich, David D Roberts
Thrombospondins are a family of five secreted proteins that have diverse roles in modulating cellular function. Thrombospondins-1 and 2 were identified as matricellular proteins based on their functional roles combined with their transient appearance or accumulation in extracellular matrix at specific times during development and in response to injury or stress in mature tissues. Thrombospondin-1 is a major component of platelet α-granules, which provides a convenient source for purification of the protein...
2018: Methods in Cell Biology
Gail Workman, Amy D Bradshaw
The matricellular protein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or as BM-40) is a collagen-binding protein with a capacity to induce cell rounding and influence proliferation in cultured cells. In mice that do not express SPARC, fibrillar collagen is reduced in some adult tissues; notably, a reduction in fibrosis is reported in response to fibrotic stimuli in lungs, heart, skin, liver, and in the eye. Recently, mutations in the gene encoding SPARC were found in patients afflicted with osteogenesis imperfecta...
2018: Methods in Cell Biology
Pyong Woo Park
Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans are composed of sulfated glycosaminoglycans (GAGs), heparan sulfate (HS) or both HS and chondroitin sulfate (CS), attached covalently to core proteins. Syndecans regulate many cellular processes, such as adhesion, proliferation, and migration. Syndecans bind and regulate molecules primarily through their HS chains, but do not bind to all HS/heparin-binding molecules. Furthermore, mice ablated for the syndecan-1 or -4 gene do not show major developmental abnormalities, but they do show striking pathological phenotypes when challenged with infectious or inflammatory stimuli and conditions, suggesting that certain functions of syndecans are specific and cannot be compensated for by other syndecans or other HSPGs...
2018: Methods in Cell Biology
Ronald J Midura, Valbona Cali, Mark E Lauer, Anthony Calabro, Vincent C Hascall
Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types...
2018: Methods in Cell Biology
David J Kirby, Marian F Young
Small leucine-rich proteoglycans (SLRPs) are a unique class of proteins that exist in the extracellular matrix, playing key roles in cell proliferation and function. In bone, SLRPs such as biglycan and decorin affect osteogenesis and bone remodeling. Their essential role in this organ system has created the need to isolate these proteins for study. Bone presents unique obstacles to the study of proteins; however, through the use of demineralizing agents, efficient methods of the purification of proteoglycans have been developed...
2018: Methods in Cell Biology
Stephen P Evanko, Christina K Chan, Pamela Y Johnson, Charles W Frevert, Thomas N Wight
Versican is a chondroitin sulfate proteoglycan found in the extracellular matrix that is important for changes in cell phenotype associated with development and disease. Versican has been shown to be involved in cardiovascular disorders, as well as lung disease and fibrosis, inflammatory bowel disease, cancer, and several other diseases that have an inflammatory component. Versican was first identified as a fibroblast proteoglycan and forms large multimolecular complexes with hyaluronan and other components of the provisional matrix during wound healing and inflammation...
2018: Methods in Cell Biology
Katsuhiro Hanada, Takako Sasaki
Functional studies of extracellular proteins are often performed using coimmunoprecipitation without purified proteins. However, in order to exclude unspecific reactions of contaminants and for quantitative analysis of specific functions, it is necessary to use purified proteins. It is usually very difficult, however, to purify sufficient amounts of reasonably pure extracellular matrix proteins from tissue samples, but the recombinant expression and purification of proteins in eukaryotic expression systems including insect cells and mammalian cells has proven an alternative powerful method...
2018: Methods in Cell Biology
Heena Kumra, Dieter P Reinhardt
Fibrillins are one of the major components of supramolecular fibrous structures in the extracellular matrix of elastic and nonelastic tissues, termed microfibrils. Microfibrils provide tensile strength in nonelastic tissues and scaffolds for the assembly of tropoelastin in elastic tissues, and act a regulator of growth factor bioavailability and activity in connective tissues. Mutations in fibrillins lead to a variety of connective tissue disorders including Marfan syndrome, stiff skin syndrome, dominant Weill-Marchesani syndrome, and others...
2018: Methods in Cell Biology
Carmen M Halabi, Robert P Mecham
The functional form of elastin is a highly cross-linked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of the mature protein is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove nonelastin "contaminates" are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification...
2018: Methods in Cell Biology
Monique Aumailley
Laminins are large glycoproteins forming structural and signaling networks with two major physiological roles: one role crucial for the formation and stability of basement membranes and the other role, as crucial as the first, in cell anchorage and signaling. Laminins come in several flavors as 16 different isoforms are known, each with both common and unique functions. Here the most current techniques for purification and identification of laminins in tissues and cultivated cells as well as for testing the cell adhesion-promoting activity of laminins will be described...
2018: Methods in Cell Biology
Sergei P Boudko, Neonila Danylevych, Billy G Hudson, Vadim K Pedchenko
Collagen IV is a major constituent of basement membranes, specialized form of extracellular matrix that provides a mechanical support for tissues, serves as a polyvalent ligand for cell adhesion receptors and as a scaffold for other proteins, and plays a key role in tissue genesis, differentiation, homeostasis, and remodeling. Collagen IV underlies the pathogenesis of several human disorders including Goodpasture's disease, Alport's syndrome, diabetic nephropathy, angiopathy, and porencephaly. While the isolation of the collagen IV molecules from tissues is an ultimate prerequisite for structural and functional studies, it has been always hampered by the protein insolubility due to extensive intermolecular crosslinking and noncovalent associations with other components of basement membranes...
2018: Methods in Cell Biology
Bianca R Tomasini-Johansson, Deane F Mosher
Fibronectin (FN) is a plasma glycoprotein produced by hepatocytes that circulates at near micromolar concentration and assembles into extracellular matrix fibrils at cell surfaces along with locally produced cellular FN. We describe two microplate assays that quantify assembly of human FN by cells in monolayer culture. One assay measures fluorescence due to incorporation of ALEXA488-plasma FN into matrices of fibroblasts and has been used successfully in high-throughput screens. The other measures fluorescence due to binding of fluorochrome-labeled antibody to the EDA domain of cellular FN synthesized and deposited by various cell types...
2018: Methods in Cell Biology
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