Browsing by Subject "Extracellular matrix"

Basement membranes (BMs) are thin, dense sheets of extracellular matrix that surround most animal tissues and provide structural support. While the role of BMs in the structural support of tissues is well established, how these matrices can structurally support tissues while accommodating dynamic tissue function is not well understood. Using C. elegans, a powerful model organism that allows for live imaging, genetic analysis, and rapid screening, I was able to utilize endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging to investigate how BM components contribute to the BM’s ability to withstand mechanical load in various circumstances. In Chapter 1, I discuss the known roles of BM, introduce BM proteins of interest, explore gaps in our understanding of BM’s function in withstanding mechanical force, and expand upon the utility of C. elegans as a model system to investigate these questions. In Chapter 2, I show that BM-to-BM linkages can function to resist the mechanical forces involved in egg-laying. In Chapter 3, I explore how BM stretches to accommodate dynamic tissue movement. In Chapter 4, I discuss future directions and the implications of these findings and in Chapter 5 I summarize my conclusions.

Basement membranes are a dense, sheet-like form of extracellular matrix that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stresses. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instruct cell behavior and morphogenetic processes. In this thesis, I discuss how changes to basement membrane affect anchor cell (AC) invasion during C. elegans uterine vulval attachment. During AC invasion, the uterine AC breaches two juxtaposed basement membranes to contact the underlying vulval epithelium. Using live-cell imaging, genetics, molecular biology and electron microscopy I identify three modifications to the BM that affect AC invasion. In Chapter 2, I describe a system for linking juxtaposed basement membranes to stably align or connect adjacent tissues. This adhesion system promotes rapid AC invasion and also regulates a more long-term connection between the uterine tissue and the hypodermal seam cell in the adult worm. Chapter 3 elucidates how the BM component SPARC promotes cell invasion. As SPARC overexpression is correlated with cancer metastasis, this aims to understand how SPARC overexpression promote invasion in a pathological situation. In Chapter 4, I discuss preliminary data showing that the AC actively secretes laminin into the basement membrane targeted for invasion. I outline how future studies could elucidate the mechanism by which AC-derived laminin might promote cell invasion. Finally, Chapter 5 discusses conclusions and future directions for these studies.

Articular cartilage is a highly specialized avascular tissue and consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. The chondrocyte morphology and extracellular matrix properties vary with the depth of cartilage. Some past studies have defined the zonal distribution of a broad range of cartilage proteins in different layers. Based on the variations within each layer, the extracellular matrix can be further distinguished to pericellular, territorial and interterritorial regions. However, most of these studies used guanidine-HCl extraction that leaves an unextracted residual with a substantial amount of collagen. The high abundance of anionic polysaccharide molecules from cartilage adversely affects the chromatographic separation. Scatter oriented chondrocytes only account for the small proportion of the whole tissue protein extraction. However, the density of the cell varies with depth of cartilage as well. Moreover, the physiological status may also altered the extracellular matrix properties. Therefore, a comprehensive strategy to solve all these difficulties are necessary to elucidate the molecular structure of cartilage.

In this study, we used quantitative and qualitative proteomic analysis to investigate various cartilage tissue processing protocols. We established a method for removing chondrocytes from cartilage sections that minimized matrix protein loss. Quantitative and qualitative proteomic analyses were used to evaluate different cartilage extraction methodologies. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. The different extraction methods yielded different protein profiles. For instance, an overwhelming number of collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction.

Subsequently we applied these methods to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic). We also utilized lase capture microscopy (LCM) to harvest cartilage sample from individual subregions (territorial and interterritorial regions). The results suggested that there is more unique proteins existed in the superficial layer. By removing the chondrocytes, we were able to identify more extracellular matrix proteins. The phenotyping of cartilage subregions provided the chance to precisely localize the protein distribution, such as clusterin protein. We observed that the guanidine-HCl extractability (guanidine-HCl/ guanidine-HCl + in situ digestion extracts) of cartilage proteins. Proteoglycans showed high extractability while collagen and non-collagenous proteins had lower extractability. We also observed that the extractability might differ with depth of cartilage and also disease states might alter the characters as well.

Laser capture microscopy provides us the access to the cartilage subregions in which only few studies have investigated because of the difficulties to separate them. We established the proteomic analysis compatible-protocol to prepare the cartilage section for LCM application. The results showed that most of the proteoglycans and other proteins were enriched in the interterritorial regions. Type III and VI collagens, and fibrillin-1 were enriched in the territorial regions. We demonstrated that this distribution difference also varied with depth of cartilage. The difference of protein abundance between subregions might be altered because of disease states.

Last we were looking for the post-transliational modification existed in these subregions of cartilage. Deamidation is one of the modification without the enzyme involved. Previous studies have showed that deamidation may accumulated in the tissue with low turnover rate. Our proteomic analysis results suggests that abundance of deamidated peptides also varied in different layers and subregions of cartilage.

We have developed the monoclonal antibody based immunoassay to quantify the deamidated cartilage oligomeric matrix protein within cartilage tissue from different joints (hip and knee) and disease states (healthy, para-lesion, and remote lesion). The results suggests that the highest concentration of deamidated COMP was identified in arthritic hip cartilage.

The results of this study generated several reliable protocols to perform cartilage matrix proteomic analysis and provided data on the cartilage matrix proteome, without confounding by intracellular proteins and an overwhelming abundance of collagens. The discovery results elucidated the molecular architecture of cartilage tissue at different joint sites and disease states. The similarities among these cartilages suggested a constitutive role of some proteins such as collagen, prolargin, biglycan and decorin. Differences in abundance or distribution patterns, for other proteins such as for cartilage oligomaric matrix protein, aggrecan and hyaluronan and proteoglycan link protein, point to intriguing biological difference by joint site and disease state. Decellularization and a combination of extraction methodologies provides a holistic approach in characterizing the cartilage extracellular matrix. Guanidine-HCl extractability is an important marker to characterize the statue of cartilage; however it has not been fully understand. The protein distributions in matrix subregions may also serve as an index to characterize the metabolic status of cartilage in different disease states. A large sample cohort will be necessary to elucidate these characters.

Basement membranes are a specialized type of extracellular matrix found covering most tissues in animals. These structures are made up of many proteins, most notably laminin and type IV collagen, which form separate polymeric networks that are the core of the BM. BMs are involved in many cell and tissue scale processes during development and homeostasis, and misregulation of BM components lies at the heart of many pathologies. Despite their importance, many of the fundamental aspects of BM biology are not well understood. For example, the mechanisms that regulate differences in BM composition, dynamics, and ultrastructure remain largely unknown. One reason for this is the lack of a model to study these processes in vivo. This has also led to BM dependent processes, such as tissue adhesion through BMs, to be largely overlooked. In Chapter 1, I summarize some of my basic knowledge of BMs, highlight important areas that require further study, and review the process of tissue adhesion through BMs. In Chapter 2, I discuss the creation of an in vivo toolkit of endogenously fluorescently labeled BM components, show how these tools can be used to address questions surrounding BM composition and dynamics, and use these tools to identify papilin as a regulator of type IV collagen network architecture in growing tissues. In Chapter three, I explore the process of tissue adhesion through BMs in greater detail, and identify an enrichment of type IV collagen mediated by tissue specific modifications of the BM that is required to maintain stable BM adhesions between tissues. In Chapter 4, I discuss these findings in more detail, their implications, and future directions based off of this work.

Intervertebral disc (IVD) disorders are a major contributor to disability and health costs. Disc disorders and resulting pain may be preceded by changes which first occur in the nucleus pulposus (NP) region of the IVD, where significant alterations in tissue cellularity, composition, and structure begin early in human life and continue with increasing age and degeneration. These changes coincide with the loss of a distinct cell population, notochordally-derived immature NP cells, which may play a key role in the generation and maintenance of this tissue. These cells reside in a gelatinous, highly-hydrated extracellular matrix (ECM) environment and exhibit in situ cell-matrix and cell-cell interactions which are quite distinct from cells in other regions of the disc or in other cartilagenous, including expression of laminin cell-matrix receptors and cell-associated laminin proteins. The ECM environment is known to be a key regulator of cellular behaviors, with ECM ligands and elasticity modulating cell adhesion, organization, differentiation, and phenotype. The primary motivating hypothesis of this thesis is that the unique ECM environment of the NP plays a key role in modulating immature NP cell behaviors, and that laminin ligands, in combination with ECM elasticity, modulate immature NP cell behaviors including adhesion, organization, and phenotype.

To investigate this hypothesis, flow cytometric analyses were performed to examine IVD cell integrin receptor expression over time in culture, including assessment of key laminin-binding integrin subunits. The roles of specific integrin receptors modulating NP cell adhesion to ECM proteins were identified in studies utilizing function-blocking antibodies. NP cell adhesion, spreading, and relative cell adhesion strength was assessed on various ECM constituents, including specific isoforms of laminin. Additionally, studies were performed to examine the roles of ECM ligand and substrate stiffness in modulating NP cellular organization, utilizing polyacrylamide gel substrates with tunable mechanical properties and functionalized with different ECM ligands. Finally, the role of ECM environment was examined on one key measure of NP cell function, proteoglycan production, over time in culture.

NP cells isolated from immature NP tissues were found to express high levels of the laminin-binding integrin subunit alpha 6 ex situ and maintain this expression over time in culture. Function blocking studies revealed this receptor to be a key regulator of NP cell adhesion to laminin, in contrast to cells from the adjacent AF region, which did not express this receptor nor adhere to laminin. Cell adhesion studies demonstrated that NP cells preferentially interact with two laminin isoforms, LM-511 and LM-332, in comparison to other ECM proteins, with enhanced cell attachment, spreading, and adhesion strength on surfaces coated with these ligands. These findings correspond with laminin isoform and receptor expression patterns identified in immature NP tissues. Additionally, NP cell-cell interactions were found to be modulated by both ECM ligand and substrate stiffness, with soft, laminin-functionalized substrates promoting self-assembly of NP cells into cell clusters with morphologies similar to those identified in immature NP tissues. Finally, culture of immature NP cells on soft, laminin-rich substrates was found to promote a key measure of NP cell function, proteoglycan synthesis.

The studies presented here demonstrate that immature NP cells interact uniquely with laminin extracellular matrix proteins, and that laminin ligands and matrix elasticity are two key regulators of NP cell organization and phenotype in the IVD. These findings suggest that alterations in one or both of these factors during IVD aging or degeneration may contribute to the differentiation or loss of this unique cell population. Additionally, these results indicate that soft, laminin-functionalized biomaterials may be appropriate for in vitro culture and expansion of immature NP cells, as well as for use in NP tissue engineering strategies.