Browsing by Subject "Basement membrane"

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 (BMs) are conserved, cell-associated, sheet-like networks of extracellular matrices that provide structural support to tissues. BMs also function as a signaling scaffold, directing cellular processes including self-renewal, adhesion, and proliferation. However, the role of BMs in mediating interactions between tissues is an active area of inquiry. Using C. elegans, which are highly amenable to genetic and visual manipulation, I investigated the role of BMs in mediating inter-tissue interactions within the reproductive system: in tissue separation (gonad and body wall muscle), in tissue linkage (uterine utse and epidermal seam), and in stem cell-niche interactions (germ stem cells and niche). To probe BM function, I used live-cell imaging of endogenous localization of BM components, conditional knockdown, RNAi screening, and genetic mutant alleles. In Chapter 1, I discuss what is known about BMs in the separation and linkages of reproductive tissues, BM function in the germ stem cell niche, and introduce the advantages of using C. elegans as a model. In Chapter 2, I explore the role of BMs in mediating niche enwrapment. In Chapter 3, I show that failed BM separation between the gonad and body wall muscle leaks germ cells into the body cavity to become enwrapped by abnormally protrusive muscle cells. In Chapter 4, I find that discoidin domain receptor-2 directs adhesion of uterine utse BM and epidermal seam BM to connect reproductive tissues. In Chapter 5, I discuss the impact of these findings.

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.

Basement membrane (BM) is the thin, dense, highly cross-linked form of extracellular matrix that underlies all epithelia and endothelia, as well as surrounds muscle, nerve and fat. These sheet-like networks function as physiological barriers to maintain tissue homeostasis. During normal developmental processes and immune surveillance, cells invade through BM to establish tissues and fight infection. Similarly, metastatic cancer cells are thought to co-opt normal programs for BM transmigration as they spread from primary tumors and colonize distant tissues. The difficulty of visualizing cell-BM interactions during invasion in vivo has left the cellular and molecular mechanisms used to breach BM undefined. Specialized F-actin-rich matrix-degrading membrane protrusions, termed invadosomes, have been described in cultured invasive cell lines for more 30 years. Invadosomes are hypothesized to mediate BM penetration during cancer metastasis. Despite promising advances in intravital imaging technologies, however, invadosomes have yet to be observed in cells transmigrating BM in vivo, leaving their physiological relevance unclear. Anchor cell invasion in C. elegans is a simple in vivo model of cell invasion that allows for combined visual and genetic analysis of BM transmigration. In this dissertation I develop high-resolution time-lapse imaging approaches to understand the dynamic interactions that occur at the AC-BM interface during invasion. Through the course of this work we identify an integrin-based mechanism that polarizes the AC towards the BM. We further discover protrusive F-actin-based invadosome structures that mediate BM breach during anchor cell (AC) invasion. We find that in most cases only one or two invadosomes penetrate the BM and then transform into an invasive protrusion that guides the AC through a single BM gap. Using genetics and quantitative single-cell image analysis we characterize several molecular regulators of invadosome formation in vivo. Our findings establish an essential role for invadosomes during BM transmigration in vivo, and support the idea that these structures are a core, conserved element of a normal invasive cellular strategy activated during cancer metastasis.

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.

Cell invasion through basement membrane (BM) barriers is necessary for development and homeostasis, and is misappropriated in diseases like cancer. Many regulators of this complex biological process have been identified by relying on studies completed in vitro and through the analysis of genetic mutants in vivo, however, these methods are unable to identify redundant and subtle regulators of invasion. Capturing the gene expression profile of a cell actively transmigrating BM in vivo remains elusive as invasion through BM is often random. However, a gene expression profile would shed light on genes and pathways that have previously gone unnoticed using traditional screening methods. Here, I use the C. elegans model of anchor cell (AC) invasion through BM as a visually and genetically tractable in vivo model utilizing forward and reverse genetic screening, transcriptome profiling, split-GFP protein tagging strategies, and live cell imaging to investigate and identify new regulators and cellular processes controlling cell invasion through BM. In Chapter 1, I review strategies and mechanisms that invasive cells employ to breach and travel through BM and present AC invasion in C. elegans as an in vivo model of cell invasion. In Chapter 2, I attempt to identify two new regulators of AC invasion identified through a large-scale forward genetic screen. In Chapter 3, by developing methods to isolate individual AC’s, I identify the AC transcriptional profile during invasion, I reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration. Chapter 4 discusses the implications of these findings and future directions.

Basement membranes (BM) are dense, highly crosslinked sheets of extracellular matrix proteins that surround and constrain cells in animal tissues. Specialized cells acquire the ability to invade through BM barriers during development and homeostasis, and aberrant BM invasion underlies many diseases. Invading cells use transient and specialized cellular protrusions to breach the BM, and the membrane dynamics and cytoskeletal rearrangements necessary to build and fuel these structures are both energy intensive and metabolically complex. Thus, it is crucial to understand how invasive cells regulate their catabolic and anabolic metabolism to drive BM invasion, but experimentally dissecting stochastic cell invasion events that occur deep within optically inaccessible tissues in vivo is challenging. Here I use the C. elegans anchor cell (AC) as an experimentally tractable and visually accessible in vivo model for cell invasion through the BM, and use 4D live cell imaging , metabolic biosensors, and RNAi-mediated screening to investigate how invading cells regulate their ATP production and lipid metabolism to drive invasion through the BM. In Chapter 1, I review the mechanisms used by cells to fuel invasion through matrix and identify gaps in our understanding of localized energy production during invasion. In Chapter 2, I discover that localized glucose import, and glycolytic processing support rapid and transient ATP production by mitochondria in the AC to fuel the invasive protrusions for BM invasion. In Chapter 3, I identify that sphingolipid biogenesis and protein prenylation support the formation of the invasive protrusion and the actin-based invasion machinery within in to breach the BM barrier. In Chapter 4, I discuss the implications of these findings on our understanding of the metabolism of cells invading through the BM.