Browsing by Subject "cell invasion"

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.

Cell invasion is a complicated process vital to tissue development, immune surveillance, and disease states such as metastatic cancer. While in vitro work has presented molecular mechanisms regulating cell invasion, visualization of the process in in vivo settings provides a deeper understanding of the cell biological events of invasion. To invade, a cell must cross dense barriers of extracellular matrix and basement membrane (BM); how an invasive cell regulates its plasma membrane to facilitate breach and removal of these barriers is a poorly-understood and underconsidered question. The developmental event of C. elegans anchor cell (AC) invasion provides an in vivo model for the visualization of cell invasion. The AC, a specialized uterine cell, creates a gap in the BM separating the uterine and vulval tissues and then expands that gap through the formation of an invasive protrusion. Using live-cell imaging, RNAi screening, genome editing, and photobleaching techniques I examined the mechanisms governing plasma membrane regulation during AC invasion. In Chapter 2, I discover that the AC rapidly expands an invasive protrusion to clear underlying BM through exocytosis of lysosomes in a netrin-dependent manner. In Chapter 3, I identify a barrier to membrane diffusion formed by the BM receptor dystroglycan as being necessary for expansion of the invasive protrusion and maintenance of polarity. Chapter 4 discusses the implications of these findings and future directions.

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.

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 are dense, sheet-like forms of extracellular matrix that

surround the epithelial tissues of metazoan organisms. While these structures are

critical for epithelial support and tissue organization, basement membranes also pose

formidable barriers to most cells. However, certain specialized cells are able to breach

these barriers and move between tissues. Acquisition of cell invasive behavior by some

tumor cells is thought be an important step in cancer progression. Due to the clear basic

and clinical importance of understanding the mechanisms underlying cell invasion

through basement membranes, cell invasive behaviors has been an area of intense study.

In this work I examine a developmentally regulated model of cell invasive behavior in

the nematode worm, C. elegans. In this system a single proto-epithelial cell remodels

basement membrane to connect two epithelial tissues, the uterus and vulva. Using this

model I identify a novel role for UNC-6/Netrin signaling during this process through basement membranes. I show that Netrin signaling is a third regulatory input for AC invasion that functions partially in parallel to fos-1a and the vulval signal. Further I link netrin signaling to the formation of invasive protrusions that penetrate basement membrane.

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.

Basement membranes (BM) are dense layers of cross-linked extracellular matrix (ECM) proteins that provide structure for tissues, as well as serving as a barrier that prevents cell movement between tissues. Despite formidable barrier properties, specialized cells have acquired the ability to invade BM during development and physiological homeostasis. Furthermore, dysregulation of invasive behavior is the root of many diseases and disorders. Cell invasion is a robust process that requires extensive signaling, cytoskeletal, and proteolytic proteins to coordinate the physical and chemical removal of BM. Therefore, it is crucial to understand how cells construct, support, and fuel machinery required for invasion. Here, I use the C. elegans anchor cell (AC), an experimentally tractable and visually accessible in vivo model for cell invasion through the BM, to investigate ribosome biogenesis, endomembrane expansion, and mitochondrial specialization in cell invasion through BM. In Chapter 1, I review AC invasion as a model of cell invasion. In Chapter 2, I identified new invasion regulators, an enrichment of ribosomal proteins, and key roles for ribosome biogenesis and endomembrane expansion to meet the heightened protein-translation demands of the cell during invasion through BM. In Chapter 3, I discover that AC basal mitochondria have a specialized electron transport chain (ETC) to produce rapid amounts of ATP to fuel cell invasion and that mitochondrial specialization is dependent on mitochondrial protein import machinery enrichment, cristae remodeling, and mitochondria-endoplasmic reticulum contact sites (MERCS). In Chapter 4, I discuss the implications of these finding on our understanding of how cells construct, support, and fuel machinery required for invasion.