Browsing by Author "Sherwood, David R"

Basement membrane (BM) is a sheet-like extracellular matrix that underlies most tissues and acts as a barrier to invading cells. Many cell types, including immune cells, cells migrating during development and morphogenesis, and metastatic cancer cells utilize F-actin-based structures called invadopodia to breach BM as they leave one tissue to enter another. Despite extensive study and interest in understanding invasion for its clinical importance, the molecular mechanisms regulating invadopodia and BM breach in vivo remain unclear. During uterine-vulval attachment in C. elegans, the specialized uterine anchor cell (AC) uses invadopodia to mediate breach of the underlying BM in order to contact the underlying vulval epithelium. The AC offers several advantages as a model, including experimental, visual, and genetic tractability, the presence of endogenous extracellular environment, and availability of the tissue targeted for invasion. In Chapter 2, I describe development of a novel technique for spatiotemporal-specific knockdown of proteins that will facilitate investigation of proteins, particularly those that are essential or required in other tissues, in the regulation of invadopodia and AC invasion. Using a sensitized genome-wide RNAi screen, classical genetics, and timelapse imaging of invadopodia at the AC-BM interface, Chapter 3 presents two in vivo invadopodia regulators that function by distinct mechanisms. This is the first in vivo evidence that the RhoGTPase CDC-42 regulates invadopodia formation through WSP-1. RabGTPase GDI-1 is a novel regulator of the unique membrane compartment required for invadopodia formation. CDC-42 and GDI-1 both function downstream of an unknown cue secreted by the cells targeted by the AC for invasion, illustrating that extracellular cues can play key roles in mediating cell invasion. The characterization of CDC-42 and GDI-1 as in vivo regulators of invadopodia is an important first step to understanding the mechanisms of this critical cellular process and we expect the AC will be an excellent model for future identification of novel regulators of BM breach.

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.

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 a dense, conserved sheet-like extracellular matrix that provides structural support, compartmentalizes tissues, and regulates cell behaviors. Despite the barrier-like properties of BM, cell invasion through BM takes place normally in many developmental and physiological processes. Deregulation of cell invasion causes a variety of human diseases, most notably, cancer metastasis. A better understanding of cell invasion would help in the design more effective therapeutic strategies for those diseases.

Cell invasion through BM is a dynamic process comprising multiple intertwined steps, including acquirement of polarized cellular morphology, BM breaching, and BM remodeling. Despite much effort on investigating cellular invasive programs used for BM penetration, little is know about how cells detect invasive cues that polarize the invasive responses. Although the establishment of invasive polarity is critical as it initiates subsequent invasive behavior, the invasion process would not be completed without effective BM remodeling. Given that BM remodeling is often an integral part of tissue morphogenesis, the underlying interactions among cells and surrounding tissues make it challenging to understand the individual contributions of cells to changes in BM structure.

To gain insight into these two questions requires simple, experimental in vivo models. Anchor cell (AC) invasion into the vulval epithelium in C. elegans provides a visually accessible and experimentally tractable invasion model that is particularly suitable for cell biological and genetic analysis of the complicated interplay among local BM, an invading cell and the surrounding tissues. Using this model, I have investigated (1) how the AC detects dynamically expressed and localized netrin, a polarizing invasive cue for the AC; (2) what the functional contribution of the AC (as an invading cell) is to BM remodeling during uterine-vulval attachment, a post-embryonic organogenesis process.

First, I found that localized netrin polarizes the cellular invasive response towards the BM by stabilizing and spatially orienting a novel receptor-induced polarity oscillation. This oscillation is characterized by periodic F-actin assembly and disassembly at random sites of the plasma membrane of the AC. I have found F-actin assembly is accompanied by the formation of cellular protrusions. Strikingly, when these protrusions contact localized netrin, they are stabilized. Thus, I propose a mechanistic model where the ligand-independent activity of the receptor generates exploratory behavior. This mechanism orients the invasive polarity of the AC towards its BM target where netrin is normally localized. Second, taking advantage of an unbiased mutagenesis screen, I characterized a mutant with defects in BM sliding, a newly uncovered BM remodeling mechanism. I found that the invading AC utilizes a conserved transcription factor to control the initiation of BM sliding, which involves the regulation of integrin-mediated cell-matrix adhesion. Thus, my study revealed a novel functional role for the AC in BM remodeling during tissue restructuring.

Niches regulate stem cell fate during development and disease. It is thus critical to understand the cellular behaviors that underlie stem cell-niche interactions. Cellular enwrapment of stem cells by their niche is observed in the C. elegans germ stem cell niche, the D. rerio hematopoietic niche, and the D. melanogaster intestinal and lymph niches. This widespread niche behavior is nevertheless difficult to dissect. In the visually and genetically tractable C. elegans germ stem cell niche, the distal tip cell (DTC) functions as a single-cell niche that elaborates to form a plexus of cellular processes enwrapping germ stem cells. Using the DTC niche as a model and tools including live imaging, genetic screens, and tissue-specific knockdown, we elucidate the regulation and function of cellular enwrapping behavior in C. elegans stem cell-niche interactions. In Chapter 2, I identify a set of DTC-autonomous genes including lin-40/MTA1 that directly promote DTC plexus formation. I also identify a set of germline-autonomous genes that indirectly promote enwrapment by the DTC plexus by supporting germ progenitor cell fate, suggesting that germ progenitor cells produce a cue for enwrapment. DTC plexus formation promotes GLP-1/Notch signaling and stem cell fate in enwrapped germ cells, suggesting a positive feedback loop between the niche and stem cells that expands the stem cell pool. In Chapter 3, I show that the adhesion molecules hmr-1/cadherin and sax-7/L1CAM promote DTC plexus formation. Substantiating the idea that germ cells produce a cue for enwrapment, I show that outside the reciprocal interactions within the germ stem cell niche, escaped germ cells induce enwrapment by other somatic tissues. Outside of importance in normal niche establishment, these observations suggest that enwrapment could be coopted in disease states. Chapter 4 discusses the implications of this work.

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 (BMs) are cell-associated extracellular matrices that support tissue integrity, signaling, and barrier properties. Type IV collagen is critical for the mechanical and signaling functions of BM. However, due to the challenge of imaging of BMs in vivo, the lethal phenotypes of null mutations of many BM components, and the expanded gene families of BM receptors and matrix components in vertebrates, how collagen is directed to BMs in vivo is not clear. Here, I exploited the visual tractability and small BM receptor and matrix families of the nematode C. elegans, using live-cell imaging of endogenous localization, conditional knockdown, misexpression, and RNAi screening techniques to investigate how the sole C. elegans type IV collagen molecule is recruited to the BMs of growing gonadal and pharyngeal organs during larval development. In Chapter 1, I review BM structure and functions, focusing on type IV collagen; identify gaps in our understanding of how collagen is directed to BMs, and introduce C. elegans as a model to study type IV collagen incorporation into BMs in vivo. In Chapter 2, I discover that the α subunits of the matrix receptor integrin dictate distinct modes of collagen IV recruitment to the C. elegans pharyngeal and gonadal BMs. In Chapter 3, I explore the roles of the matricellular proteins nidogen, agrin, perlecan, and SPARC in the targeting of collagen to pharyngeal and gonadal BMs. In Chapter 4, I identify potentially novel regulators of type IV collagen incorporation into BMs through a genome-scale RNAi screen. Finally, in Chapter 5, I discuss the implications of these findings to our understanding of type IV collagen incorporation into BMs in vivo, relating them to both established and emerging functions of type IV collagen in BMs.

Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism’s genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. Morphogenesis is a complex orchestration of movements by cells that are specified early in development.

The activation of an upstream GRN is crucial in order to orchestrate downstream morphogenetic events. In the sea urchin, activation of the endomesoderm GRN occurs after the asymmetric 4th cleavage. Embryonic asymmetric cell divisions often are accompanied by differential segregation of fate-determinants into one of two daughter cells. That asymmetric cleavage of the sea urchin micromeres leads to a differential animal-vegetal (A/V) nuclear accumulation of cell fate determinants, β-Catenin and SoxB1. Β-Catenin protein is localized into the nuclei of micromeres and activates the endomesoderm gene regulatory network, while SoxB1 is excluded from micromeres and enters the nucleus of the macromeres, the large progeny of the unequal 4th cleavage. Although nuclear localization of β-Catenin and SoxB1 shows dependence on the asymmetric cleavage, the mechanics behind the asymmetrical division has not been demonstrated. In Chapter 3, we show that micromere formation requires the small RhoGTPase, Cdc42 by directing the apical/basal orientation of the mitotic spindle at the apical cortex. By attenuating or augmenting sea urchin Cdc42 function, micromere divisions became defective and failed to correctly localize asymmetrically distributed determinants. As a consequence, cell fates were altered and multiple A/V axes were produced resulting in a “Siamese-twinning” phenotype that occurred with increasing frequency depending on the quantitative level of perturbation. Our findings show that Cdc42 plays a pivotal role in the asymmetric division of the micromeres, endomesoderm fate-determinant segregation, and A/V axis formation.

This dissertation also characterizes, at high resolution, the repertoire of cellular movements contributing to three different morphogenetic processes of sea urchin development: the elongation of gut, the formation of the primary mouth, and the migration of the small micromeres (the presumptive primordial germ cells) in the sea urchin, Lytechinus variegatus. Descriptive studies of the cellular processes during the different morphogenetic movements allow us to begin investigating their molecular control.

In Chapter 4, we dissected the series of complex events that coordinate gut and mouth morphogenesis. Until now, it was thought that lateral rearrangement of endoderm cells by convergent extension was the main contributor to sea urchin archenteron elongation and that cell divisions were minimal during elongation. We performed cell transplantations to live image and analyze a subset of labeled endoderm cells at high-resolution in the optically clear sea urchin embryo. We found that the endomesoderm cells that initially invaginate into the sea urchin blastocoel remained contiguous throughout extension, so that, if convergent extension were present, it was not a major contributor to elongation. We also found a prevalence of cell divisions throughout archenteron elongation that increased the number of cells within the gut linearly over time; however, we showed that the proliferation did not contribute to growth, and their spindle orientations were randomized during divisions and therefore did not selectively contribute to the final gut length. When cell divisions were inhibited, we saw no difference in the ability of the cells within the gut to migrate in order to elongate. Also in Chapter 4, we describe our observations of the cell biological processes underlying primary mouth formation at the end of gastrulation. Using time-lapse microscopy, photo-convertible Kaede, and an assay of the basement membrane remodeling, we describe a sequential orchestration of events that leads to the fusion of the oral ectoderm and the foregut endoderm. Our work characterizes, at higher resolution than previously recorded, the temporal sequence and repertoire of the cellular movements contributing to the length of the sea urchin larval gut and tissue fusion with the larval primary mouth.

In Chapter 5, the migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a “coherent feed-forward” transcriptional subcircuit composed of Pax6, Six3, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.

The sea urchin gene regulatory network (GRN) describes the cell fate specification of the developing embryo; however, the GRN does not describe specific cell biological events driving the three distinct sequences of cell movements. Our ability to connect the GRN to the morphogenetic events of gastrulation, primary mouth formation, and small micromere migration will provide a framework for characterizing these remarkable sequences of cell movements in the simplest of deuterostome models at an unprecedented scale.

The basement membrane (BM) is a highly conserved form of extracellular matrix that underlies or surrounds and supports most animal tissues. BMs are crossed by cells during various remodeling events in development, immune surveillance, or during cancer metastasis. Because BMs are dense and not easily penetrable, most of these cells must open a gap in order to facilitate their migration. The mechanisms by which cells execute these changes are poorly understood. A developmental event that requires the opening of a BM gap is C. elegans uterine-vulval connection. The anchor cell (AC), a specialized uterine cell, creates a de novo BM gap. Subsequent widening of the BM gap involves the underlying vulval precursor cells (VPCs) and the π cells, uterine neighbors of the AC through non-proteolytic BM sliding. Using forward and reverse genetic screening, transcriptome profiling, and live-cell imaging, I investigated how the cells in these tissues accomplish BM gap formation. In Chapter 2, I identify two potentially novel regulators of BM breaching, isolated through a large-scale forward genetic screen and characterize the invasion defect in these mutants. In Chapter 3, I describe single-cell transcriptome sequencing of the invasive AC. In Chapter 4, I describe the role of the π cells in opening the nascent BM gap. A complete developmental pathway for this process has been elucidated: the AC induces the π fate through Notch signaling, after which the π cells upregulate the Sec14 family protein CTG-1, which in turn restricts the trafficking of DGN-1 (dystroglycan), a laminin receptor, allowing the BM to slide. Chapter 5 outlines the implications of these discoveries.

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 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.