David Sherwood, Ph.D. - US grants

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the genetic networks that direct cell invasion through basement membranes, the dense, sheet-like extracellular matrix that surrounds most tissues. The mechanisms that cells employ to cross basement membranes in vivo remain poorly understood, as these invasions most often occur in complex environments that are difficult to study. We are thus dissecting the process of anchor-cell (AC) invasion into the vulval epithelium in the visually and genetically accessible model organism Caenorhabiditis elegans. AC invasion involves: (1) the attachment of the AC to the basement membrane, (2) its polarization towards the basement membrane, (3) the generation and reception of a chemotactic signal(s) that stimulates invasion, (4) the precise removal of the basement membrane and (5) transit through the basement membrane. We have discovered a novel role for the netrin pathway in directing the polarization of the AC's invasive cellular processes towards the basement membrane. We have also identified a specific isoform of the C. elegans fos transcription factor, fos-1b, which inhibits AC invasion, perhaps by blocking fos-1a activity, an isoform that promotes basement membrane removal during AC invasion. Finally, we have conducted a pilot screen using a database generated from previous whole genome RNAi screens and identified five additional genes that promote AC invasion, four of which have not previously been implicated in regulating cell invasion. Integrating cellular, genetic, and molecular approaches, our proposed work will: 1) elucidate a new role for netrin signaling in polarizing an invasive cell, 2) determine the mechanisms by which fos-1b inhibits AC invasion, and 3) characterize the function of new genes identified in our RNAi database screen that specifically promote removal of the basement membrane during AC invasion. Cell invasions through basement membranes play crucial roles during normal development and are essential for leukocyte trafficking to sites of infection and injury. Uncontrolled cell- invasive activity is also associated with a number of deadly diseases, including cancer and rheumatoid arthritis. The proposed work will advance our understanding of the fundamental mechanisms controlling cell- invasive behavior and thus has a strong potential to lead to new treatment strategies for a number of human diseases associated with unregulated cell-invasive activity.

DESCRIPTION (provided by applicant): Basement membrane is a dense, highly cross-linked form of extracellular matrix that surrounds most tissues. During development and immune surveillance, specialized cells acquire the ability to breach basement membrane to disperse and traffic to sites of infection and injury. The cell invasion program is also co-opted or misregulate during many diseases, including asthma, arthritis, the pregnancy disorder pre-eclampsia, and cancer. Understanding how cells invade through basement membrane is thus of great importance to human health. Cell invasion involves dynamic interactions between the invading cell, the tissue being invaded, and the basement membrane separating them. Owing to an inability to recapitulate these complex interactions in vitro, and the challenge of experimentally examining invasion in vivo, the key mechanisms underlying cell invasive behavior remain poorly understood. Anchor cell invasion in C. elegans is an experimentally accessible in vivo model of cell invasion that uniquely combines single cell visual analysis with powerful genetic and genomic approaches. Using these strengths, we have identified two conserved transcription factors that regulate distinct steps in acquiring an invasive cell fate. NHR-67, an ortholog of the vertebrate Tailless protein, maintains the anchor cell in a post-mitotic state. Exit from the cell cycle appears necessary to then permit the C. elegans Fos family transcription factor ortholog FOS-1A to initiate the invasion program. Preliminary data indicate that FOS-1A regulates the expression of three matrix metalloproteinases (MMPs) in the anchor cell, implicating a specific pathway that controls basement membrane removal. Finally, we have found that elevated levels of the extracellular matrix protein SPARC, which is overexpressed in most advanced cancer malignancies, decreases type IV collagen levels in basement membrane, and dramatically enhances anchor cell invasion. The goal of this proposal is to use live-cell imaging with genetic and molecular analysis to determine: (1) How NHR-67 maintains the anchor cell in a post-mitotic state and allows the cell invasion program to initiate, (2) the role of FOS-1A in regulatin MMP expression and the function of MMPs in breaching the BM, (3) the role of SPARC in enhancing cell invasion. These studies are relevant to NIH's mission as they will lead to new insights into the importance of cell cycle exit for invasion, the specific role of MMPs in breachin basement membrane and the role of SPARC in facilitating the invasive process, thus allowing the development of better therapeutic strategies to limit invasive behavior in human diseases such as cancer.

DESCRIPTION (provided by applicant): Basement membrane is a thin, dense, sheet-like extracellular matrix that encircles most tissues and provides structural support for epithelial and endothelial cells. Understanding how specialized invasive cells cross basement membrane is of profound importance to human health: Basement membrane transmigration underlies the dispersal of cells in many developmental processes, is required in leukocytes for immune surveillance and inappropriate manifestation of cell-invasive behavior underpins the development of metastatic cancers. F-actin-rich, cell membrane associated structures, termed invadosomes, were identified over three decades ago in normally invasive cell types and metastatic cancer cell lines. Invadosomes are thought to mediate the ability of cells to invade through basement membrane barriers. Owing to the difficulty of examining cell-invasive behavior in vivo, invadosomes have only been studied in vitro, where cell culture conditions do not recapitulate native environmental signals or matrix conditions. As a result, the relevance, regulation and potential functions of invadosomes in vivo remain one of the most critical gaps in our understanding of cell- invasive behavior. Anchor cell invasion in C. elegans is a simple, highly stereotyped in vivo model of cell invasion through basement membrane that uniquely combines single-cell visual and genetic analysis. Through the development of live-cell imaging approaches, our group has recently identified dynamic invadosome structures within the anchor cell that breach the basement membrane. We find that when one invadosome penetrates the BM, new invadosome formation ceases, and a single invasive protrusion matures from the infiltrated invadosome. Using optical highlighting of basement membrane components, we have found that this protrusion pushes the basement membrane aside to clear a path for invasion. The goal of this application is to uncover the mechanistic details of how these invadosomes are used to breach basement membrane. Our proposed research will combine live-cell imaging with genetic and molecular analysis to determine: (1) how key pathways that regulate anchor cell invasion coordinately regulate the formation, dynamics and function of invadosomes prior to and during BM penetration, (2) the role of netrin signaling in selecting a single invadosome for basement membrane invasion, (3) the function of the actin regulator Ena/VASP in promoting basement membrane gap expansion at the anchor cell-basement membrane contact points after invadosome penetration. Together, this work will identify new mechanisms underlying cell invasion and elucidate how they function together to breach basement membrane. This project is relevant to NIH's mission because it will lead to specific therapeutic strategies to control invasive behavior in diseases such as metastatic cancer.

? DESCRIPTION (provided by applicant): Basement membrane is a dense, highly cross-linked form of extracellular matrix that surrounds most tissues. During development and immune surveillance, specialized cells acquire the ability to breach basement membrane to disperse and traffic to sites of infection and injury. The cell invasion program is misregulated during many diseases, including asthma, arthritis, multiple sclerosis, and pre-eclampsia. The inappropriate acquisition of invasive behavior also underlies the spread of cancer, which accounts for 90% of all cancer- related deaths. Understanding how cells invade through basement membrane is thus of great importance to human health. Cell invasion involves dynamic interactions between the invading cell, the tissue being invaded, and the basement membrane separating them. Owing to an inability to recapitulate these complex interactions in vitro, and the challenge of experimentally examining invasion in vivo in vertebrates, the mechanisms underlying cell invasive behavior remain poorly understood. Anchor cell invasion in C. elegans is an experimentally accessible in vivo model of cell invasion that uniquely combines subcellular visual analysis of cell-basement membrane interactions with powerful forward genetic and functional genomic approaches. Using these strengths, our work will characterize a newly identified cellular structure-the invasive protrusion, a specialized membrane domain that both degrades and physically displaces basement membrane during invasion. We will also determine how secretion of the basement membrane structural protein laminin by the invading anchor cell facilitates invasion. Most metastatic tumors overexpress laminin, and we expect this work to have wide relevance to understanding cancer progression. Our studies have also unexpectedly revealed that the anchor cell can invade basement in the absence of matrix metalloproteinases (MMPs) by physically displacing the basement membrane. This finding might explain why inhibition of MMPs in clinical trials of metastatic cancer patients failed. Our work will determine how the anchor cell alters its invasion mode and investigate an increased requirement for mitochondrial generated ATP to compensate for the loss of MMPs. These findings will begin a new research area in energy requirements during cell invasion and inform better approaches to target invasion with MMP inhibitors. Finally, our work will characterize a nascent transcriptional regulatory network that specifies invasion, thus addressing the crucial question of how cells are programmed to be invasive. These integrative studies spanning specialized cellular invasive machinery, basement membrane remodeling and transcriptional regulation are relevant to NIH's mission as they will lead to a deep understanding of the fundamental biological process of cell invasive behavior, thus allowing the development of better therapeutic strategies to limit invasion in human diseases such as cancer.

Visualizing and elucidating the role of force on type IV collagen in development PROJECT SUMMARY Basement membranes are highly conserved, dense, sheet-like extracellular matrices that surround most tissues and organs. Basement membranes provide mechanical strength to developing tissues, and loss or mutations in basement membrane components results in embryonic lethality, developmental defects, and numerous human diseases. Type IV collagen has been proposed to be the key structural component in basement membranes that provides mechanical stability, and mutations in collagen result in devastating developmental disorders that affect multiple dynamically growing and mechanically active tissues, including the vasculature, muscles, and brain. Owing to the difficulty of visualizing and experimentally examining type IV collagen dynamics and basement membrane components in complex vertebrate tissues in vivo, however, it is unknown how type IV collagen is assembled in basement membranes and whether it directly bears load. We have made C. elegans strains expressing functional GFP, Venus, and mCherry tagged versions of collagen and most other basement membrane proteins and receptors. We have also developed a photoconvertible Dendra-tagged collagen strain to optical highlight and track collagen deposited in basement membrane. C. elegans encodes all major basement membrane components with only a single gene representing each family, making it a powerful experimental model to dissect type IV collagen function and basement membrane regulation in vivo. The C. elegans pharynx is encased in a BM and is a rapidly growing contractile organ that initiates pumping in the embryo. The posterior terminal pharyngeal bulb is the site of food grinding, a region of high mechanical activity. The C. elegans pharynx is first covered with basement membrane during early embryogenesis, prior to pharyngeal pumping. We have found that type IV collagen is initially localized uniformly around the developing pharynx in the embryo, but after the pharynx initiates pumping, becomes enriched specifically around the terminal bulb. Loss of type IV collagen leads to pharyngeal pumping and morphological defects, indicating a critical role for collagen in pharyngeal development and function. The goal of this proposal is to combine live-cell imaging of type IV collagen with genetic analysis, RNAi knockdown, and force manipulations and development of a new collagen Fluorescence Energy Transfer (FRET)-based force sensor to: (1) Elucidate the biochemical and biophysical mechanisms of collagen addition to the basement membrane of the growing pharynx and the role of mechanical force in collagen recruitment; and (2) Visualize the load on type IV collagen in situ and determine if collagen is preferentially recruited to BM in areas of high mechanical activity. These studies are relevant to NIH's mission as they will lead to new mechanistic insights into the function, regulation, and assembly of type IV collagen in BMs, thus allowing a better understanding of the basis of human developmental disorders that result from defects in type IV collagen.

The purpose of the Developmental and Stem Cell Biology Training Program (DSCB) is to prepare Ph.D. candidates for participation as active scientists in disciplines having an emphasis on developmental and stem cell biology. To accomplish this goal, the training program provides education in core principles of development, genetics, cell biology, and molecular biology. Of special importance is the preparation of critical and creative minds. In addition to didactic training, the Program provides and encourages participation in seminars, journal clubs, and colloquia that foster discussion, perspective and critical review of the literature. Extensive laboratory training provides each student with a special expertise in his or her chosen specialty, and University-wide resources help students develop their full potential to pursue a diversity of careers.

PROJECT SUMMARY Basement membranes (BMs) are dense, sheet-like extracellular matrices that surround most animal tissues and provide mechanical strength and signaling cues that control growth, differentiation, and survival. Genetic and regulatory disruptions in BM components underlie numerous diseases, including cancer, fibrosis, and diabetes as well as eye, kidney, vasculature, and skin disorders. Despite the importance of BM to human health, there are currently no animal models that allow comprehensive real time visualization of BM components in vivo, which limits the understanding of fundamental properties of BMs and hinders development of therapies to treat BM disorders. The overall objective of this proposal is to create a complete toolkit of endogenously fluorescently tagged BM components in C. elegans with methodologies to advance our understanding of BM regulation and renewal in normal and disease states. These tagged proteins can be visualized in vivo because C. elegans is optically clear. C. elegans also has single genes encoding most major BM protein families and receptors and conditional knockdown approaches to disrupt their activity. Preliminary work has pioneered Cas9-mediated homologous recombination to insert the mNeonGreen fluorophore in-frame with 27 BM-associated genes, including all core matrix components, most matricellular proteins and all BM-associated receptors. These strains have been verified for full length protein expression, BM localization and viability. To complete the objective of developing a new BM toolkit animal model the following two specific aims will establish methods: (1) to quantify dynamic alterations of BM components and receptors in distinct tissues, and (2) to follow BM component turnover for the first time in vivo. Under Aim 1, experimental approaches will be developed to reveal BM component variation in different organs during development and aging, how BM composition variation is mediated by BM receptors, and how BMs adapt after loss of a key component (modeling human disease). In Aim 2, matrix components tagged with the photoconvertible fluorophore mEos2 will be made and fluorescent recovery after photobleaching (FRAP) and optical highlighting techniques will be developed to determine how BMs are renewed during normal maintenance and rapid growth. The proposed study will powerfully advance our understanding of BMs by providing the first model to dynamically track BM levels to understand how BMs are assembled, adapt in disease states, and change over time. Further, preliminary FRAP and photoconversion studies of BM components are already revealing that most matrix components are remarkably dynamic and some even flow along the BM. This unexpected dynamic nature implies that BM structural and signaling properties can be altered on the order of minutes?a profound change in our view of BM regulation. The proposed research is thus significant, as the tools and methods created by completion of this work will not only advance our understanding of BMs during normal and disease states, but also open a new field of study into the dynamic nature of BMs.