Richard Fehon - US grants

Cell-cell interactions play an important role in defining the fates of individual cells as they begin to differentiate. While much has been learned about the cell surface proteins that are necessary for these interactions, relatively little is known about cytoplasmic components of this process. The overall goal of this research is to identify and functionally characterize Drosophila cytoplasmic proteins that are important for developmentally crucial signals. Specifically, these experiments will examine the functions of the Drosophila homolog of the vertebrate cytoskeletal protein 4.1 (D4.1). The goals of this proposal are therefore to: 1) Examine the cellular and developmental effects of D4.1 mutations, 2) Characterize the major D4.1 alternative splice forms and determine the genomic organization of the gene that encodes them, and 3) Learn the precise expression patterns of the major D4.1 protein isoforms at the cellular and subcellular levels, and determine the cellular and developmental effects of ectopic D4.1 expression. These experiments are expected to provide insights into the function of both the Drosophila and the human 4.1 genes. Therefore these studies should contribute significantly to work on the role of cellular interactions in regulating cell growth and determining cell fate during development.

membrane proteins; cytoskeletal proteins; protein structure function; neurofibromatosis; Drosophilidae; endocytosis; intermolecular interaction; tumor suppressor genes; gene mutation; cell growth regulation; chimeric proteins; animal genetic material tag; transfection; larva; site directed mutagenesis; tissue /cell culture; human genetic material tag;

Neurofibromatosis type 2 (NF2), a dominantly inherited disease, has been shown to be caused by mutations in Merlin (Schwannomin), a member of the protein 4.1 superfamily. Symptoms of NF2, which usually appear by early adult life, are caused by the formation of bilateral vestibular Schwannomas and other benign tumors. The cellular functions of Merlin and its role in tumor suppression are still largely unknown. Identifying specific proteins and signal transduction pathways with Merlin interacts is especially important because these partners may act as genetic modifiers of NF2 disease phenotypes and provide potential targets for therapeutic agents. The common fruit fly, Drosophila, has proven to be a useful model system for identifying genetic modifiers of gene function. The overall goal of this proposal is therefore to examine the cellular functions of Merlin and Drosophila, identify the proteins with which it interacts, and examine the relationship between Merlin and the closely related Ezrin/Radixin/Moesin proteins. In the next funding period, we plan to continue our studies of Merlin function in developing organisms in individual cells. Specifically, the proposed experiments will: 1) Examine the mechanisms by which Merlin function is regulate using a combination of in vitro mutagenesis and cellular nad biochemical analysis of protein function. 2) Identify second site modifiers of Merlin function by screening for mutations that enhance or suppress the phenotypes of dominant-negative Merlin transgenes. 3) Elucidate the functions of ERM proteins and their functional relationship with Merlin by isolating and characterizing mutations in the Drosophila Moesin gene. These experiments are expected to provide insights into the functions of Merlin and the ERM proteins. Thus they will contribute to our understanding of human NF2, tumor suppression in general, and carcinogenesis. In addition, the proposed experiments should lead to a better understanding of cellular processes that occur in the apical junctional domain. Finally, these studies should contribute to work on the mechanisms by which cellular interactions function to control cell growth and determine cell fate during development.

A central problem in the development and function of epithelial cells is the process by which specialized membrane domains are formed and maintained. These domains, including the apical and basolateral membranes, the junctional complex, and other subdomains, are important in organizing and compartmentalizing membrane-related functions. Intercellular junctions, including adherens, tight, and septate junctions, have well-defined structural functions in intercellular adhesion and preventing paracellular flow. In addition, there is an increasing body of evidence that these junctions, and other specialized membrane domains, play an essential role in regulating signaling pathways. The overall goal of this proposal is to better understand how specialized membrane domains, including intercellular junctions and signaling complexes, are established and regulated in developing epithelial cells. More specifically, we plan to elucidate the functions of the epithelial septate junction, a key part of the junctional complex that forms between Drosophila epithelial cells. Septate junctions are structurally and molecularly homologous to the mammalian paranodal junction that separates the node of Ranvier from the myelinated portion of the axon. Studies in our laboratory and others have identified a handful of septate junction components, but we still have a very limited understanding of either its structure or function. To shed light on these important questions, we plan to: 1) Identify novel septate junction components using a genetic and cellular approaches. 2) Study the functions of Ankyrin, a membrane associated protein that is believed to interact with some septate junction components, in organizing the basolateral membrane. 3) Examine functional interactions between the septate junction and mechanisms that establish and maintain apical/basal epithelial polarity. These experiments are expected to provide insights into questions of fundamental importance in all developing tissues and in particular epithelia. Knowing more about the mechanisms by which epithelial cells establish and maintain contacts, regulate paracellular diffusion, and form specialized membrane domains is important not only for understanding normal physiology and development, but also for elucidating processes associated with a variety of human diseases.

DESCRIPTION (provided by applicant): The department of Molecular Genetics and Cell Biology is applying to the NIGMS for a Recovery Act grant (RFA-OD-09-005) to recruit a tenure track Assistant Professor in the area of Biochemistry of Chromatin Structure and Function. The department had determined the lack of this important discipline at the University and had obtained permission from Dean James Madara in September 2008 to recruit in this area, novel for the Department and the University. Subsequently, Dean Madara requested that we stop this search because of a shortage of funds. Recruitment will be a coordinated effort led by the Department of Molecular Genetics and Cell Biology (MGCB), together with the Institute of Genomics and Systems Biology (IGSB) and the Department of Biochemistry and Molecular Biology (BMB). The recruit will have a primary appointment in MGCB with the potential for membership in IGSB depending on interests. A core search committee has been formed consisting of key faculty in MGCB, BMB, and IGSB with interests in chromatin and gene expression. The position will be advertised in major scientific journals and will emphasize that the University is interested in applications from ethnic minorities and women. The new investigator will have a Ph.D. or M.D. degree, an outstanding publication record and several years of postdoctoral experience in a laboratory that is a pioneer in chromatin biochemistry/proteomics. The potential recruit will be housed in completely renovated space within MGCB. Up-to-date research facilities that cover essentially every current methodology will be available to the young investigator within the Division of Biological Sciences. A mentoring plan for junior faculty is in place in MGCB and will be streamlined for the needs of the recruit in consultation with the chair. If NIH grants us an ARRA to recruit, the Dean of the Division of Biological Sciences is committed to supplement the first two years and to extend funding for this tenure track position for additional two years and beyond.

DESCRIPTION (provided by applicant): The ability to form specialized membrane domains composed of unique sets of transmembrane proteins, associated cytoplasmic proteins, and phospholipids, is a fundamental property of polarized epithelial cells. Membrane domains, such as the apical surface or junctional complex, allow spatial segregation of functions at the plasma membrane that are essential for polarized epithelia. Central to this process is the formation of protein complexes on the cytoplasmic side of the membrane that localize transmembrane proteins, regulate their signaling output and control their abundance via regulated endocytosis. The Ezrin, Radixin, Moesin (ERM) proteins organize a key role in this process. In this proposal we describe experiments designed to take advantage of the combined expertise of two investigators, Andrea McClatchey (Harvard/MGH) and Richard Fehon (University of Chicago), to extend our understanding of ERM function. The investigators and their laboratories bring together expertise in two powerful experimental systems, the mouse and Drosophila. The proposed research utilizes a multifaceted approach, including genetics, biochemistry, cell biology and proteomics to better understand the functions of these highly conserved proteins. Specifically, we plan to: 1) Determine the molecular mechanisms that link the ERM proteins to the activation state of Rho in developing epithelial cells. 2) Examine the molecular mechanisms that regulate ERM activity, particularly in the context of how ERM activity is dynamically regulated. 3) Delineate the function of the ERM proteins in cell:cell junction remodeling. 4) To build an integrated model of ERM-mediated complex formation. These experiments are expected to provide novel insight into the functions of ERM proteins in biological processes such as apical-basal polarity cytoskeletal regulation, intestinal lumen formation and homeostasis, and metastasis. They should also yield a better understanding of the cellular processes that establish specialized membrane domains in polarized cells, and inform our understanding of cytoskeletal and junctional dynamics during morphogenesis and in disease. PUBLIC HEALTH RELEVANCE: We will carry out complementary and synergistic studies in two powerful model systems - Drosophila and the mouse - to examine the function of ERM proteins in epithelial morphogenesis. Specifically, we will test the hypothesis that ERM proteins provide local regulation of RhoGTPase activity in response to upstream signals/receptors using molecular tools and genetic models that we have developed in each system. We will also delineate the function of ERM proteins in cell junction remodeling/stability as suggested by the phenotypes of ERM loss in both flies and mammals.

Project Summary/Abstract Neurofibromatosis type 2 (NF2), a dominantly inherited disease characterized by the formation of bilateral vestibular Schwannomas (resulting in deafness) and other tumors, is caused by loss of the tumor suppressor protein Merlin, a member of the FERM domain superfamily. Studies using the fruit fly Drosophila and subsequently confirmed in mammalian systems indicate that Merlin is an upstream component of the Hippo/Salvador/Warts (HSW) pathway, a conserved signal transduction pathway that regulates tissue growth. Mutations in Merlin and other HSW pathway components are believed to cause tumors because they cause activation of an oncogenic protein Yorkie/YAP and increased expression of growth promoting genes. Identifying specific proteins and signal transduction pathways with which Merlin interacts is especially important because these partners may act as genetic modifiers of NF2 disease phenotypes and provide potential targets for therapeutic agents. We seek to understand how Merlin and the other HSW components are organized into a signaling complex at the cell cortex, what controls the activity of this complex, and how feedback regulation operates within the pathway. We propose that Merlin and Kibra nucleate formation of a signaling complex at a site separate from intercellular junctions, and thus that these proteins can function in parallel to another upstream regulator, Expanded. We also plan to study how cortical tension regulates pathway activity, and in turn how pathway activity might control cortical tension. To address these questions, we have developed tools and techniques that allow us to examine the localization and dynamics of HSW pathway proteins expressed at endogenous levels in living tissues. Using with the exquisite genetic tools available in Drosophila, we can now elucidate the role of each pathway component in assembling and activating the HSW pathway. These experiments are expected to provide insights into NF2, tumor suppression in general, and the normal cellular processes that establish specialized membrane domains in epithelial cells and neurons. Finally, these studies should contribute to work on the mechanisms by which cellular interactions function to control tissue growth and determine cell fate during development and regeneration.

Neurofibromatosis type 2 (NF2), a dominantly inherited disease characterized by the formation of bilateral vestibular Schwannomas (resulting in deafness) and other tumors, is caused by loss of the tumor suppressor protein Merlin, a member of the FERM domain superfamily. Studies using the fruit fly Drosophila and subsequently confirmed in mammalian systems indicate that Merlin is an upstream component of the Hippo/Salvador/Warts (HSW) pathway, a conserved signal transduction pathway that regulates tissue growth. Mutations in Merlin and other HSW pathway components are believed to cause tumors because they cause activation of an oncogenic protein Yorkie/YAP and increased expression of growth promoting genes. Identifying specific proteins and signal transduction pathways with which Merlin interacts is especially important because these partners may act as genetic modifiers of NF2 disease phenotypes and provide potential targets for therapeutic agents. We seek to understand how Merlin and the other HSW components are organized into a signaling complex at the cell cortex, what controls the activity of this complex, and how feedback regulation operates within the pathway. We propose that Merlin and Kibra nucleate formation of a signaling complex at a site separate from intercellular junctions, and thus that these proteins can function in parallel to another upstream regulator, Expanded. We also plan to study how cortical tension regulates pathway activity, and in turn how pathway activity might control cortical tension. To address these questions, we have developed tools and techniques that allow us to examine the localization and dynamics of HSW pathway proteins expressed at endogenous levels in living tissues. Using with the exquisite genetic tools available in Drosophila, we can now elucidate the role of each pathway component in assembling and activating the HSW pathway. These experiments are expected to provide insights into NF2, tumor suppression in general, and the normal cellular processes that establish specialized membrane domains in epithelial cells and neurons. Finally, these studies should contribute to work on the mechanisms by which cellular interactions function to control tissue growth and determine cell fate during development and regeneration.

Project Summary/Abstract Proper coordination of tissue growth is of fundamental importance both in normal development and in many disease processes, including cancer. Studies in Drosophila and mammalian systems over the past 20 years or so have identified the Hippo pathway as a central regulator of tissue growth. As with many signaling pathways, Hippo components include transmembrane proteins, a cytoplasmic kinase cascade, and output mediated through nuclear transcription factors. However, regulation of Hippo signaling is unusually complex, involving mechanical tension, apical-basal polarity, feedback loops, and regulated assembly of different pathway components at spatially distinct regions of the cell cortex. In addition, further pathway control is exerted by components of the planar cell polarity mechanism, specifically by output from Fat/Dachsous signaling. Fat and Dachsous are both large transmembrane protocadherins that function to control the abundance and localization of an atypical myosin named Dachs. Dachs does not appear be a functional motor protein, but instead functions at the cell cortex to regulate pathway activity at the level of the kinase cascade. Studying the Hippo pathway, and in particular Fat/Dachsous signaling, offers an extraordinary opportunity to learn how multiple inputs that are sensing distinct environmental cues are coordinated to control the output of an important developmental and disease-related growth control pathway. In this proposal, we describe experiments designed to unravel some of the complexity underlying Fat- Dachsous signaling so that we can elucidate the underlying molecular principles essential for its function. First, we propose the notion of a 'core complex' of proteins that interact with and recruit Dachs to the cell cortex where it functions to promote growth. In the absence of upstream regulation, these proteins are maximally active. We describe experiments designed to better understand how this complex forms and what factors regulate its ability to assemble at the cell cortex. Second, we propose the notion that Fat and Dachsous together function to repress activity of the core complex, and thereby control tissue growth. Based on previously published data and our own unpublished observations, we propose a previously unrecognized role for Dachsous in removing Dachs from the cell cortex via endocytosis, and describe an experimental plan to rigorously test this hypothesis. Together, the results of these studies have the potential to fundamentally change our mechanistic understanding of Fat/Dachsous signaling in the Hippo tissue growth control pathway.