1999 — 2001 |
Fay, David S |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Control of the Ras Signaling Pathway in C Elegans @ University of Colorado At Boulder
Ras-mediated signal transduction pathways are highly conserved among metazoan and play key roles in controlling cell proliferation and differentiation. Mutant forms of two members of the pathway, ras and raf, are among the most commonly seen oncogenes involved in many types of cancers. Through a genetic suppresser-of-ras screen in the nematode C. elegans, several conserved regulators of Ras-Raf activation were discovered. One such gene, sur-8, encodes a conserved Leucine-Rich Repeat protein that binds directly to Ras and plays an important role in Ras regulation. I propose to study the regulation of signaling activity transmitted by Ras by focusing on two genes which have been shown to act positively to regulate Ras-Raf activation. C. elegans genetics, in vitro biochemistry, and possibly mammalian cell culture systems will be used. I will study the role of the sur-8 gene in mediating ras signal transduction by examining the nature of SUR-8 interactions with both Ras and Raf. I will also use molecular methods including two-hybrid techniques to identify other protein(s) that I may interact and function with SUR-8 to facilitate Ras-pathway activation. I will also genetically and molecularly characterize another suppresser gene, sur-7, which appears to also function as a positive regulator in the pathway. Work in the past several years demonstrated that vulval induction in C. elegans provides a specific assay system to identify new genes and elucidate their functions in signal transduction.
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0.964 |
2004 — 2010 |
Fay, David S |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Developmental Function of Rb Family Proteins
DESCRIPTION (provided by applicant): Among the many pathways controlling cell proliferation and differentiation, genes of the retinoblastoma protein (Rb) regulatory network stand out as frequent if not obligatory targets for mutation or deregulation during tumorigenesis. Although biochemical and tissue culture studies have implicated Rb family members in a wide range of cellular activities, the bona fide functions of Rb in vivo and during normal development are not well understood. Our long-term objective is to understand the cellular, and developmental functions of Rb family proteins at the molecular level. To this end, we have devised a genetic strategy that has allowed us to identify genes and pathways that function coordinately with the C. elegans Rb homolog, lin-35, to control essential developmental processes. Using this system, we have demonstrated canonical cell cycle functions for LIN-35 as well as a novel role for this protein in organ morphogenesis. We have also uncovered a complementary pathway that acts to control organ morphogenesis through UBC-18/UbcH7, a conserved ubiquitin-conjugating enzyme involved in the targeting of proteins for degradation. The proposed experiments are designed to uncover the underlying mechanism by which LIN-35, acting in conjunction with one or more parallel pathways, regulates organ morphogenesis in C. elegans. Our main objectives fall into two categories. One broad aim is to identify additional factors that function cooperatively with LIN-35 and UBC-18 to control organogenesis. These studies will include the cloning and characterization of sir-9, a gene that, like ubc-18, functions redundantly with lin-35 to control organ morphogenesis; the execution of a two-hybrid screen to identify UBC-18-interacting proteins; and a directed RNAi feeding screen using known or putative ubiquitin pathway components. Our second objective is to identify functionally relevant downstream targets for regulation by LIN-35 and UBC-18. These studies will include genetic selections to isolate mutations that suppress the lethality of lin-35; ubc-18 double mutants; microarray analyses to identify the complete spectrum of LIN-35-regulated transcripts; and additional two-hybrid screens using co-factors of UBC-18 identified through earlier two-hybrid or RNAi-feeding screens. The successful completion of these studies will greatly enhance our general understanding of Rb family functions and will provide detailed mechanistic knowledge of this novel role for Rb proteins in morphogenesis.
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2006 — 2009 |
Fay, David S |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
A Novel Genetic Approach For Elucidating Glycopeptide Hormone Functions |
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2008 |
Fay, David S |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
Celegans as a Model to Study Viral Anti-Apoptotic Genes
Animal Model; Animal Models and Related Studies; Apoptotic; Biological Function; Biological Process; C elegans; C.elegans; CRISP; Caenorhabditis elegans; Cell Communication and Signaling; Cell Signaling; Cell Survival; Cell Viability; Chemotherapy-Hormones/Steroids; Computer Retrieval of Information on Scientific Projects Database; Development; Developmental Biology; Endocrine Gland Secretion; Funding; Gametes; Genes; Genetic; Germ Cells; Germ-Line Cells; Glycopeptides; Goals; Grant; Hormones; Inhibition of Apoptosis; Institution; Intracellular Communication and Signaling; Investigators; Knowledge; Maintenance; Maintenances; Mediating; Modeling; Mother Cells; NIH; National Institutes of Health; National Institutes of Health (U.S.); Oocytes; Ortholog; Orthologous Gene; Ovocytes; Population; Position; Positioning Attribute; Progenitor Cells; Receptors, Thyroid Stimulating Hormone; Regulation; Reproductive Cells; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Sex Cell; Signal Transduction; Signal Transduction Systems; Signaling; Source; Stem cells; TSH Receptors; Therapeutic Hormone; Thyrotropin Receptor; United States National Institutes of Health; Viral; biological signal transduction; initial cell; model organism; novel; reproductive function; sexual cell; social role
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2011 — 2014 |
Fay, David S |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Developmental Functions of Rb Family Proteins
DESCRIPTION (provided by applicant): Among the many pathways controlling cell proliferation and differentiation, genes of the retinoblastoma protein (pRb) regulatory network stand out as frequent, if not obligatory, targets for mutation or deregulation during human tumorigenesis. Although biochemical, tissue culture, and transgenic studies in mice have implicated pRb family members in a wide range of activities, the full spectrum of authentic pRb functions during normal development has yet to be resolved. In addition, new studies suggest that pRb may exert its anti-oncogenic effects through a variety of distinct mechanisms, although the critical functions of pRb in tumor suppression remain to be determined. Our long-term objectives are to understand the molecular, cellular, and developmental functions of pRb family proteins and to mechanistically link the pRb pathway to other cellular networks. To this end, we have used a range of genetic and molecular methods to characterize novel functions of lin-35, the sole Rb ortholog in C. elegans, including roles in organ morphogenesis, intestinal homeostasis, and cell fate determination. In addition, using transcriptome profiling, we have identified many LIN-35 target genes that rep- resent novel candidate cell cycle regulators. Our main objectives fall into three categories. In Aim 1, we will fo- cus on further elucidating the LIN-35 regulatory network controlling pharyngeal morphogenesis. Specifically, we will explore the roles of several new genes that we have implicated as functioning within this pathway and will identify additional components through established screening methods. In Aim 2, we will study the roles of LIN-35 and SLR-2, a Zn-finger protein, in regulating intestinal function and gene expression. This will be accomplished using forward genetics and molecular approaches and will include directed studies to examine the functional connection between LIN-35 and genes identified in a genome-wide RNAi suppressor screen. In Aim 3, we will follow up on microarray and bioinformatical analyses to identify novel cell cycle components. Functions for candidate genes will be determined using a variety of sensitized genetic backgrounds and in vivo as- says, which will allow us to determine roles for these genes in cell cycle progression and regulation. The successful completion of these aims will enhance our basic understanding of both cell cycle and non-cell cycle functions for pRb family members and will provide mechanistic insights into the roles of LIN-35 and associated pathways in controlling the formation and function of two principal organs involved in nutrient uptake and utilization. This proposed research will also support efforts to define authentic in vivo activities for pRb family members and to identify genetic modifiers that may augment or diminish the phenotypic effects of Rb mutations. As such, these studies have strong relevance to understanding the tumor-suppressing functions of pRb and the many cellular factors that comprise the greater pRb regulatory network in humans. Our published findings and extensive preliminary results provide a solid foundation and logical framework for carrying out the proposed experiments. PUBLIC HEALTH RELEVANCE: The goal of our research is to understand the biological functions of the Retinoblastoma protein (pRb), which normally protects against tumor formation in humans. Because a protein that is closely related to pRb is present in the nematode model organism C. elegans, we will use this powerful system to study the functions of pRb family proteins in order to learn more about their roles during normal development. This will lead to a better understanding of the anti-cancer activities carried out by pRb in humans and may suggest new approaches for anti-cancer therapies.
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2016 — 2019 |
Fay, David S |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterizing Novel Functions of Conserved Nima Family Kinases
? DESCRIPTION (provided by applicant) Although members of the NIMA-related kinase (NEK) family have been implicated extensively in human dis- eases including cancer and renal and cardiovascular defects, their physiological substrates are largely un- known. Moreover, little is known about the upstream and downstream components of NEK signaling pathways or about the proteins that modulate NEK activities. This has led to a fundamental gap in our knowledge concerning the molecular and cellular functions of this biomedically relevant and highly conserved family of protein kinases. The long-term goal is to characterize a recently discovered and potentially widespread function for NEKs in intracellular trafficking and extracellular matrix remodeling. The objective of this application isto characterize the trafficking functions of NEKL-2/NEK8 and NEKL-3/NEK6/7 during C. elegans development and in human cell culture systems. The central hypothesis is that NEKs regulate endocytosis and vesicle trafficking through interactions with components of the cytoskeleton including microtubule (MT)-associated proteins, molecular motors, and regulators of the acting network. Strong preliminary data support the two specific aims: 1) Elucidate the mechanisms by which NEKL-2 and NEKL-3 control trafficking; and 2) Identify molecular targets and components of the NEKL signaling pathway. Under Aim 1, the localization of NEKLs to intracellular traf- ficking and cytoskeletal compartments will be characterized, and the specific cellular functions of NEKLs in regulating vesicular trafficking and cytoskeletal organization and dynamics will be determined. In addition, the hypothesis that ankyrin repeat (AR) binding partners of NEKLs act as signaling scaffolds will be tested. Finally, a panel of assays will be carried out to test the rles of NEK6/7, NEK8, and associated AR proteins in trafficking functions in mammalian cells. Under Aim 2, proven RNAi-feeding screens will be carried out to identify components and candidate targets of the NEKL signaling networks. In addition, a recently developed powerful chemical genetic-proteomic strategy, which is well supported by preliminary data, will be used to directly identify in vivo substrates of the NEKLs. Follow-up studies will be carried out on select validated targets to deter- mine their functions in trafficking and cytoskeletal organization downstream of the NEKs. The approach is in- novative because other groups have not applied powerful forward genetic methods to study NEK kinases and because the function of NEKs in intracellular trafficking is entirely unknown. In addition the proposed chemical- genetic strategy to identify substrates has not been previously applied to C. elegans or to the study of NEKs in any system. The proposed research is significant because human NEKs are widely implicated in human dis- eases and have been suggested as drug targets, but their roles during interphase are not well understood nor, in most cases, are their physiological substrates known. As an additional benefit, these studies will lead to the characterization of previously unknown but conserved regulators of intracellular trafficking and extracellular matrix remodeling, both of which are highly relevant to human development, health, and disease.
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2017 — 2019 |
Fay, David S |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms Controlling Tissue Morphogenesis, Architecture, and the Response to Mechanical Forces in C. Elegans.
The role of the apical extracellular matrix (aECM) as an important mediator of animal development is only just beginning to be recognized and explored, and few studies have used intact systems to examine how the aECM affects the response of cells and tissues to mechanical forces. This knowledge gap limits the understanding of fundamental biological processes, which when misregulated can lead to diverse developmental and adult- onset diseases. Recent studies uncovered mechanical forces acting on epidermal cells adjacent to the devel- oping anterior foregut in C. elegans embryos. These studies also identified highly conserved proteins that are required to mediate the response to this force, including FBN-1, a fibrillin-related aECM protein; MEC-8, a splicing factor that regulates FBN-1; and SYM-3 and SYM-4, candidate RAB-11?associated factors implicated in apical protein trafficking. The principal objectives of this application are to understand how the epidermal aECM is generated and how the aECM facilitates morphogenesis, cell organization, and resistance to mechan- ical stress. These objectives will be met by pursuing three specific aims. Aim 1 will identify and further charac- terize key components of the aECM, including FBN-1, and will determine how the aECM is physically attached to the underlying epidermis. Aim 2 will test the hypothesis that SYM-3 and SYM-4 are RAB-11 cofactors and will determine the molecular functions of these proteins and other conserved trafficking components in the transport of protein cargos to the apical membrane. Aim 3 will extend preliminary studies to understand the mechanisms by which diverse embryonic epithelial cells are organized into a rosette structure that must resist mechanical forces to form a normal foregut lumen. We will test the hypothesis that the aECM, acting with the cytoskeleton and adherens junction proteins, maintains the structural integrity of the rosette, a widespread but little-studied embryonic structure. In addition, these studies will reveal the locations, magnitude, and dynamics of mechanical forces acting at cell?cell and cell?ECM junctions and within the cytoskeletal network during C. elegans embryonic development. This research is significant because it will (1) characterize physical forces in an intact developing system and reveal mechanisms governing how cells and tissues respond to mechanical stress; (2) lead to the discovery of novel aECM components and regulators of apical protein trafficking; (3) pro- vide insights into how diverse epithelial cells underlying the aECM organize into rosette structures and resolve to form lumens; and (4) identify genetic modifiers of a fibrillin-related protein, which are clinically important in Marfan syndrome but have not been previously characterized. This research is innovative because (1) it is based on the discovery of a previously unrecognized biomechanical force in C. elegans embryos; (2) it is the first study to directly visualize mechanical forces in developing C. elegans embryos using FRET-based tension sensors; and (3) the phenotypes under study are generally penetrant only when two or more genes have been inactivated, thereby serving as a model for redundant control mechanisms and complex disease traits.
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2020 — 2021 |
Fay, David S |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
In Vivo Regulation of the Extracellular Matrix
Summary. Animals are composed of living cells and the 3-diminsional network of molecules that surrounds them, the extracellular matrix (ECM). In addition to its structural and protective functions, the ECM is an important regulator of cell organization, differentiation, morphogenesis, and physiology. Previous ECM studies have focused largely on basement membranes, the ECM that contacts the basal surface of polarized cells. Much less is known about the apical ECM (aECM), which resides within epithelial, mesothelial, and endothelial lumens and on the surface of epidermal cells. Recent studies have implicated the aECM in the control of cell shape, tissue morphogenesis, and tube formation, leading to a new appreciation of aECM impacts on development and disease. At present, little is known about the regulation of the aECM, including the pathways that control its deposition, organization, and remodeling. The proposed studies will address these gaps by investigating aECM regulation in two distinct contexts: C. elegans (1) embryonic morphogenesis and (2) larval molting. These separate lines of investigation recently converged with the discovery that intracellular trafficking factors play a crucial role in aECM regulation at both stages. In the case of embryogenesis, two conserved but previously uncharacterized proteins, SYM-3/FAM102A and SYM-4/WDR44, enable the nascent epidermis to resist deformation by biomechanical forces. Current data suggest that SYMs partner with multiple endocytic factors, including RAB-11, to control trafficking and aECM integrity. In the case of larval molting, conserved members of the NEK family of protein kinases, NEKL-2/NEK8/9 and NEKL-3/NEK6/7, are required at each molt to facilitate remodeling of the cuticle, an aECM derived from the epidermis. Current data indicate that NEKLs regulate trafficking in close association with AP2, a core component of clathrin-coated vesicles, and through the control of endocytic actin. Future studies on SYMs and NEKLs will combine genetics, cell biological, biochemical, and omics-based approaches to understand their specific functions in trafficking and to link these activities to effects on the aECM. To broaden impact, analyses will incorporate mammalian cell culture systems, as current data indicate that NEKL and SYM functions are conserved. Beyond elucidating aECM biology, these investigations will characterize mechanisms of apical trafficking, which is poorly understood and differs substantially from endocytosis at non-polarized or basolateral membranes. Work on NEKLs will also address the role of phosphorylation in regulating components of the endocytic machinery, which is thought to be pervasive but remains largely uncharacterized. Moreover, whereas the vast majority of trafficking studies have used in vitro cell culture systems, work on the NEKLs and SYMs will take advantage of the ability to study trafficking within an intact developing organism. Finally, proposed studies will yield insights into the roles of trafficking, signaling, and ECM remodeling in nematode molting, an understudied process with relevance to human biology and health. Collectively, this work will impact the fields of intracellular trafficking, ECM biology, signaling, and development.
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1 |
2020 — 2021 |
Fay, David S |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
Wyoming Inbre Developmental Research Project Program
Wyoming INBRE Developmental Research Projects Program (DRPP) Project Summary. The Developmental Research Project Program (DRPP) of the Wyoming INBRE will carry out the central function of allocating funds to support biomedical research throughout the Wyoming network including the University of Wyoming (UW) and the state?s 7 community colleges. The DRPP will manage internal grant competitions in the INBRE-4 thematic areas of (1) Cell and Molecular Biology and (2) Methods for Chronic Disease Research and Therapies. These themes are a natural outgrowth of INBRE-3 focus areas and are designed to capture the widest possible field of active biomedical researchers within the Wyoming network. Other keys functions of the DRPP will be to oversee the advising and mentoring of supported faculty, to monitor progression toward project goals and funding independence, and to ensure standards of compliance. In carrying out its functions the DRPP will continue to work closely with the Administrative Core, Bioinformatics Core, and INBRE Student Programs. Since the start of INBRE-3 (2015?present), the DRPP has allocated $3M, or 95% of its total budget, to support 45 different research projects within the Wyoming network. This includes Thematic and Pilot grants, which are used to support research-intensive faculty at UW. A key litmus test for Pilot and Thematic projects is that they must have the potential to be developed into independent R-type or similar awards. In addition, 10% of Thematic and Pilot awards are used to promote UW?community college interactions, thus strengthening the network. The DRPP also funds Collaborative grants, which support extensive scientific collaborations between UW and community college investigators. Collaborative projects emphasize student training and network development but also encourage the publication of results. During INBRE-4, the DRPP will continue to work with the Administration Core to allocate graduate assistantships and shared equipment grants, as well as other initiatives. Our central aims will be to: (1) Collaborate with other INBRE cores to support biomedical research projects, training opportunities, and critical infrastructure throughout the Wyoming network; (2) Administer a fair and rigorous system for the evaluation of INBRE research and training proposals; (3) Ensure appropriate advising, oversight, and assessment for faculty and students engaged in Wyoming INBRE projects; and (4) Promote inter-institutional biomedical research, training, and interactions within the Wyoming network and western region of the Regional Alliance of INBRE Networks. These goals are supported by a strong foundation laid during INBRE-3, which included the rigorous external review of Pilot and Thematic awards. In addition, during INBRE-3 the DRPP introduced five new training initiatives including (1) Faculty Chalk Talks, (2) External Consulting, and (3) Grant and Manuscript Editing. During INBRE-4 we will continue with these successfully reviewed activities and further enhance our efforts to aid faculty with grantsmanship and publication output. A long-term objective of the INBRE/DRPP is to substantially expand biomedical research, training opportunities, and technology development within Wyoming.
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