Jason M. Haugh - US grants

Much effort has been devoted in the last decade to elucidate signal transduction mechanisms initiated by receptor tyrosine kinases. For example, these receptors mediate phosphoinositide hydrolysis and phosphorylation by modulating the activities of phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3-kinase (PI 3-kinase), respectively, and they stimulate the activation of the p21 ras GTPase by assembling multiprotein complexes with Ras-guanine nucleotide exchange activity. Given the current appreciation of complex feedback and crosstalk interactions within and among these signaling cascades, quantitative experimental and analytical methodologies are lacking. In the proposed study, I will develop a relatively high-throughput array platform for imaging membrane recruitment of fluorescent biosensors, allowing two signaling pathways to be monitored simultaneously in living cells. This imaging setup, coupled with an engineering analysis of the data, will allow multiple signaling variables to be related.

The ultimate goal is to build a mathematical model that captures the cross-talk between cell proliferation and cell death signaling pathways. The focus of the proposed work is on the Ras/Raf and P13K/Akt pathways. Preliminary data will be gathered that investigates and illustrates the cross-talk between these pathways. With an improved understanding and a mathematical description in-hand, an improved base for optimizing cell culture or developing pharmaceutical compounds can be envisioned.

Proposal Title: PECASE: Intracellular Signaling Networks in the Immune Response
Institution: North Carolina State University

The objective of the proposed research is to study T lymphocyte (T cell) regulation at the intracellular level, with a focus on molecular signaling processes initiated by the cell surface receptors for the cytocines, interleukins (IL)-2 and (IL)-4. The specific objectives of this research include: (1) the quantitative analysis of pathway crosstalk interactions in IL-2 receptor signaling, using interventions that target specific intracellular molecules, (2) the characterization of the cooperation of IL-2 receptor-mediated signaling pathways in mediating T cell life (proliferation) and death (apoptosis) decision making, and (3) the elucidation of the synergistic effect of IL-2 and IL-4 on T cell expansion at the level of signaling network interactions. This research could provide new information to help modulate cell function, with potential application being the elucidation of therapeutic strategies.

This project was originally funded as a CAREER award, and was converted to a Presidential Early Career Award for Engineers and Scientists (PECASE) award in May 2004.

DESCRIPTION (provided by applicant): The most widely studied signaling system in cell physiology is arguably the regulatory switch governing proliferation and programmed cell death. Not surprisingly, mitogens such as platelet-derived growth factor (PDGF) trigger proliferation, through the Ras/Erk pathway, as well as protection from cell death, through the phosphoinositide 3-kinase (PI3K)/Akt pathway. Confounding the analysis of these pathways are the numerous crosstalk interactions between them, which suggest that cell life and death are co-regulated. A quantitative understanding of crosstalk in signaling networks is thought to be a major hurdle in the design of molecular therapeutics targeting intracellular signaling proteins, with implications for cancer, wound healing, and immune cell regulation. Employing PDGF-stimulated signaling in NIH 3T3 fibroblasts as a model system, the magnitudes and kinetics of Ras/Erk and PI3K/Akt signaling will be manipulated through specific genetic and pharmacological interventions, and crosstalk will be assessed by measuring the sensitivities of other intermediates to those changes for various levels of receptor stimulation. This general strategy is termed crosstalk titration. Central to these efforts are quantitative, high-throughput cell biochemical assays for the levels of PDGF beta-receptor phosphorylation, Ras-GTP, Akt kinase activity, and Erk kinase activity. With extensive signaling data for various PDGF concentrations and intracellular manipulations, a mathematical model of the signaling network will be formulated to predict the outcomes of more complex intervention strategies. Finally, for conditions found to perturb the signaling network, the corresponding effects on cell proliferation and survival will be assessed.

DESCRIPTION (provided by applicant): The most widely studied signaling system in cell physiology is arguably the regulatory switch governing proliferation and programmed cell death. Not surprisingly, mitogens such as platelet-derived growth factor (PDGF) trigger proliferation, through the Ras/Erk pathway, as well as protection from cell death, through the phosphoinositide 3-kinase (PI3K)/Akt pathway. Confounding the analysis of these pathways are the numerous crosstalk interactions between them, which suggest that cell life and death are co-regulated. A quantitative understanding of crosstalk in signaling networks is thought to be a major hurdle in the design of molecular therapeutics targeting intracellular signaling proteins, with implications for cancer, wound healing, and immune cell regulation. Employing PDGF-stimulated signaling in NIH 3T3 fibroblasts as a model system, the magnitudes and kinetics of Ras/Erk and PI3K/Akt signaling will be manipulated through specific genetic and pharmacological interventions, and crosstalk will be assessed by measuring the sensitivities of other intermediates to those changes for various levels of receptor stimulation. This general strategy is termed crosstalk titration. Central to these efforts are quantitative, high-throughput cell biochemical assays for the levels of PDGF beta-receptor phosphorylation, Ras-GTP, Akt kinase activity, and Erk kinase activity. With extensive signaling data for various PDGF concentrations and intracellular manipulations, a mathematical model of the signaling network will be formulated to predict the outcomes of more complex intervention strategies. Finally, for conditions found to perturb the signaling network, the corresponding effects on cell proliferation and survival will be assessed.

[unreadable] DESCRIPTION (provided by applicant): Wound healing is a complex process involving multiple cell types, but invasion of nearby fibroblasts into the clot is considered the rate-limiting step. The rate of fibroblast invasion is in turn controlled, to a significant extent, by the action of platelet-derived growth factor (PDGF) released by platelets and macrophages in the clot. The basis of this proposal is to explore the prospect that mathematical modeling can be used to bridge the fundamental gap between in vitro studies of cell signaling at the molecular/cellular levels, performed under controlled experimental conditions, and physiological settings where cell population behaviors evolve dynamically. In general, targeted molecular therapeutic strategies will have the most benefit if they are informed by molecular-, cellular-, and tissue-level design principles. Based on indications from previous and preliminary work, we have formulated a new, hypothetical model with the potential for redefining the mechanisms by which migration of fibroblasts and similar cell types is spatially directed. We aim to explore this and alternative models in quantitative terms through a combination of experimental and computational approaches. Intracellular signaling through the PI 3-kinase pathway will be monitored in living fibroblasts, using total internal reflection fluorescence (TIRF) microscopy, as the cells sense and respond to PDGF gradients. In these experiments, morphological polarity and cell migration will also be tracked, in an effort to quantitatively relate migration speed and turning behavior to the pattern of PI 3-kinase activation. Preliminary work has shown that cell polarity imposes an intrinsic bias in PI 3-kinase signaling, which is reinforced by PDGF when cells migrate up-gradient. To further test this mechanism, cell polarity will be disrupted using a variety of molecular perturbations, and the effects on PI 3-kinase signaling will be analyzed. Finally, the relationships among PI 3-kinase signaling, cell polarity, and cell migration will be incorporated in a biased random walk model, which will ultimately define the cell flux term in a model of fibroblast invasion. These efforts will serve as a foundation for more in-depth studies of molecular mechanisms governing physiological processes. [unreadable] [unreadable] [unreadable]

In the first funding phase, the Modeling Initiative constructed a number of relational and biophysical models of mechanics and molecular phenomena related to cell migration and started to develop migration related capabilities within the Virtual Cell (VC) software. This activity can be considered as the last step in the reductionism agenda - in silico reconstitution of a simplified motile system using mathematical representation combining biological knowledge and hypotheses, with determination of the consequences of these hypotheses facilitated beyond human reasoning by means of computer-generated numerical calculations. These models and software development enabled the exciting possibility to make a large, critical step in our quantitative understanding of cell migration from the point of view of systems biology. The models will be standardized from the technical point of view, integrated, comprehensive and predictive. A crucial feature of our endeavor, absolutely required for validating such models and using them for hypothesis prediction-test efforts, is that no modeling is undertaken absent direct input from experimental collaborators. We will describe below the mechanism by which this requirement will be consistently met. The Modeling Initiative will investigate migration mechanisms at the systems-level with a long term goal of developing a comprehensive model of cell migration. This model will have a modular character combining deterministic and stochastic components. Our approach is to develop models for each of the component processes that drive cell migration, e.g., development of polarity, protrusion, adhesion, and contraction and rear release, and then integrate them into a comprehensive model. For each of these processes, a 'Process Team'that includes both computational biologists and experimental biologists (in a few cases, these capabilities reside within the same laboratory ), will work together to develop a 'Process Model'capable of capturing dynamic behavior in terms of molecular properties (protein levels, states, locations, and activities). It is through this collaborative team that data will be produced, analyzed, and modeled iteratively with a goal of developing additional data from model predictions and using these data to refine the models.

CBET-0828936
Jason M Haugh, North Carolina State University

Intellectual merit

This project offers a new conceptual framework for thinking about control of fibroblast migration at the level of intracellular signal transduction and novel tools for quantitatively analyzing it. If successful, the proposed work would more generally unify distinct cell migration stimuli and behaviors in terms of common signaling dynamics, providing a blueprint for more ambitious studies with other cell types, environmental contexts, and molecular-level perturbations of the underlying signal transduction network.

Broader impacts

The project would contribute to the training of researchers in the cross-disciplinary areas of engineering analysis, cell biology, fluorescence microscopy, image analysis, and kinetic modeling. In all of these endeavors, the PI will maintain an inclusive research environment that embraces diversity on all levels. The results of the proposed research will be published in archived, peer reviewed journals, and all MATLAB codes will be freely available via the PI's website.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Driven by imaging data from existing and planned imaging experiments in our laboratory and through collaborations within the Cell Migration Consortium, we are building a spatial model of PI3K/RFG co-regulation at the whole-cell level. A phenomenological model of signaling-protrusion coupling will be constructed, guided by analysis of live-cell imaging experiments. To the extent possible, we will develop the model, or modules thereof, in the Virtual Cell environment. The Haugh group has previously made use of the new membrane diffusion capabilities of VCell in our work on gradient sensing, and these models are publicly available to other VCell users. Cell mechanical modeling capabilities, currently under development by the VCell team in collaboration with the CMC modeling initiative, will be utilized in this project as they become available. These will depend on the elliptic solver planned for the next NRCAM project period. Incorporation of mechanics into the VCell framework will then be facilitated by the newly proposed multiphysics layer and plug-in architecture.

DESCRIPTION (provided by applicant): In mammalian cell biology, an ongoing challenge is to bridge the gaps in our understanding of processes at the molecular, cellular, and tissue levels. Central to this hierarchy of biological complexity is the field of signal transduction, which deals with the biochemical mechanisms and pathways by which cells respond to external stimuli. The over-arching goal of this project is to move the signal transduction field from a linear, pathway-centric framework to a network-centric one;to do this;we are quantifying the complexities of feedback regulation and crosstalk interactions, having demonstrated our approach in elucidating dynamical system features of growth factor receptor-mediated signaling in fibroblasts. Quantitative experiments canvassing an array of cell stimulation and molecular perturbation conditions, together with computational modeling, have comprehensively elucidated the dynamic features of Ras- and phosphoinositide 3-kinase (PI3K)-dependent signaling integrated by ERK, the best-characterized mitogen- activated protein kinase (MAPK) in mammalian cells. Certain challenges remain and will be addressed in the proposed effort using molecular and computational approaches: 1) Mapping the molecular determinants of crosstalk and regulatory feedback onto dynamic features of the signaling network;2) Probing the diversity of PI3K/Erk signaling responses at the single-cell level;3) Comparative analysis of signaling networks among receptor and cell systems;and 4) Elucidating mechanisms of chronically perturbed signaling networks in cells harboring oncogenes. PUBLIC HEALTH RELEVANCE: The goals of this project are to study the complex interactions between specific biochemical pathways that control cell growth and survival during wound healing and which contribute to the progression of cancer. By analyzing these mechanisms quantitatively and using mathematical models, we hope to be able to predict the outcomes of interventions targeting the molecular players in these pathways.

The overall goal of the proposed project is to elucidate the fundamental physical mechanisms governing the reorientation of cell migration polarity, which affects how persistently the cells maintain their directionality of movement and their ability to follow spatial cues during physiological processes such as wound healing, the immune response, and embryonic development. The phosphoinositide 3-kinase (PI3K) signaling pathway has been implicated in the control of cell reorientation, which is achieved by stabilizing the branching of existing cell protrusions activity. Using an approach integrating live-cell fluorescence microscopy, image analysis, and computational modeling, the team will characterize and manipulate the dynamics of signal transduction pathways, focal adhesions, and the actin cytoskeleton during protrusion branching. The hallmarks of cell reorientation thusly identified will be re-evaluated in PI3K-inhibited cells to evaluate if the dynamics are altered and how. These research objectives are to be integrated with educational activities, and, through the support and training of graduate students and the participation of undergraduate students, the project will contribute to the training of researchers in the cross-disciplinary areas of engineering analysis and cell biology.

DESCRIPTION (provided by applicant): Live-cell fluorescence microscopy provides spatially resolved kinetic information about dynamic processes as they occur in cells, and therefore it has unique potential for the quantitative measurement of signal transduction and other intracellular processes. This approach has not yet lived up to its full potential, however, because it is currently limited by the availability of well-characterized biosensor molecules that can be either genetically encoded or microinjected into cells and which recognize intracellular target molecules of interest. What is not appreciated by most practitioners of live-cell imaging is that the binding affinity of a suitable biosensor for its target(s) must lie within a relatively narrow range, and the reliance on modular binding domains derived from naturally occurring proteins presents a major constraint in that regard. Herein, we propose a collaborative effort to develop a novel and general approach enabling de novo design of intracellular biosensors with prescribed properties for live-cell imaging. To do this, it is clear that we must move away from binding domains derived from naturally occurring proteins. Instead, our starting point is an ensemble of 7 different proteins from hyperthermophilic archaea and bacteria (which thrive at extreme temperatures) as templates, or scaffolds, for engineering biosensors. These scaffolds have several favorable properties; they are low in molecular weight (~100 amino acids or less), lack disulfide bonds, and are functionally inert in mammalian cells. Proteins with desired binding specificity will be screened from a large (> 108) combinatorial collection of mutant proteins that we have successfully generated through randomization of surface-accessible residues on the protein scaffolds. Because the protein engineering strategy uses multiple scaffolds, we refer to the repertoire of variants as a super library, which possesses greater theoretical diversity than a library of the same size derived from any single scaffold. We propose to screen the super library for biosensors recognizing specific phosphorylation sites and characterize their binding affinities (Aim 2). Subsequently, we will validate the identified biosensors by characterizing thei translocation in cells expressing wild-type or mutant Epidermal Growth Factor Receptor (EGFR) (Aim 2). Finally, we propose a pilot study to assess the kinetics of site-specific EGFR phosphorylation in human mammary epithelial cells (Aim 3).

DESCRIPTION (provided by applicant): Chronic wounds are a major threat to public health and the economy and present as a comorbid complication with major diseases in humans. Although the proper healing of cutaneous wounds requires collective and coordinated behaviors of multiple cell types, the rate-determining step is the recruitment and function of dermal fibroblasts, which are directed to invade the wound by a gradient in the concentration of platelet-derived growth factor (PDGF). A great deal is known about the signal transduction pathways activated by PDGF receptors and other receptor tyrosine kinases; yet mechanistic insights about how those pathways are spatially organized to bias the dynamics of the actin cytoskeleton and the directionality of cell migration are still emerging. A still larger fundamental gap lies inthe integration of molecular, supramolecular, cellular, and tissue-level dynamics of wound healing, which span disparate time (seconds to weeks) and spatial (nm to cm) scales. To advance this field, novel approaches are needed to fuse experimental and observational scales that are relatively data-rich (signaling, cytoskeletal dynamics) and data-poor (in vivo dynamics). To that end, we propose to develop a predictive, multiscale model of the proliferative phase of wound healing, incorporating 1) receptor-mediated signal transduction (molecular scale), 2) self-assembly of contractile actomyosin structures (supramolecular scale), 3) morphodynamics and statistics of cell migration (cellular scale), and 4) collective cell behavior in vivo (tissue scal). Our partnership combines expertise in experimental cell biology and biophysical modeling, and model development will be guided by new, quantitative measurements at every scale of biological abstraction.

DESCRIPTION (provided by applicant): Cells in a variety of contexts migrate towards soluble chemical cues in a process known as chemotaxis. Despite nearly a century of study, the mechanistic underpinnings of chemotaxis remain incompletely understood. Spatial gradients of growth factors direct the movements of mesenchymal cells in tissues to coordinate and accelerate physiologically important processes such as wound healing, and mesenchymal chemotaxis has been implicated in pathological conditions such as cardiovascular and fibrotic diseases. Yet, the vast majority of chemotaxis studies have focused on leukocytes and other fast-moving, amoeboid cells. Mesenchymal chemotaxis has been prohibitively difficult to study, because it requires maintenance of stable gradients for many hours. Traditional methods such as transwell assays provide little or no dynamic information and poorly discriminate effects on the efficiency of motility from actual directional sensing. To overcome these technical limitations we recently established a microfluidic chemotaxis assay that allows direct observation of mesenchymal cells in stable, linear gradients over many hours, allowing both single-cell tracking and high-resolution live-cell imaging approaches such as TIRF microscopy. Our preliminary data indicate that the growth factor receptor, PDGF-R controls mesenchymal chemotaxis by a PLC > PKC > Myosin II pathway and requires the coordination of signaling events and cytoskeletal organization. We propose to elucidate the mechanisms of mesenchymal chemotaxis by 1) Dissecting the spatio- temporal nature of chemotactic signaling in mesenchymal cells 2) Understanding the dynamic organization of the cytoskeleton during chemotaxis in this cell type and 3) Delineating the coordination of signaling and cytoskeletal events that lead to complex chemotactic behaviors such as re-orientation to new cues and chemotaxis in 3D environments. These studies will directly contribute to our understanding the physiological basis of disease states such tumor metastasis, fibrosis and cardiovascular disease, as well as our understanding of physiological processes such as wound healing.

An award is made to North Carolina State University (NCSU) to acquire a state-of the art confocal microscope in order train a well-prepared and knowledgeable workforce, as well as to conduct world-class research. This instrument will be used to train undergraduates, graduate students and postdocs in quantitative fluorescence microscopy applications and single molecule counting. The microscope will be located within the Cellular and Molecular Imaging Facility (CMIF), a University-administered core research facility that serves researchers from six NCSU colleges and from neighboring institutions and local industries. This system will expose students to state-of-the-art imaging applications via a new course (Quantitative Imaging and Dynamics in Biology), other established courses (BIT595 Confocal Microscopy and BIT 478/578 Mapping the Brain), and during direct undergraduate research experiences. In addition, a new and innovative Light Microscopy Workshop will serve under-represented students from local Historically Black Colleges and Universities (HBCUs) and a local women's college in North Carolina. By exposing traditionally underrepresented students to cutting edge research and technology, NCSU intends to broaden interest in and to improve STEM (Science, Technology, Engineering, Mathematics) education. This innovative program will i) expose students across the region to advanced imaging technologies that are not available at their home institutions; ii) introduce students at institutions lacking comprehensive research programs to ongoing research at NCSU; and iii) integrate and strengthen existing collaborations between NCSU and partnering institutions. NCSU will partner with the NC Museum of Natural Sciences to share imaging results via presentations and hands-on educational activities led by Workshop participants as well as students and postdocs from NCSU laboratories.

The state of the confocal microscope will impact research and training programs across six NCSU colleges and numerous disciplines, including Cell and Molecular Biology, Biochemistry, Animal and Plant Developmental Biology, Plant Pathology, and Bio-Engineering, among others. This instrument will enable NCSU faculty and students to i) perform measurements with increased sensitivity and discrimination; ii) obtain fast temporal and spatial data; and iii) image live biological samples for longer times with reduced phototoxicity. These capabilities will impact, among others, research programs in plant vacuole biogenesis and dynamics, stem cell maintenance in plants, long-range signaling in the Drosophila embryo, and cell signaling during animal cell migration.

Cell migration is an important step in many processes in the body, such as the healing of wounds and the response to infection. The cells are in contact with a matrix that allows them to crawl and explore. To do so, cells must actively sense matrix properties by touch and feel. An overarching goal of this collaborative project is to unify or distinguish cell sensing of the quantity (stickiness by touch) versus quality (stiffness by feel) of contacts with the matrix. The multidisciplinary research will be integrated with graduate student training, teaching of engineering courses, and outreach that will foster science communication.


Cell migration is a fundamental process in tissue development and homeostasis. The molecular determinants of chemotaxis, cell migration biased by a gradient of a soluble attractant, are reasonably well understood, and a paradigm has emerged characterizing certain intracellular signaling pathways as a molecular "compass". In contrast, haptotaxis and durotaxis, migration directed by gradients of immobilized ligand density and of mechanical stiffness, respectively, are poorly understood and require a new paradigm, considering that cells must actively encounter and respond to physical cues (i.e., by "feel") rather than by passive sensing of diffusible ligands. Under Objective 1, the aim is to define feedback mechanisms that control coupled cytoskeletal dynamics in migrating cells. It is hypothesized that adhesion-mediated signaling pathways control the duration of lamellipodial protrusion and are spatially coordinated by F-actin bundles. High-resolution, live-cell microscopy and molecular perturbations of the putative signaling and cytoskeletal processes will be applied to systematically relate perturbations of putative mechanisms regulating the actin cytoskeleton to changes in lamellipodial protrusion dynamics. These studies are expected to show how cell motility is biased by physical cues. Under Objective 2, it is proposed to elucidate the nature of haptotactic bias at the level of adhesion, signaling, and cytoskeletal dynamics. It is hypothesized that F-actin bundles/filopodia direct, and lamellipodia propagate, haptotactic exploration. A related hypothesis is that the haptotactic bias (comparing up- vs. down-gradient) manifests as differences in signaling and/or cytoskeletal dynamics, integrated by adhesions. It is proposed to analyze the dynamics of adhesion, signaling, and cytoskeletal structures during migration on haptotactic gradients generated using microfluidic devices. These studies will characterize leading-edge motility dynamics during haptotaxis, a poorly understood mode of directed migration that is distinct from chemotaxis. Under Objective 3, the proposed work will elucidate the molecular and biophysical determinants of durotaxis and define features that are common or distinct between haptotaxis and durotaxis. Hydrogels with gradients of crosslinking will be generated and integrated with traction force microscopy, a method for measuring local stress in an elastic gel. These studies will test the role of fascin-rich filopodia as sensory organelles guiding durotactic migration. The results will further elucidate the relationship between cell protrusion, focal adhesion dynamics, and traction force exerted by durotaxing cells. The interdisciplinary nature of the project offers a unique environment for the training of two graduate students, who will be engaged in research and an outreach activity fostering science communication.

Project Summary Training in molecular biotechnology is essential for an expanding list of disciplines that have found modern biology?based skills of critical importance in pursuing research goals in areas ranging from biochemistry to chemical engineering to plant biology. Recognizing this, NC State University has created a core education facility that serves campus?wide needs for graduate students requiring laboratory?based training in aspects of modern biology. This not only facilitates completion of the students' dissertation research, but also lays the basis for career opportunities in academic, government, and industrial research settings. Using this campus educational resource as a framework, NC State University proposes a Molecular Biotechnology Training Program (MBTP) to foster graduate-level training in modern biology that will involve students from at least four Colleges and 13 university Departments/Programs. An Executive Committee chaired by the Directors will lead the operation of the MBTP and oversee sub-committees focusing on program elements. The specific objectives of the training program are: 1) Ensure technical proficiency and training in responsible and rigorous science; 2) Provide an educational and professional experience that satisfies graduates' expectations; and 3) Foster robust PhD graduation outcomes. Ten trainee slots are requested that will be augmented by four slots funded from university resources. The program requirements include: a graduate-level, laboratory minor in Molecular Biotechnology; an off?campus, 3-month industrial internship; a capstone biotechnology design course; a course in professional development; courses in research ethics; a course in scientific rigor and reproducibility; an annual research symposium; and a biotechnology ? related outreach project. These requirements are in addition to those associated with the student's particular Department or Program for the doctoral degree. This program will also provide a central focus for faculty of the various disciplines involved in this training effort to seek out new opportunities for formal and informal research collaboration.