Klaus Michael Hahn, PhD - US grants

DESCRIPTION (provide by applicant): This proposal concerns the MAP Kinase Erk 2, a critical regulator of multiple cell behaviors, including motility, apoptosis, and proliferation. We will produce novel fluorescent biosensors to study the activation of Erk in individual, living cells. This will be accomplished by developing new, generally applicable methodologies that can report the dynamics of many different protein behaviors in vivo. Erk activation is controlled both spatially and kinetically, but it has not been possible to understand this level of regulation using biochemical techniques applied to cell populations or fixed cells. The much greater spatial and temporal resolution of the new biosensors will enable us to understand how localized, transient activation of Erk generates rapid changes in cell morphology, and how spatio-temporal control is used to activate Erk differently to produce either motility or apoptosis. We will study the roles of upstream molecules in regulating this previously inaccessible mechanism of Erk regulation, focusing on phosphorylation of the focal adhesion protein vinculin, sumoylation of the upstream kinase MEK, and regulation of EGF receptor trafficking. The fundamental processes controlled by Erk, including motility, apoptosis, and proliferation, are critical to normal homeostasis and relevant to many diseases and immune function.

DESCRIPTION (provided by applicant): New approaches will be developed to examine the dynamics of signaling protein activation in living cells. These methods will be based upon novel, environmentally-sensitive fluorescent dyes designed to respond to protein conformational changes in vivo. Generally applicable methods and dyes will be developed by designing biosensors for the nucleotide state of Cdc42, Rac, and Rho. The new approaches will provide the ability to study endogenous, unlabeled proteins in living cells, to study activation of multiple proteins in the same cell, and to examine proteins buried within multiprotein complexes. The dyes will be very bright and undergo large fluorescence changes, enabling us to use low biosensor concentrations for minimum perturbation of normal cell physiology. The dyes can be used to obtain many images before photobleaching, providing excellent temporal resolution, and quantitation of changes in protein activation level over time in individual cells. Biosensors of Rac and Cdc42 will be imaged in the same cell, to examine how Cdc42 activation of Rac coordinates the activity of the two GTPases for motility. Finally, we will develop means to convert antibody fragments into biosensors, providing access to many protein activities. The new dyes will make it possible to use highly reversible antibody binding, to minimally perturb cell behavior. A monoclonal antibody specific for phosphorylated alpha4 integrin will be used as a model for development of the approach, and to study alpha4 regulation of Rac in directed cell movement.

bioimaging /biomedical imaging; biosensor device; fluorescent dye /probe; imaging /visualization /scanning; technology /technique development

[unreadable] DESCRIPTION (provided by applicant): This proposal presents a new approach to produce fluorescent biosensors, capable of reporting the spatio-temporal dynamics of endogenous protein activation in living cells. The biosensors consist of an 'affinity reagent' which binds specifically to the activated form of the target protein, coupled to a novel dye designed for live cell imaging. The dye undergoes a fluorescence change suitable for ratio imaging when the affinity reagent finds and binds the activated target. Libraries of affinity reagents will be displayed on the surface of phage, enabling selection of biosensors for specific targets using high throughput screening. This approach can produce biosensors for previously inaccessible targets, because it does not rely on identifying naturally occurring protein domains that bind activated target, or known target substrates. The approach is less perturbing than other current biosensor designs both because endogenous proteins can be examined, and because the bright dyes are directly excited for enhanced sensitivity. Phage display can be used to 'fine tune' the affinity and reversibility of the biosensor. Focused libraries will be produced to target a particular type of protein target, thus improving the efficiency of screening and the binding characteristics of the biosensor. Through computation and protein modeling, variable regions will be introduced into naturally occurring domains already targeted to phosphoproteins, and protein scaffolds will be engineered for use as biosensors. Computation and protein modeling will be used to improve the intracellular stability, labeling and expression of the sensors, and to restrict variability to the most productive regions of the structure. We will target two broadly relevant mechanisms of signaling regulation: phosphorylation and intramolecular interaction of autoinhibitory and kinase domains. Biosensors for Src, PAK, JNK, and ERK2 will be targeted as examples of such regulation, and because biosensors of these molecules will enable us to address an important biological question, for a 'real world' test of this new class of biosensor reagents. PAK, JNK, and ERK2 are each on a different, parallel pathway downstream of Src. The spatio-temporal regulation of this signaling network to produce different cellular responses will be examined. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Proteins that control cell behavior can be activated (i.e. phosphorylated, undergo conformational changes) in different locations within the cell or with different kinetics to produce essentially opposing behaviors. Deciphering the spatio-temporal control of signaling is essential to understanding normal cellular homeostasis and its perturbation in many diseases. Although we have made tremendous strides in our ability to study the activity of single proteins in living cells, it remains difficult to characterize the coordination of more than one activity, critically important in rapid morphological changes and in the interaction of different signaling pathways. Biosensors based on environment-sensing dyes, which have valuable advantages over other approaches, offer an opportunity for ready multiplex imaging using equipment available on even basic live cell imaging microscopes. Biosensors based on environment sensing dyes consist of a `recognition element', a small protein fragment that binds only to the activated state of the target protein, coupled to a bright fluorescent dye that changes fluorescence when the biosensor binds its target. This design enables study of endogenous, untagged target proteins, and provides high sensitivity because bright dyes can be directly excited. Structural changes in these dyes to enhance brightness, photostability, water solubility etc. often require compromises, as structural changes affecting one property adversely affect another. We have studied the mechanisms of dye photobleaching and response to solvent polarity, and devised novel approaches to enhance water solubility. Based on this we will design here a new generation of biosensor dyes, shifting their wavelengths to permit multiplex imaging, while maintaining the photophysical features that confer advantages on dye-based biosensors. Using the new dyes, we will build `multiplexing biosensors'to quantify activation of Cdc42 or Src simultaneously with activation of either RhoA or Rac1. These new biosensors will be used to characterize the spatio-temporal coordination of Src and Rho family activation as they generate cytoskeletal changes during macropinocytosis and transendothelial migration. This proposal will develop new methods to study the cellular `circuitry'that determines how a cell responds to its environment. Such circuits consist of complex networks of interacting proteins which can be activated in different positions within a cell to produce different cell behaviors. It is currently difficult to study the activation of more than one such protein in the same cell, especially for rapid events. The new technique enables us to better understand how circuit components interact by enabling visualization of multiple circuit components, even for rapid activation events. After the technique is developed, it will be applied to study cell engulfment of other bodies, a ubiquitous response that plays an important role in many diseases (i.e. immune cells engulf invaders, white blood cells are engulfed by blood vessel walls as they pass through them during inflammation, and cells move similarly across the blood brain barrier). Engulfment requires precise orchestration of protein interactions in time and space, an ideal challenge for the new tools.

DESCRIPTION (provided by applicant): This is a proposal to establish a new paradigm for the study of cellular signal transduction. Most of our current understanding of signal transduction pathways relies on solution biochemical analyses of protein interactions and on bulk measurement of select pathways averaged over large cell populations. The sensitivity of these measurements is severely limited by the averaging of heterogeneous signaling states in asynchronous cell populations. To obtain robust responses strong stimuli are usually applied to homogenize and synchronize the signaling activities across cell populations. However, such stimulation tends to activate pathways far outside the normal range of operation. As a result, the balance between different network branches is distorted and feedback/feedforward interactions between pathway nodes are obfuscated. Hence, for many pathways, our current knowledge lacks the level of detail required to pinpoint key differences in signaling between physiological and pathophysiological conditions. Many pathways, especially those controlling cell morphogenic functions, are regulated over a time scale of seconds and on subcellular length scales. Thus, single cell measurements, e.g. by multi-parameter flow cytometry, tend to be incomplete as well. We argue that the use of emerging biosensor technology, capable of indicating protein activity at resolution levels matching the spatiotemporal regulation of cellular signal transduction, would be key to producing conclusive models of cellular signaling. However, at present biosensor imaging is employed mostly to visualize the activity of an isolated node in a signaling network and with little quantitation of the image dynamics. Coupled transformational changes in biosensor engineering and image analysis are required to provide more than a phenomenological view of one aspect of a pathway. In recognizing this need, we bring here together the expertise of the Hahn lab in biosensor design and live cell imaging and the expertise of the Danuser lab in image analysis of dynamic cellular processes, to establish systematic and quantitative imaging of large signal transduction networks. We propose developments using uniform, engineered biosensor scaffolds to generate new biosensor approaches that enable sensitive multiplex imaging, in living cells, of currently inaccessible network nodes. We also propose developments of computational methods to extract from these data the direction, efficiency, and kinetics of signal transduction between concurrently imaged network nodes and to compile the data from many experiments into a single concise pathway model, despite cell to cell heterogeneity. Hence pathways with tens to hundreds of nodes can be probed despite the spectral constrictions of biosensors, which can be foreseen to image at most 4 - 5 nodes simultaneously (current technology is reaching towards imaging only two activities). Furthermore, the new computational methods will provide the ability to identify feedback/feedforward interactions between observed nodes, determine spatial cues in signal transduction, and predict feedbacks involving as yet unobserved nodes. Central to our approach is exploiting the high sensitivity of the new biosensor technology to probe networks based on the propagation of constitutive signaling fluctuations between nodes. Thus, we can avoid massive stimulation of the imaged network and instead generate a relevant reconstruction of signal transduction at physiological activation levels. Our developments will be driven by investigation of Rho GTPase coordination, a network with many suspected feedback interactions centrally implicated in the regulation of cell migration. PUBLIC HEALTH RELEVANCE: This project will bundle expertise of the Hahn lab at UNC Chapel Hill in biosensor design and live cell imaging with expertise of the Danuser lab at the Scripps Research Institute in image analysis, to produce a transformative approach to studying cellular signal transduction and decision processes. Novel, versatile biosensor technology will enable direct visualization of a wide range of signaling events in living cells. Novel mathematical methods will enable inference from live cell images of the wiring and dynamics of signal transduction through large networks, including the identification of feedback/feedforward interactions between network nodes and of subcellular regions with distinct transduction regimes. In combination, these advances promise a breakthrough in our ability to probe complex, spatially and temporally organized signaling processes in cell models of normal and diseased physiology. Our developments will be driven by investigations of the balance between Rho-family GTPases in multiple nested feedback interactions, a network centrally implicated in the regulation of the mechanics of cell migration.

DESCRIPTION (provided by applicant): The recruitment of neutrophils out of the blood and into surrounding lung tissues is a critical event in pulmonary inflammation. For this to occur, neutrophils must first adhere to cell adhesion molecules (particularly E-selectin and ICAM-1) expressed on the surface of endothelial cells lining blood vessels. These adhesive interactions provide attachment and allow neutrophils to generate traction on the endothelial surface, so that they can migrate over it as they probe for endothelial junctions or other sites where they can cross the endothelial barrier. Engagement of these adhesion molecules also triggers signaling pathways in the endothelial cells that promote transmigration. Of the many signaling pathways that have been identified downstream from E-selectin and ICAM-1, several Rho family GTPases have been implicated in mediating the changes in the cytoskeleton and cell junctions that allow neutrophil passage. Little is known about the co-ordination of the different Rho proteins and how they become activated in response to E-selectin and ICAM-1 ligation. Additionally, it is not known whether tractional forces exerted by neutrophils on these adhesion molecules affect their signaling pathways to promote neutrophil transit across the endothelium. However, these processes are highly co- ordinated and tightly regulated to maximize the benefits of host defense and minimize the injury resulting from endothelial cell damage, particularly in the lungs where edema interferes with gas exchange. To tackle these questions, we propose the following specific aims. Aim 1 will examine the dynamics of activation of key Rho proteins (RhoA, Rac1, RhoG and Cdc42) in response to engagement and crosslinking of E-selectin and ICAM- 1 on lung microvascular endothelial cells. FRET-based biosensors for each Rho GTPase will be used to follow the time and location of their activation. Novel photomanipulation techniques will be used to activate or inhibit specific GTPases at precise times and places to examine how interactions of the GTPases affect neutrophil transmigration. Aim 2 will identify and manipulate guanine nucleotide exchange factors (GEFs) that are downstream of E-selectin and ICAM-1 and that regulate Rho protein activity. Aim 3 will determine whether tension on E-selectin and ICAM-1 initiates activation of Rho proteins. Using 3D force microscopy, we will examine whether mimicking the tension applied by neutrophils on E-selectin and ICAM-1 initiates or modulates signaling to Rho GTPases. Aim 4 will determine how neutrophil migration over endothelial cell surfaces induces tension along and across endothelial cells through E-selectin and ICAM-1, and whether their ligation modulates disassembly of VE-cadherin complexes. Taken together, the proposed studies address important signaling pathways that regulate neutrophil passage across the endothelium during inflammation. They will contribute to an integrated model of endothelial adhesion molecule signaling, incorporating spatial and temporal control that is novel and important to a comprehensive understanding of inflammation. These studies may identify new targets for therapies in the treatment of inflammatory diseases.

Guanine nucleotide exchange factors, or GEFs, activate GTPases to control a broad array of cellular behaviors. They integrate diverse cell stimuli and coordinate the localized dynamics of multiple subcellular systems, including the cytoskeleton, adhesion complexes, transcription machinery, and trafficking compartments. GEFs are capable of such diverse roles because they have complex overlapping specificities for downstream GTPases, with interactions controlled by the shifting localization of both the GEFs and GTPases, and by GEF activation kinetics. To fully understand GEF function and specificity, one must study the spatio-temporal dynamics of GEF activation, and transient GEF interactions with specific targets, in living cells. Here we will make this possible by generating the first fluorescent biosensors of GEF proteins. We are purposefully targeting GEFs representative of different structural classes, to develop generalizable biosensor designs that can open the door to biosensors for many different GEFs. We will explore new biosensor technologies, including genetically encoded biosensors that report activation of endogenous GEFs, and complementary biosensors that can report either the overall activation of a GEF or activation only by specific stimuli. The complementary biosensors will enable us to dissect out the contributions of different upstream pathways activating the same GEF for different purposes in LPA-induced cell motility. Each project in the PPG will use the biosensors, together with computational and modeling approaches developed by Project 2 (Danuser/Hahn), to study the coordinated activation of Dbl family GEFs and Rho family GTPases in different biological contexts.

DESCRIPTION (provided by applicant): Rho family GTPases is ubiquitous molecular switches that control extraordinarily diverse cellular processes. They are activated by guanine nucleotide exchange factors (GEFs) that are roughly 5-fold more numerous than the GTPases themselves and integrate the many cellular inputs controlling GTPase function. GEFs and GTPases form complex networks that are constituted transiently and locally for specific purposes. Biochemical, genetic, molecular, and structural analyses have unraveled a great deal about these critically important pathways, but the most important functional property, their spatio-temporal regulation, can only be fully understood in the context of intact cells. This PPG brings together team members with diverse expertise to develop innovative technologies enabling the study of GEF/GTPase networks in vivo, computational tools to extract network architecture and signaling kinetics from imaging data, and in-depth knowledge of cell behaviors critically dependent on GEF/GTPase dynamics: (Project 1- Hahn) will deliver GEF biosensors based on designs addressing different GEF structural classes. In a collaborative effort with Sondek, expert in GEF structure, different biosensor designs will report GEF activation by specific upstream inputs, and activation of endogenous GEFs. (Project 2- Danuser) will develop the ability to simultaneously image and/or photomanipulate the activity of any pair of GEFs and GTPases, for high resolution studies of GEF/GTPase spatio-temporal coordination. New computational tools will combine data from different experiments to model large networks, and to extract network architecture and signaling kinetics from imaging data. These methods will be tested in studies of complex GEF-GTPase feedback interactions. (Project 3- Hall): This biologically focused project will extend our work to multicellular systems. We will focus on GEF activation in cell-cell junctions and cryptic lamellipodia, and identify GEFs regulating collective migration. (Project 4- Burridge) will address the role of GEF/GTPase netvvorks in mechanotransduction, exploring novel findings regarding the mechanical regulation of RhoA signaling at cell-matrix and cell-cell adhesions during initiation of protrusions, and in the nucleus.

DESCRIPTION (provided by applicant): Abstract Gene activation and silencing via epigenetic modifications is important for determining cell fate and is critical for the proper development of multicellular organisms. Determining the relative importance of various epigenetic markers in specific developmental processes can be challenging because these modifications are often dynamic and vary between cell and tissue types. Common approaches such as a gene knockout of an enzyme required for epigenetic modifications can be used to assess the global importance of the modification, but do not provide a straightforward approach for testing the importance of the modification at specific loci, cell types and developmental time points. Here, we aim to create a general approach for the control of epigenetic modifications that is reversible and can be applied at specific times in development to a specified set of cells. In preliminary studies we have developed a light activatable protein that localizes to the cytoplasm in the dark, but enters the nucleus when the cell is illuminated with blue light. Our hypothesis is that this switch, called LANS for Light Activatable Nuclear Shuttle, can be used to control the activity of proteins that must be in the nucleus to be functional. As proof of concept, we have shown that we can use LANS to control the activity of a transcription factor in yeast with light. Here, we wil explore if light activated nuclear localization can be used to control enzymes and scaffolding proteins required for histone and DNA methylation and acetylation. To target predetermined loci, we will fuse LANS with naturally occurring and engineered DNA binding domains. We will test the LANS switch in mammalian cell culture, and we will explore whether the switch can be used to manipulate cell fate in C. elegans via light mediated control of a histone deacetylase.

DESCRIPTION (provided by applicant): Recent exome sequencing of pancreatic ductal adenocarcinoma (PDAC) determined that aside from the near 100% mutational activation of KRAS, no other oncoproteins are mutationally activated beyond single digit percentages. This has renewed interest in efforts to make undruggable K-Ras druggable. The most promising direction involves inhibitors of K-Ras effector signaling, prompting current clinical evaluation of the Raf-MEK- ERK cascade and the phosphatidylinositol 3-kinase (PI3K)-AKT-mTOR signaling network. However, to date, when applied as monotherapy, or with limited combination approaches, these inhibitors have shown little to no clinical efficacy for RAS mutant cancers. Two key issues contribute to this failure. First, kinome reprogramming mechanisms drive resistance mechanisms that reactivate the pathway downstream of the inhibitor block point. Second, it is clear that cancer cell dependency on mutant K-Ras cannot be attributed to the Raf and PI3K effectors alone, prompting efforts to validate noncanonical effectors for anti-K-Ras drug discovery. We propose that therapeutic targeting of the lesser studied Rac small GTPase effector pathway and its key effector, the Group I PAK serine/threonine kinases will address both issues. To accomplish this, we propose the application of three innovative tools to interrogate the role and mechanism by which the Rac-PAK effector network contributes to K-Ras-driven cancer growth. Specifically, our studies will focus on two immerging themes in signal transduction targeted therapies: (i) dynamic signal reprogramming mechanisms that drive de novo or acquired resistance to limit the therapeutic activity of signaling inhibitors and (ii) the cancer driver function of a signaling protein is strongly dependent on subcellular location. We have assembled a team of researchers with diverse and complementary expertise to (1) define the mechanisms of PAK1 activation by aberrant K-Ras- Rac1 signaling and the driver functions of plasma membrane-associated, cytoplasmic and nuclear PAK1, (2) identify the spatio-temporal phosphorylation events essential for aberrant PAK1 activation and PAK1- dependent cancer growth, (3) profile kinome reprogramming to identify the compensatory protein kinases that overcome PAK1 inhibition to promote cancer cell resistance, and (4) determine if Group I PAK suppression enhances PDAC sensitivity to inhibitors of the Raf or PI3K effector pathways.

ABSTRACT Modern microscopy and image analysis, together with fluorescent probe technology, has evolved to quantify signaling in living cells and animals with seconds and microns resolution. More recently, optogenetics and chemogenetics have made it possible to control signaling in vivo, and thereby explore causal relationships among signaling molecules as they are regulated by spatio-temporal dynamics. We propose here to combine protein visualization and control in the same cell, for unprecedented quantitative accuracy in studying how Rho GTPase signals are coordinated by feed-back and feed-forward relationships. To generate proteins controlled by light or small molecules, we will use novel approaches that provide ready access to many different structures and minimize perturbation of living cells. These include dye-based biosensors of endogenous protein conformation, engineered allosteric control for inhibition or activation by light, and the use of photoresponsive protein analogs that can serve as substitutes for endogenous proteins. We will study ?frustrated phagocytosis?, a system where the complex dynamics driving phagocytosis are preserved, but are restricted to two dimensions and occur in precise geometries generated by patterned substrates. We will examine communication between spatially restricted zones of signaling using single molecule microscopy of protein conformational change. Precise control of activation gradients, kinetics and localization will be used to inform mathematical models examining how precisely segregated signaling domains are maintained. In a second project, we will work with our collaborators Eric Betzig and Leong Chew of Janelia Farm to adapt biosensor and optogenetic technologies to lattice light sheet microscopy, for visualization and control of the complex morphological changes megakaryocytes undergo as they produce platelets. There our ultimate goal will be optogenetic modification of signaling to enhance platelet production. We will focus on enabling technologies to generate minimally perturbing biosensors and optogenetic tools that can be applied by other researchers in a wide range of fields.

PROJECT SUMMARY Megakaryocytes (MKs) are specialized blood cells that produce all of the platelets found in the human body. They reside primarily in the bone marrow, where they undergo endomitosis, synthesis of granules, membrane invagination, and finally proplatelet formation (PPF), a process characterized by dramatic changes to the cytoskeleton. Impaired platelet production leads to thrombocytopenia (low platelet count), which can cause life- threatening bleeding complications. Treating thrombocytopenia requires millions of platelet transfusions annually in the US alone. The high demand for platelet concentrates presents a significant problem in transfusion medicine, as platelets have a short shelf-life so must be supplied frequently by volunteer donors. Multifaceted approaches are under investigation to address this problem by producing platelets in vitro, but the low efficiency of PPF and the limited quality of the platelets produced remain major obstacles. The overarching goal of this proposal is to elucidate the mechanism of PPF, and thereby develop strategies for carefully timed perturbations of Rap and/or Rho GTPase activity as a means to significantly improve PPF efficiency and the quality of the platelet product. Rho-family GTPases are master regulators of the cytoskeleton that control morphodynamics through localized, precisely timed activation events. It has recently become clear that they are important regulators of PPF, but very little is known about their spatio-temporal dynamics or coordination. In addition, we here present the first direct proof that PPF also requires signaling by the Ras-family GTPases Rap1A and Rap1B; mice deficient in both Rap1 isoforms in MKs show significant thrombocytopenia and a near complete loss of PPF in vitro. To elucidate how GTPases orchestrate the complex morphological changes of PPF, we have assembled an interdisciplinary team of investigators with expert knowledge in platelet/megakaryocyte biology, small GTPase signaling, and the design of molecules to visualize and photo- manipulate GTPase activity in living cells. We will define the contribution of Rap1 isoforms and their regulators to MK development and PPF (Aim 1), establish GTPase ?activity signatures? and network connections critical to PPF (Aims 2 & 3), and establish proof-of-principle that precisely targeted perturbation of GTPase activity is a viable strategy to optimize in vitro platelet production (Aim 3). Successful completion of these studies will provide critical novel insights into the cell biology of PPF and may have important implications for improving transfusion medicine.