Tobias Meyer - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The goal of the proposed research is to understand the signal transduction pathway mediated by receptor-triggered calcium spiking. Calcium spikes or oscillations are repetitive transient increases in cytosolic calcium concentration that are induced by constant extracellular stimuli such as hormones or neurotransmitters. The intensity of the stimulus often determines the spike frequency. We will study how calcium spikes are generated and whether the spike frequency can control the activity of calcium-dependent enzymes. Calcium/calmodulin dependent protein kinase II (CaM kinase) is chosen as a model enzyme because of its ubiquitous distribution and its involvement in important cell functions such as the regulation of ion-channels, carbohydrate metabolism, cytoskeletal organization and neurotransmitter release. The specific aims of the proposed research are based on our preliminary findings that different mechanisms generate localized and long-ranged calcium spikes and that the activation of CaM kinase by calcium spikes is associated with a calmodulin trapping process. Calmodulin trapping is mediated by autophosphorylation of CaM kinase and leads to a 1000-fold increase in calmodulin binding affinity of CaM kinase. The following two hypothesis will be tested: (i) Localized subcellular calcium spikes are generated by calcium-gated opening of calcium channels and long-ranged spikes are mediated by IP3 diffusion and by coupling of IP3 and calcium concentration. This hypothesis is tested by measuring calcium and IP3 diffusion in PC12 cells using confocal calcium imaging. The gating of IP3- receptor and ryanodine receptor calcium channels by calcium will be investigated by 45Ca flux studies. (ii) The activation of CaM kinase by calcium spikes is enhanced by calmodulin trapping, a process that may be involved in the decoding of the frequency of calcium spiking. This hypothesis is tested by investigating the activation of recombinant and rat brain CaM kinase by fluorescence anisotropy measurements of calmodulin trapping, by photobleaching measurements of calmodulin diffusion and by peptide mapping of autophosphorylated CaM kinase. The calcium and calmodulin dependence of these processes are of importance for the understanding of CaM kinase activation by calcium spikes. The proposed study of calcium-mediated signal transduction may contribute to the understanding of a number of disorders such as hypertension which can be treated with, calcium channel blockers or manic depressive illness which responds to lithium, an inhibitor of the inositolphosphate metabolism.

DESCRIPTION: Biochemical studies have shown that the enzymatic activity of PKC isoforms can be regulated by diacylglycerol (DG), free fatty acids (FFA), ceramide and Ca2+. At the same time, these messengers control the translocation of PKC to isoform specific cellular sites. The precise cellular localization of PKC is important for the specificity of substrate phosphorylation. The key structural determinants for Ca2+ and lipid messenger mediated PKC translocation are thought to reside in Cys- and C2-domains. More recently, these domains were also proposed to be essential for the function of a large number of signaling proteins including Raf, GAP, PLC, Vav, myosin, Unc-13 and synaptotagmin. The goal of the proposed research is to understand Cys- and C2-domains in the cellular context. This study involves the hypothesis that Cys-domains target signaling proteins to different sites of action by binding DG, FFA or ceramide and C2-domains by binding Ca2+. It has been demonstrated that individual Cys-domains tagged with green fluorescent protein (GFP) can be used to study receptor-mediated translocation processes and to measure binding parameters in intact cells. These measurements were made possible by (1) the development of an efficient RNA transfection method for mammalian cells, (2) identification of a GFP mutant that is ideally suited for use as a fusion tag, and (3) analysis procedures for measuring diffusion and binding parameters of GFP-tagged protein domains in intact cells. The use of the preceding in vivo approaches to understand distinct roles of Cys- and C2-domains from different proteins is proposed. The localization and binding parameters of individual Cys- and C2-domains in response to different stimuli will be investigated. The role of CysCysC2 domain motifs of PKC in isoform specific localization will be examined in parallel experiments. The results from these measurements will be integrated into a model for the role of lipid messengers and Ca2+ in Cys- and C2-domain function.

DESCRIPTION Calcium signals have important roles in many neuronal processes, including the release of neurotransmitter, gene expression as well as different aspects of neuronal plasticity. However, the mechanisms by which different intracellular calcium release mechanisms and different calcium influx pathways can selectively activate down-stream targets are not yet understood. Localization of calcium signals and the localized activation of distinct calcium effectors are thought to be important means through which specificity can be achieved. The objective of this grant is to define, in living cells, to which extent calcium signals are localized and to determine how localized calcium signals are used for the specific activation of calmodulin and of Ca2+/calmodulin-dependent protein kinase II (CaMkinase). A better understanding of the pleiotropic functions of calcium signals is of fundamental importance for a large number of neuronal diseases. During the last funding period, the applicants have developed several new fluorescent methods to attack their objective. In particular, they have developed an efficient"microporation" technique to introduce RNA and other macromolecules into different neuronal and non-neuronal cell models. RNA transfection has significant advantages over DNA transfection for the rapid and efficient expression of GFP-tagged proteins in different cell types. The applicants will study the local control of depolarization-induced calcium influx and local IP3-gated calcium release by using newly developed localized calcium indicators, localized markers for calcium stores as well as GFP-based fluorescent probes to measure calmodulin binding. Specifically, they will investigate: 1. whether localized calcium signals are an essential part of calcium-mediated signal transduction processes, 2. whether calmodulin is a local mediator of signaling transduction and 3. which mechanism is responsible for the localization of CaMkinase to different cellular sites. The results from these experiments will be integrated into a model of localized neuronal signal transduction mediated by calcium, calmodulin and CaMkinase.

Cancer-relevant signal transduction involves a large number of signaling proteins with many parallel steps and interconnected feedback mechanisms. These properties of signal transduction processes cannot readily be measured by current techniques, which limits researchers ability to validate each of the more than 10,000 human signaling proteins as potential drug targets for cancer therapy. Over the last several years, my laboratory has made three separate developments that can now be integrated into a technology to systematically characterize complex signal transduction networks. In this approach, cell arrays will be made for simultaneously monitoring a large number of signaling processes. The proposed Evanescent Wave Cell Array Technology (ECAT) incorporates: 1) a microvolume electroporation method for RNA transfection, 2) a set of single cell GFP-tagged biosensors (such as Akt, PKC, Grb-2, GAP and Raf isoforms) that can monitor diverse signaling processes by their plasma membrane translocation or dissociation, and 3) an evanescent wave microscope setup to quantitatively monitor these plasma membrane translocation and dissociation events. In Phase I of the project, we will develop and test a 4 x 3 prototype cell array and, in Phase II, we will expand the cell array to 15 x 10. These two arrays will be used to simultaneously monitor different receptor or transformation induced signaling events in each of the 12, or 150, separate cell array segments on the same glass slide. While phase I includes a test of principle using existing biosensors and dominant negative and constitutively active interference proteins, Phase II will develop a library of such fluorescent translocation biosensors. Furthermore, we will develop two libraries of interference proteins by mutating a large number of serine/threonine kinases and small GTP-binding proteins into constitutively active and dominant negative constructs. As a test of the usefulness and the limitations of the ECAT method, we will determine the role of each member of these two interference libraries by testing them in the context of different receptor stimuli using the existing and newly developed fluorescent biosensors. Overall, this proposal will provide a new approach to signal transduction research and will provide a technological platform for the validation of cancer drug targets and the advancement of drug discovery.

DESCRIPTION (provided by applicant): The long term goal of the proposed project is to understand how Ca2+ and lipid second messenger signals are used by neurons and astrocytes to regulate signaling responses. The lipid second messengers phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-triphosphate (PIP3), diacyiglycerol (DAG) and phosphatidic acid (PA) as well as Ca2+ signals are connected by cross-talk and feedback loops and can be considered as key components of a complex "signaling network." The understanding of how different Ca2+ and lipid second messenger signaling systems work in neurons and astrocytes will likely lead to the identification of new classes of drug targets for different brain diseases. Our laboratory has developed a fluorescent microscopy strategy for monitoring local Ca2+ and lipid second messenger signals and we are now in a good position to ask questions on how and where positive and negative feedback loops and cross-talk is operational within the two cell types. We will be using lipid binding domains conjugated with the GFP color variants CFP and YFP as translocation biosensors and total internal reflection fluorescence (TIRF) and confocal microscopy to simultaneously monitor local changes in Ca2+ and lipid second messenger concentration in astrocytes and in the dendrites of hippocampal neurons as a function of time. Using these strategies, we will 1. test the model that glutamate stimulated calcium waves and oscillations in astrocytes require DAG and investigatorP2 oscillations, 2. use a new wide-field TIRF system that we developed to test the hypothesis that crosstalk and positive and negative feedback processes between Ca2+ and lipid second messengers define an interconnected second messenger signaling network in astrocytes, 3. test the hypothesis of a "geometric delay mechanism" and other spatial and temporal control mechanisms that are thought to regulate translocating signaling proteins in the dendrites of hippocampal neurons, and 4. investigate local PIP3 and other positive feedback mechanisms and determine whether localized second messenger signals occur at postsynaptic terminals. Furthermore, we will test if such local signals define where and when dendritic branches and synapses are formed. The overall goal of these aims is to obtain a model that describes the key features of the Ca2+ and lipid second messenger signaling networks of astrocytes and neurons and their regulation by different electrical and receptor stimuli.

enzyme activity; calmodulin; calmodulin dependent protein kinase; neurotransmitters; isozymes; neurotransmitter transport; neuroanatomy; enzyme inhibitors; hippocampus; RNA splicing; synapsins; phosphorylation; calcineurin; potassium channel; in situ hybridization; Chelonia; laboratory rat; laboratory mouse;

DESCRIPTION (provided by applicant): Single cell and collective cell migration are fundamental processes that enable human development, immune responses and wound healing while also playing a key function in cancer progression. Our overall goal in this project is to understand the fundamental molecular and cellular mechanisms of how single cells and groups of human cells migrate. We will be focusing on two in vitro cell models, human umbilical vasculature endothelial cells (HUVEC) to study collective directed migration in response to growth factor and a cell model for neutrophils, differentiated HL-60 cells, to investigate single cell chemotaxis. These model systems were chosen since they allow us to use automated imaging of important functional migration parameters to explore the role of a large numbers of migration related genes using siRNAs knockdown and to perform a phenotypic classification. Our project is taking a systems approach, using small interference RNAs to perturb different parts of the cell migration machinery as well as computational modeling approaches. We are also employing rapid chemical perturbation methods of the PI3K and other pathways that our laboratory developed as well as high resolution fluorescent imaging using biosensors and markers for cell migration. By perturbing and monitoring local signaling events, we will be exploring different hypotheses of how cells polarize, steer their front and migrate with the goal to generate a quantitative molecular and mechanistic model for directed migration in these cell models. Insights into new regulators of migration and the roles that different regulators play in the overall endothelial and leukocyte migration processes will likely lead to the identification of new drug targets relevant for vasculature, immune diseases or cancer. PUBLIC HEALTH RELEVANCE: Single cell and collective cell migration are fundamental processes that enable human development, immune responses and wound healing while also playing a necessary function in cancer progression. Our overall goal in this grant is to understand the key molecular and cellular mechanisms of how single cells and groups of human cells migrate.

DESCRIPTION (provided by applicant): Quantitative and chemical approaches to biomedical science are becoming increasingly important in the post-genomic era. Through the completion of multiple sequencing efforts, the scientific community has a panoramic view of nature's genetic 'parts', and it is now challenged with acquiring a functional knowledge of this complex machinery. Achieving this goal will require innovative approaches to biomedical research, and our next generation of scientists will therefore need training that seamlessly integrates quantitative, chemical, and biological methods. This application describes plans by Stanford University to create the Quantitative Chemical Biology (QCB) Program, an interschool initiative that will train predoctoral and postdoctoral students in interdisciplinary research. The QCB Program will be organized around several modules that include an interdepartmental graduate training program, a QCB Fellows program, specialized coursework, seminars and symposia, and two QCB faculty-directed facilities that will support post-genomic research projects. These activities and resources will establish a new paradigm for training young scientists, while synergizing with current programs at Stanford, such as the Bio-X initiative for multidisciplinary research. The University's ultimate goal is the creation of an interdepartmental, degree-granting QCB Program. In the interim, the Program will target students admitted to the Departments of Molecular Pharmacology and Chemistry who demonstrate an interest in interdisciplinary research, providing them with unrestricted access to nineteen QCB-affiliated laboratories in six academic departments. The QCB Program will also sponsor postdoctoral fellows who work collaboratively with two or more QCB-affiliated faculty members. Through these mechanisms, the QCB Training Program will support a diverse community that fosters interdisciplinary research and collaborative discovery, preparing future scientists for a scientific landscape without boundaries.

DESCRIPTION (provided by applicant): The objective of this proposal is to create a new high-throughput screening (HTS) facility at Stanford University. Recent advances in genetics and chemical synthesis have fostered novel strategies for biomedical research, and high-throughput liquid-handling and data acquisition instrumentation will enable Stanford faculty and students to exploit these new scientific approaches. For example, the sequencing of entire genomes and the development of techniques to selectively alter gene expression now permit the rapid functional evaluation of essentially any gene product. The screening of natural products and synthetic chemicals for biologically active compounds is also emerging as a powerful complementary approach for deciphering biological processes. The proposed Biomek FX robotic liquid-handling workstation and EnVision multi-label microplate reader will constitute the primary instrumentation systems of this facility, and with this equipment, Stanford researchers will be able to conduct systematic and comprehensive screens of biological systems involving genetic and/or chemical perturbations. Possible approaches include the use of: (1) cDNA libraries for in vivo or in vitro protein expression; (2) siRNA libraries for targeted 'knock-downs' of protein expression; and (3) chemical libraries for the identification of small molecule modulators of specific biological processes. The proposed HTS facility will be developed and managed by the Department of Molecular Pharmacology, and the research projects specifically outlined in this proposal involve studies by its faculty members that pertain to cardiac hypertrophy, neuronal function, drug delivery strategies, embryonic signaling pathways, and mechanisms of oncogenesis. It should be emphasized, however, that these investigations are merely representative of the diverse research activities that the HTS facility will support. Available to the entire Stanford research community and its neighboring institutions, the proposed instrumentation will broadly advance our molecular and cellular understanding of human health and disease by promoting the use of genome-wide approaches in the biomedical sciences and the advancement of chemical biology research.

DESCRIPTION (provided by applicant): Glucose uptake by muscle and fat tissue is a primary mechanism for energy storage and represents a main control mechanism for glucose concentration in the circulation. A detailed understanding of the intracellular signaling system that mediates insulin-triggered translocation of the glucose transporter (GLUT4) will likely provide new findings for attacking diseases such as diabetes and obesity. The main goal of the proposal is to develop a kinetic screen to identify adipocyte regulatory proteins that control dynamic parameters for PIP3 signaling and for GLUT4 translocation and endocytosis. We will establish a wide-field TIRF system to measure the translocation and endocytosis rates of GLUT4 transporters, as well as the generation and degradation rate of PI3P lipids, in individual cells under 12 different perturbation conditions in parallel. We will create an RNAi library to target the approximately 800 adipocyte signaling/secretory proteins and a library of 800 expressed dominant negative and constitutively active expression constructs for complementary pertubation studies. In contrast to earlier approaches in which screens were based on end-point assays in fixed cells, the live-cell strategy proposed here focuses instead on the identification of adipocyte signaling and secretory proteins that control important kinetic parameters in insulin triggered PIP3 signaling as well as in GLUT4 translocation. In the second part, we will execute, analyze and interpret a screen of the insulin-PIP3-GLUT4 signaling network with this new approach and will also perform a screen of cross-talk between insulin and TNF-alpha signaling. These screens will be used to identify new players in the insulin-GLUT4 signaling network and to gain a better understanding of kinetic control mechanisms. The project will also provide a test case for a kinetic screening strategy and a test case for a combined screen using RNAi, as well as DN and CA expression constructs.

Quantitative and chemical approaches to biomedical science are becoming increasingly important in the post-genomic era. Through the completion of multiple sequencing efforts, the scientific community has a panoramic view of nature's genetic 'parts', and it is now challenged with acquiring a functional knowledge of this complex machinery. Achieving this goal will require innovative approaches to biomedical research, and our next generation of scientists will therefore need training that seamlessly integrates quantitative, chemical, and biological methods. This application describes plans by Stanford University to create the Quantitative Chemical Biology (QCB) Program, an interschool initiative that will train predoctoral and postdoctoral students in interdisciplinary research. The QCB Program will be organized around several modules that include an interdepartmental graduate training program, a QCB Fellows program, specialized coursework, seminars and symposia, and two QCB faculty-directed facilities that will support post-genomic research projects. These activities and resources will establish a new paradigm for training young scientists, while synergizing with current programs at Stanford, such as the Bio-X initiative for multidisciplinary research. The University's ultimate goal is the creation of an interdepartmental, degree-granting QCB Program. In the interim, the Program will target students admitted to the Departments of Molecular Pharmacology and Chemistry who demonstrate an interest in interdisciplinary research, providing them with unrestricted access to nineteen QCB-affiliated laboratories in six academic departments. The QCB Program will also sponsor postdoctoral fellows who work collaboratively with two or more QCB-affiliated faculty members. Through these mechanisms, the QCB Training Program will support a diverse community that fosters interdisciplinary research and collaborative discovery, preparing future scientists for a scientific landscape without boundaries.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The development of human cancer is a multistep process in which future cancer cells acquire mutant alleles of proto-oncogenes, tumor-suppressor genes, and other regulatory genes. Many or most of these genes are signaling related proteins and we are focusing here on the design principles of signaling networks that control the cancer related processes of proliferation, migration and endocytosis. We will test the key questions of 1) whether these cancer related signaling networks have a modular structure and 2) whether cancer cells have missing or added signaling modules that cannot be observed in normal cells. [unreadable] [unreadable] We have made significant advances to answer these questions by developing a method to create 2304 in vitro Dicer generated siRNAs against a core set of human signaling proteins. Using these siRNAs, we have already discovered the function of STIM1, a Ca2+ sensor in the ER lumen that controls Ca2+ influx into cells, and which also acts as a tumor suppressor. We have also developed quantitative microscopy- based measurement tools to track signaling processes and cell functions. Phase 1 of the proposal will demonstrate the overall feasibility of using a microscopy-based siRNA strategy to investigate multiple cancer-related cell functions. Phase 2 will address the questions posed above using an expanded set of 6000 siRNAs and a focus on six cell-types, 3 non-transformed and three breast cancer epithelial cell lines. We will screen to identify signaling siRNAs that alter proliferation, cell migration or endocytosis and then utilize follow-up studies with live cell biosensors that we developed to measure the duration of different cell cycle phases, as well as migration velocity and other kinetic parameters. We will then link genes that alter these cell functions to a subset of cancer-relevant signaling pathways using secondary siRNA screens. [unreadable] [unreadable] Based on these functional and signaling datasets, we will create a modular map of signaling systems using clustering methods. We will experimentally test the predictive power of modular maps using perturbations with pairs of effective siRNAs. We will show if and how modularity in a signaling system can be used to predict how cell functions can be manipulated using combinations of siRNAs and learn if and what distinguishing features exist that define modularity of signaling systems in cancer versus non-cancer cells. This will likely lead to the identification of new cancer drug targets and new therapeutic strategies. [unreadable] [unreadable] [unreadable] [unreadable]

Quantitative and chemical approaches to biomedical science are becoming increasingly important in the post-genomic era. Through the completion of multiple sequencing efforts, the scientific community has a panoramic view of nature's genetic 'parts', and it is now challenged with acquiring a functional knowledge of this complex machinery. Achieving this goal will require innovative approaches to biomedical research, and our next generation of scientists will therefore need training that seamlessly integrates quantitative, chemical, and biological methods. This application describes plans by Stanford University to create the Quantitative Chemical Biology (QCB) Program, an interschool initiative that will train predoctoral and postdoctoral students in interdisciplinary research. The QCB Program will be organized around several modules that include an interdepartmental graduate training program, a QCB Fellows program, specialized coursework, seminars and symposia, and two QCB faculty-directed facilities that will support post-genomic research projects. These activities and resources will establish a new paradigm for training young scientists, while synergizing with current programs at Stanford, such as the Bio-X initiative for multidisciplinary research. The University's ultimate goal is the creation of an interdepartmental, degree-granting QCB Program. In the interim, the Program will target students admitted to the Departments of Molecular Pharmacology and Chemistry who demonstrate an interest in interdisciplinary research, providing them with unrestricted access to nineteen QCB-affiliated laboratories in six academic departments. The QCB Program will also sponsor postdoctoral fellows who work collaboratively with two or more QCB-affiliated faculty members. Through these mechanisms, the QCB Training Program will support a diverse community that fosters interdisciplinary research and collaborative discovery, preparing future scientists for a scientific landscape without boundaries.

Receptor-triggered cytosolic calcium (Ca2+) signals are ubiquitously used in signal transduction. In most cells, an initial Ca2+ signals can be induced by receptor-mediated activation of phospholipase C, the production of the second messenger IPS, and the release of Ca2+ from ER Ca2+-stores. Nevertheless, this transient IPS-mediated Ca2+-signal is typically not sufficient to induce long-term regulation of transcription, secretion and other important cellular processes. In addition, cell activation requires a Ca2+-store depletion triggered opening of plasma membrane Ca2+ channels. Our grant investigates this important but poorly understood "store-operated Ca2+ signaling pathway" (SOC). Previous experimental evidence suggested that this ubiquitous signaling pathway begins in the lumen of the ER where an unkown Ca2+-sensor would monitor the loss of ER Ca2+ and then would signal to the plasma membrane to open more Ca2+-channels. During the last funding period, we discovered that STIM1 and STIM2 proteins function as Ca2+-sensors in the ER and that they are necessary and sufficient for signaling to the plasma membrane and opening SOC Ca2+-influx channels. This is the first molecular component that has been identified in this important cell signaling pathway. Our grant is focusing on these STIM proteins and on other regulators of the SOC signaling pathway that we have identified in subsequent siRNA screens using a human cell line. We will be using calcium imaging, fluorescence microscopy of protein distribution, biochemical approaches and mutant constructs of STIM and other identified regulators to dissect the mechanism of action of STIM proteins and other identified regulators in the regulation of SOC. We will also investigate the broader question of how this SOC signaling pathway participates in the balance of plasma membrane Ca2+-influx and Ca2+-export. Finally, we will develop a quantitative model of the global Ca2+ signaling network that integrates this new signaling pathway with other Ca2+-regulatory mechanisms. We will test our model by using siRNA perturbations and will compare the model predictions against measurements of the resulting changes in Ca2+-f luxes. Since this SOC influx pathway has been shown to be essential for many cellular processes such as T- and B-cell activation, mast cell activation and osteoclast differentiation, it is likely that our findings of new signaling components and regulatory processes in the SOC pathway will offer opportunities for developing novel drugs that improve pathological conditions such as transplant rejection, inflammation, allergies and autoimmune diseases.

DESCRIPTION (provided by applicant): The goal of our grant is to understand the molecular mechanisms by which the lipid second messengers phosphoinositol(4,5)bisphosphate (PIP2) and phosphoinositol(3,4,5)trisphosphate (PIPS) cooperate with small GTPases to promote the axonal-dendritic polarization of neurons. We developed a quantitative working model for axonal-dendritic polarization that involves PIPS and Ras family small GTPases and we will test this model using primary cultured rat hippocampal neurons as a model system. These neurons form a single axon from an initially unpolarized precursor cell and can be used as a prototype neuronal self-polarization process. Our proposed study builds on advances that we made during the last funding period: We used a genomic approach to characterize how small GTPases induce morphology changes (Heo et a!., 2003) and how they are targeted to the plasma membrane (Fivaz et al., 2005;Heo et al., 2006). We also discovered a "geometric attraction" principles that causes a delayed enhancement of plasma membrane targeted proteins in neurites (Craske et al., 2005). We further showed that local PIPS signals are linked to local lamellipod extension, providing a driving mechanism for growth cone extension (Arrieumerlou et al, 2005). In order to overcome a key technical limitation, we developed a chemically-induced enzyme activation method that allows us to rapidly manipulate PIP2 and PIPS lipids as well as small GTPases in the proposed study (Inoue et al., 2005, Shu et al., 2006;Heo et al., 2006). Finally, using a theoretical approach, we found evidence that robust cell polarization requires two interlinked positive feedback loops (Brandman et al., 2005). Specifically, our project makes use of site-directed mutagenesis, live-cell tracking of signaling proteins as well as fluorescence resonance energy transfer measurements to investigate PIP2, PIPS and small GTPase signaling during neuronal polarization. The results form our proposed study will increase our understanding of the molecular mechanisms by which phosphoinositides reversibly target signaling proteins with polybasic clusters and pleckstrin homology (PH)-domains to the plasma membrane and how PIPS signals and small GTPases become polarized when a single axons is specified. Using experimental and quantitative modeling approaches, we will focus on two processes that we discovered: a growth cone restricted positive feedback between HRas and PIPS as well as a directed transport process that enriches HRas in growth cones over the somatodendritic region. We will bring these results together to generate a comprehensive quantitative model of the neuronal polarization process. Neuronal polarization is a fundamental process in the conversion of unpolarized precursor cells to functioning neurons and an understanding of how neurons reliably generate a single axon will provide insights that may advance the development of cures for neurodegenerative diseases.

DESCRIPTION (provided by applicant): The goal of the proposed work is to systematically explore whether and how proteins that sense and shape the curvature of plasma membranes are responsible for building the intricate dendritic and axonal arbors that distinguish neurons from other cell types. The formation of complex 3-dimensional branched membrane structures is one of the most fundamental properties of neurons that enable them to transmit information between neurons and from neurons to other cell types. The ability of selected proteins to sense membrane curvature during this differentiation process is important as defects in proteins, such as Oligophrenin and srGAP2, that can bind to and shape lipid membranes cause neurodegenerative diseases. Our proposal aims to develop and execute a scalable experimental strategy to understand the process of arbor formation by focusing on a family of Bar domain containing proteins that are known from in vitro studies to be able to bind to and shape curved membranes. We will systematically investigate their function in generating the branched extended plasma membrane architecture of neurons. Currently available in vitro assays and structural studies of proteins with membrane binding domains can determine the radius of the membrane curvature that results from the formation of oligomers by curvature sensing proteins. Using this approach, proteins have been identified that sense and shape membranes with positive and negative curvatures. Nevertheless, it is difficult from these assays to know to which curved intracellular membranes these proteins may bind, and if or how they act dynamically to generate distinct types of curved plasma membranes in a living cell. We developed a novel assay to investigate curvature dependent processes that is based on fabricated nanostructures that trigger plasma membrane curvature in living cells. Specifically, our project will deliver a new scalable assay based on these nanostructures that allows one to measure in living cells the intracellular membrane localization and the curvature preference as well as the dynamic assembly, disassembly and exchange rate of curvature sensing membrane binding proteins. Our initial studies already identified and characterized a key regulator that binds to positively curved plasma membranes and is critically involved in controlling neuronal architecture. We have combined this approach with parallel high-throughput live-cell imaging and automated image analysis of cultured hippocampal neurons that enables us to systematically analyze the cellular roles of these same Bar domain binding proteins in controlling the neuronal architecture. At the center of our work is the development of this synergistic dual experimental approach that can ultimately be used as an unbiased and systematic platform to investigate the neuronal roles of a large number of putative neuronal membrane binding proteins. Together, our project will provide a molecular framework to understand the program used by neurons to create the vast repertoire of different neuronal architectures. PUBLIC HEALTH RELEVANCE: Project Narrative The long term goal of the project is to understand the cellular mechanism of how neurons build the intricate dendritic and axonal branch structure that distinguishes them from other cell types. We will develop and execute a scalable experimental strategy to understand how large families of proteins that sense and change the curvature of the plasma membrane synergistically control the neuronal architecture.

PROJECT SUMMARY/ABSTRACT This proposal requests funds to purchase a cutting edge Wako CV7000 High-Content Confocal Imaging system to be shared by eleven NIH-funded Principal Investigators at Stanford University School of Medicine. The new system has two purposes: it replaces a leased automated epifluorescence imaging system ImageXpress micro XL that is heavily used and it adds a critical new capability to the Stanford campus to do automated confocal imaging. The laboratories of Tobias Meyer, James Ferrell, Kang Shen, Thomas Wandless, Steven Artandi, Marius Wernig, Karlene Cimprich, Thomas Quertermous, Michael Lin, Rajat Rohatgi, and Matthew Scott are located in close proximity and share a common interest in microscopy-based dynamic analysis of cellular systems to uncover and understand the cell cycle, cell differentiation, cell damage, cell migration and other dynamic cellular processes. Central to the research in our laboratories is the use of long-term, live-cell, fluorescence microscopy to perturb, monitor and quantify dynamic changes in cellular processes. Specifically, we use methods that require automated tracking of cells, monitoring of changes in cell shape and cell polarization, of organelle distribution and of chromatin remodeling and of changes in local signaling. As part of our projects, we also need to visualize and quantify induced expression and degradation of regulatory and marker proteins as well as the movement of signaling proteins and biosensors to and from the nucleus, plasma membrane and other cellular compartments. Last year, when the manufacturer discontinued all service support for our two heavily-used, 12- year old automated ImageXpress imaging systems, we were able to obtain a one-time lease on a newer-model ImageXpress Micro XL until August 2013. This loaner system is heavily used every day 24/7 since many of our experiments take 1-3 days of continuous multi-well imaging. The advanced high-throughput, live-cell imaging capabilities of the Wako CV7000 will replace this loaned ImageXpress Micro XL and, with the critical addition of confocal capabilities, also significantly enhance the NIH-supported research in our laboratories by providing a shared resource to address our increasing microscopy needs.

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is employed in controlling cell fates in most developing tissues and organs, as well as during many regeneration events. Defects in Hh signaling lead to birth defects and cancer. Many mechanistic mysteries remain regarding how an Hh signal is transduced. Using high-throughput RNAi screening, we identified Neuropilins (Nrp) 1 and 2 as novel, specific regulators of vertebrate Hh signaling. Nrps are single-pass transmembrane proteins implicated in the reception of a diverse set of secreted ligands, including Semaphorins and VEGF165 and in cell adhesion and cell migration. In fibroblasts the inhibition of Hh signal transduction resulting from blocking Nrps is as strong as the effect of blocking cilia formation or blocking Smoothened function. Conversely, over-production of either Nrp sensitizes cells to Hh signals. New components of the Hh pathway are uncovered infrequently; the Nrps were probably missed due to their partial redundancy. Our discovery of two proteins whose functions are required by this important morphogenic pathway has the potential to bring fundamental changes to current models of Hh signaling and to enlarge the understanding of Nrp functions in other signaling pathways. Aim 1. Determine the mechanism by which Nrps regulate Hh signal transduction. Nrps could influence Hh signal transduction by directly associating with known Hh pathway components, or mediating other signals that converge with Hh transduction, or by altering cell properties or processes that are required for Hh transduction. We will investigate each of these possibilities by determining which steps in Hh signaling are affected, whether cell adhesion or migration changes are involved in the effect of Nrps upon Hh signaling, and what proteins interact directly with Nrps. Aim 2. Determine which domains of Nrp are needed to support Hh signaling, and whether known Nrp co-receptors, ligands, or effector molecules are capable of Hh pathway cross- regulation. We will investigate which domains contribute to Hh signal transduction in two ways: engineered domain deletions, and a high-throughput screen for point mutations that interfere with Nrp support of Hh signal transduction. Nrps transduce VEGF and Semaphorin signals, acting as co-receptors for VEGF receptors and Plexins, respectively. We will test whether VEGF, VEGF receptor, Plexin receptors, or Semaphorins are involved in the effect of Nrps upon Hh signal transduction. Aim 3. Investigate how Nrps influence Hh- dependent development and tumorigenesis. Using mice that carry mutations in both Nrp genes, we will control temporal and tissue-specific removal of Nrp functions to test their involvement in several Hh-dependent developmental processes in vivo. Using newly created lentiviruses, which encode specific inhibiting RNAs that block the nrp genes, we will infect primary cultures of Hh-responsive cells and monitor effects on Hh target gene expression.

? DESCRIPTION (provided by applicant): Diseases associated with cancer, immune responses, wound healing, and neurodegeneration are often caused by insufficient or excessive proliferation of particular human cell types. Under normal conditions, such cells have to robustly control if and when they proliferate to maintain and repair functioning tissues. While little is known about how cells exit the cell cycle, the decision to enter the cell cycle is often referred t as a restriction point, a point where growth factors can be removed and cells still enter and complete the cell cycle. However, our recent single cell data analysis suggests that this fundamentally important decision is made by a different mechanism involving a cell cycle priming and a final cell cycle commitment step. Our proposed work will make use of automated single live cell and fixed cell analysis using biosensor and activity selective antibodies to measure key cell cycle regulatory events and to dissect the control circuits of cell cycle entry and exit. Our final goal is to develop and validate a quantitative model for cell cycle entry and exit. The outcome of the proposed work will be the identification, characterization and modeling of critical proliferation control points that can be exploited therapeutically to treat diseases suh as cancer, immune responses, wound healing, as well as neurodegenerative diseases. For many growth associated diseases, treatment will likely involve strategies to regulate the rate of proliferation of specific cell types. In addition, our work will provide the cell cycle and cancer research communities with experimental and modeling tools to investigate cell specific cell cycle control in different cell types.