Leslie Loew, Ph.D. - US grants

The objective of this project is to combine the tools of theoretical, physical and synthetic organic chemistry to produce and characterize electrochromic molecular probes of membrane potential. We plan to produce probes which absorb in the 500-700 nm region so as to increase the usefulness of the methodology in applications to various problems in electrophysiology. We also plan to produce probes which can label specific receptor sites or which can covalently label membrane components in an effort to determine localized potential changes mediating specific events in the cell. Several analogues of the 4-(p-aminostryryl)pyridinium (ASP) chromophore with more extended conjugated Pi-systems will be examined with molecular orbital theory and synthetically incorporated into probes. The spectral properties and responses to membrane potential of the probes will be characterized and calibrated with phospholipid vesicles and a hemispherical lipid bilayer system. The probes will the be screened and further characterized with squid axon and red blood cell preparations. More complex experiments are planned to monitor electrical activity in an awake animal brain and to determine the fast kinetic details of membrane potential changes mediated by the acetylcholine receptor. These two examples illustrate how the probes can be used to help solve basic research problems in physiology, on the one hand, and how they may eventually be applied to clinical diagnostic procedures on the other.

Invasive amoebiasis is a major health problem in developing countries and within certain segments of the U.S. population. It is characterized by the massive destruction of host tissues by the parasite Entamoeba hystolytica. It appears that simple phagocytosis is preceded by a contact-dependent cytolethal stage which may involve the transfer of a membrane active toxin from the amoeba to its target cell. This toxin produces large membrane pores, depolarizing and killing the cell. The purpose of this proposed research is to characterize the action of this material in model membranes and cells, to probe the details of the contact-killing process, and, thereby, to establish whether this material is indeed a central factor underlying the disease. The material will be purified and antibodies grown to it. The purified material will be tested for pore-forming activity in lipid vesicles using a newly developed method employing voltage sensitive fluorescent dyes. This will allow determination of the rate of insertion of the pore into the membrane and provide an assay for pore-forming activity as a function of the membrane lipid composition, the presence of antibody, and the virulence of the strain of amoeba from which the toxin was isolated. The effect of the toxin on a variety of cells will be similarly assessed using a series of newly proposed voltage sensitive fluorescent probes which are designed to be used in microfluorometry. Finally, these results can be compared with those obtained from microfluorometric investigations of the contact-killing process itself. In addition to following membrane potential in these experiments, the transfer and interaction of membrane components will be monitored. The methods developed to elucidate the details of contact-dependent cytolethal effects should be generally applicable to a variety of problems in cell biology.

New potentiometric probes are proposed based on the concepts of electrochromism, charge-transfer, and thermochromism. Synthetic schemes and methodologies for the characterization of the probes are outlined. Several new strategies are described for the design of experiments aimed at measuring potential changes in single cells and in cells in suspension. These will allow the determination of rapid changes in cell suspensions and the detection of variations in single cells that are not triggered by a controlled external stimulus; currently, neither of these situations are generally accessible to the probe methodology. In addition, the temporal and spatial distribution of membrane potential induced by an external electric field on a variety of cells will be examined with the help of the potentiometric dyes. The following specific applications of these methodologies are proposed: 1) The place of the known membrane potential change within the cascade of biochemical and biophysical events accompanying the stimulus-response in neutrophils will be defined. 2) The kinetics for insertion of a variety of membrane permeabilizing agent into lipid vesicle and cell membranes will be characterized; among the agents to be examined is cytolysin, a cytotoxic factor derived from natural killer cells. 3) The possibility of an altered potential on the membrane of cells undergoing mitosis will be explored and correlated with other variations in cell surface properties. 4) Based on changing patterns of ion permeability during spermatogenesis, we will probe for expected variations of the potential along the mature sperm membrane.

This instrumentation grant proposal describes the needs for a dual wavelength excitation macro/micro-fluorescence spectrometer capable of quantitating cellular variables such as (Ca2+), pH, and membrane potential. The core of the requested system is a Fluoroplex III Spectrofluorometer system; it consists of a dual wavelength excitation source which can be used to excite fluorescence in a cuvette or in a microscopic specimen. For the former, a spectrograph and an intensified photodiode array detector capture the emission spectra. For the latter, an image processor produces ratiometric images of the fluorescence from the microscopic specimen. The Fluoroplex III also includes a photon counting photomultiplier which can replace the photodiode array for more sensitivity, albeit at a single wavelength, or the microscope camera for faster measurements, albeit at a sacrifice of spatial information. The other two essential components of the system are a Nikon Diaphot inverted microscope (which offers the most versatility for the variety of experiments planned by the users group) and a Hamamatsu C-2400-211ow-light level video camera. The research described by the primary users group includes investigation of the following: pH regulation of microtubule assembly; signal transduction in melanoma cells; the role of inositol triphosphate in calcium mobilization; acid/base secretion in kidney epithelia; endosomal acidification in the mechanism of iron uptake by reticulocytes; the role of Ca2+ and electric fields in cell motility; development of fluorescent probes of membrane potential; the role of membrane potential gradients in the reorganization of cell surface molecules; stimulus-response coupling in neutrophils; identification and characterization of cells with specialized transport functions in turtle urinary bladder. A plan for the short and long term operation of the equipment has been developed and commitments for the support of this plan have been secured.

Electric fields can have profound effects on biological systems, influencing processes such as differentiation, directed cell and neurite motility, and wound healing. Although it is widely appreciated that the fields can produce small asymmetrically distributed changes in membrane potential, it is not understood how these are amplified and translated into biochemical and biological responses at the level of the cell. This application proposes to test the hypothesis that the field induces a redistributed molecules and Ca2+ channels, clustered this way, produce foci of increased calcium activity which mediate cellular responses. To test these hypotheses, 2 cell types will be used. N1E-115 neuroblastoma cells have calcium channels which are heterogeneously distributed and appear to be involved in neurite outgrowth; they will be used as a model for galvanotropism. The relationship of the dynamics of cell surface molecules and calcium levels to the movement of fish keratocytes has been studied; they also exhibit directed motility in electric fields. Field effects on individual cells will be examined with digital imaging microscopy to study: membrane potential distribution (using potentiometric dyes), calcium channel distribution (using fluorescence and autoradiography with specific antibody or toxin probes), local changes in intracellular calcium levels (using FURA-2), cell and cell-surface particle tracking (using differential interference contrast), and actin cycling (using fluorescent actin and fluorescence recovery after photobleaching techniques).

The investigators propose to develop "Virtual Cell," a simulation framework for building complex cell models based on a hierarchical assembly of molecules, reaction mechanisms, and their associated intracellular structures. The numerical simulation is based on the finite volume method which permits a spatially heterogenous collection volume elements (compartments). The geometries of the compartments are derived directly from experimental microscope images. The partial differential equations of electrodiffusion, plus source terms corresponding to reactions, are solved for each chemical species in each volume element. The framework integrates continuous and discrete mathematical treatments. Comparison of simulation results and experimentally observed behavior is facilitated because the microscopy data and simulation model are mapped onto the same geometry. The three specific aims of the computational infrastructure project are to 1) develop continuous and discrete modeling descriptions o f cell processes and geometries, integrated in a single computational framework, 2) build a phenomenon-level web-based user interface, and 3) parallelize the code and investigate efficiency and portability issues.

This application requests support for the establishment of the National Resource for Cell Analysis and Modeling. The Resource will be housed within, and will be the principal venture, of the Center for Biomedical Imaging Technology (CBIT) at the University of Connecticut Health Center. The Resource will contain state of the art facilities for studying living cells and will develop a new technology, the Virtual Cell, for analyzing and synthesizing this knowledge. The Virtual Cell is a general framework for modeling cell biological processes. It approaches the problem by associating biochemical and electrophysiological data describing individual reactions, with experiment microscopic image data describing their subcellular locations. Individual processes are integrated within a physical and computational infrastructure that will accommodate any molecular mechanism. This will computational technology will be developed within a research center that is fully equipped for microscopic studies of living cells, will assure that experiment and theory will drive each other synergistically. Consistent with this philosophy, the research plan for the technology development is divided into two projects centered on theory and two that are primarily experimental. Computational Infrastructure addressed the issues of: (1) layering the cell biological models on a transparent physical and mathematical foundation; (2) implementing the numerical methods in a distributed parallel computing environment; (3) creating a user interface for model building that is modular, remotely accessible, and readily adaptable to a database of images and reaction mechanisms. Mathematic and Physical Analysis of Cell Biological Processes has 3 goals: the formulation of generalized mathematical descriptions of the elementary physical processes underlying the myriad of cell biological mechanisms; the development and validation of efficient numerical methods, development of rigorous methods for building and refining models from experimental data. Calcium Dynamics in Differentiated Neuroblastoma Cells and Cardiomyocytes aims to elucidate intracellular calcium signaling in these cells via a systematical marriage of experiment and modeling approaches: these studies will be used as a testbed for the Virtual Cell technology and will drive improvements and expansions. The last project, Intracellular RNA Trafficking, aims to use microscopic imaging and modeling to understand the series of events in which RNA is exported from the nucleus, packaged into granules, transported to remote regions of a cell, and translocated by the protein synthetic apparatus; this project will require new extensions of the Virtual Cell to incorporate the dynamics of intracellular supramolecular structures within the overall physical framework. A series of 11 collaborative projects are described with investigators from around the U.S. and Europe. Service, training, and dissemination activities of the Resource will all build upon the pre- existing facilities and track record of CBIT.

The Center for Biomedical Imaging Technology is active in training and dissemination related to biological images. They offer one-day workshops on "image processing" and "atomic force microscopy." They also offer a three-day short course an optical microscopy co-sponsored by Zeiss, with registrations of 40-50 students for the last two years. A full semester lab course entitled "Live Cell Imaging" and coordinated by Dr. Carson is team-taught by faculty associates of CBIT, and was taught to 12 students in 1996. They are also developing web site tutorials, and a matching Quicktime demonstration is available on CD-ROM. Workshops and short courses are organized by Dr. Cowan, with technical training by the system and Laboratory managers.

The Center for Biomedical Imaging Technology (CBIT) is already active in providing imaging services and collaborative activities. Current service is primarily to UCHC, with a list of over 80 users identifying medical and dental faculty from departments of neurology, physiology, pharmacology, microbiology, biochemistry, rheumatology, pediatrics, hematology/oncology, biomaterials, orthopaedics, infectious diseases, surgery, pathology, anatomy, biostructure & function, OB/GYN, cardiology, nuclear medicine, gastroenterology, periontology, oral diagnosis and pediatric dentistry. A number of industrial users are also involved. A fee schedule is provided which implies that the microscopes and SGI imaging workstations are available for unlimited amounts of time for a yearly fee. Fees for equipment use, training sessions and CBIT consultation have been kept low to encourage growth of the user base.

This project aims to design, synthesize and use fluorescent dyes that can sense changes in membrane potential. These dyes are highly polar or charged and therefore require FAB/MS analysis.

The objective of this project is to develop a comprehensive model of the spatio-temporal aspects of Ca2+ signals in cells within the"Virtual Cell," a computational programming environment. The plan is to incorporate morphological features of the cells and kinetic processes involved in the generation of Ca2+ signals in order to develop a precise understanding of Ca2+ signals in discrete local areas of cells and to describe how these local signals interact to produce the complex 3 dimensional spatio-temporal patterns in Ca2+ signals observed in different types of cells.

This project addresses the computational difficulties that arise when models are in the form of nonlinear, partial differential equations. It is an essential part of the proposed "Virtual Cell" realization, which comprises both stochastic and deterministic formulations to be solved simultaneously (and their outputs integrated), in addition to irregular boundaries as geometric constraints. There are some discontinuities at membrane boundaries to be included, as well.

The investigators propose to develop "Virtual Cell," a simulation framework for building complex cell models based on a hierarchical assembly of molecules, reaction mechanisms, and their associated intracellular structures. The numerical simulation is based on the finite volume method which permits a spatially heterogenous collection volume elements (compartments). The geometries of the compartments are derived directly from experimental microscope images. The partial differential equations of electrodiffusion, plus source terms corresponding to reactions, are solved for each chemical species in each volume element. The framework integrates continuous and discrete mathematical treatments. Comparison of simulation results and experimentally observed behavior is facilitated because the microscopy data and simulation model are mapped onto the same geometry. The three specific aims of the computational infrastructure project are to 1) develop continuous and discrete modeling descriptions o f cell processes and geometries, integrated in a single computational framework, 2) build a phenomenon-level web-based user interface, and 3) parallelize the code and investigate efficiency and portability issues.

The objective of this project is to develop a comprehensive model of the spatio-temporal aspects of Ca2+ signals in cells within the"Virtual Cell," a computational programming environment. The plan is to incorporate morphological features of the cells and kinetic processes involved in the generation of Ca2+ signals in order to develop a precise understanding of Ca2+ signals in discrete local areas of cells and to describe how these local signals interact to produce the complex 3 dimensional spatio-temporal patterns in Ca2+ signals observed in different types of cells.

Funds are requested to purchase a commercial non-linear optical microscope (NLOM) system dedicated for use in cell biological imaging experiments. The NLO system will be based on a Zeiss LSM510 microscope equipped with a Coherent titanium-sapphire femtosecond laser. The instrument will be housed at the Center for Biomedical Imaging Technology (CBIT) at the University of Connecticut Health Center. This research center offers use of high level fluorescence microscopy instrumentation and image analysis resources to the research community both at the UCHC and from outside academic institutions and industry through a well established user facility. It is expected that many of the current 30 laboratories and over 55 graduate and postdoctoral students using the facilities at CBIT will employ the capabilities of the NLO microscope in a wide range research programs. Projects from 6 major users with well developed projects at UCHC are described here. All of these require the deep optical sectioning capabilities characteristic of NLOM and benefit from the suitability of NLOM for imaging living specimens. Dr. Leslie Loew will use NLOM to perform optical recording of membrane electrical properties of neuronal cells in acute brain slice preparations. Dr. John Carson will use NLOM for three dimensional visualization of RNA trafficking in oligodendrocytes in intact optic nerve preparations. Dr. Elizabeth Eipper will follow the expression of GFP-tagged proteins involved in regulated secretion in hippocampal or cerebellar slice preparations. Dr. William Mohler will use two photon imaging to visualize and dissect individual cell fusion events in intact C. elegans embryos. Dr. David Papermaster will study photoreceptor development in transgenic Xenopus tadpoles by following GFP tagged proteins in the living tadpole eye using NLOM. Dr. Mark Terasaki will use NLOM to follow nuclear envelope breakdown in living starfish oocytes, combining high spatial and temporal resolution imaging with photobleaching experiments to assess changes in nuclear envelope and nuclear pore structures that are the antecedents of nuclear envelop breakdown.

This grant provides partial support for the First International Symposium on Computational Cell
Biology to be held March 4-6, 2001 in Lenox, MA. Computational Cell Biology is an emerging interdisciplinary field that responds to the need for computational methods to analyze and organize the abundance of experimental data on the structure and function of the cell. Whereas other meetings have been held that focus on bioinformatics and molecular and structural biology, this will be the first meeting geared to the use of computational modeling applications in cell biology, and will be primarily targeted to cell
biologists. The major focus of the meeting will be on areas of cell biology for which modeling approaches are currently being developed, or that are ripe for computational modeling approaches. A key goal is to bring together cell biologists whose research addresses quantitative aspects of cellular mechanisms with computer scientists and mathematicians who can provide the computational tools. The symposium is being organized by The National Resource for Cell Analysis and Modeling (NRCAM), located in the Center for Biomedical Imaging Technology (CBIT) at the University of Connecticut Health Center. The organizing committee consists of
Drs. Leslie M. Loew, Director of NRCAM and CBIT, John Carson, co-Director of NRCAM and Vladimir Rodionov, associate faculty of CBIT, and is chaired by Ann Cowan, Deputy Director of CBIT and head of dissemination and training for NRCAM. The members of the organizing committee feel that the time is ripe to bring together cell biologists, mathematicians, and computer scientists to develop a true community of scientists sharing the common goal of developing computational tools for cell biological modeling and simulation. Topics at the meetings will encompass a range of cellular mechanisms including regulation of the cytoskeleton and molecular motors, membrane and protein trafficking, regulation of calcium dynamics, signal transduction pathways, and cell cycle control. In each of these areas key researchers who utilize highly quantitative experimental approaches and/or are applying mathematical modeling approaches will be invited to speak. The meeting will be an intensive mix of platform sessions, poster sessions and small workshops and tutorials. The venue for the meeting is a resort in the Berkshire mountains that is limited to a maximum of 125 participants. This setting and meeting size will be ideal for encouraging individual interactions between this diverse group of scientists. It will also serve as an excellent educational activity for graduate and
postdoctoral students working in this young and expanding field.

This award supports student participation in the "Second International Symposium on Computational Cell Biology" to be held March 23-25, 2003 at the Cranwell Resort and Golf Club in Lenox, MA. Computational Cell Biology is an emerging interdisciplinary field that responds to the need for computational methods to analyze and organize complex experimental data on the structure and function of the cell. Whereas other meetings in computational biology focus on bioinformatics and structural biology, this is the only meeting geared specifically to the use of computational modeling applications in cell biology and that is primarily targeted to cell biologists. The symposium is being organized by The National Resource for Cell Analysis and Modeling (NRCAM), located in the Center for Biomedical Imaging Technology (CBIT) at the University of Connecticut Health Center. The organizing committee consists of Drs. Leslie M. Loew, Director of NRCAM and CBIT, John Carson, co-Director of NRCAM, and Ann Cowan, Deputy Director of CBIT and head of dissemination and training for NRCAM, who chairs the committee. The first such symposium, held in March 2001, provided a venue for cell biologists to interact with theoreticians and computer scientists, and was highly successful both in its scientific content and in its goal of initiating a new level of communication among different groups of scientists. A major goal of this second meeting is to solidify the community of scientists in this young and expanding field. The scientific focus of the meeting is on areas of cell biology for which modeling approaches are currently being developed, or that are ripe for computational modeling approaches. Specific topics at the meetings will include a range of cellular mechanisms such as regulation of the cytoskeleton and molecular motors, membrane and protein trafficking, regulation of calcium dynamics, signal transduction pathways, and cell cycle control. In each of these areas, researcher using highly quantitative experimental approaches and/or applying mathematical modeling approaches will be invited to speak. The meeting format includes platform sessions, poster sessions and small workshops and tutorials. The venue for the meeting is a small resort in the Berkshire Mountains that is limited to a maximum of 125 on-site participants. This setting and meeting size is ideal for encouraging individual interactions, and also encourages active participation by graduate and postdoctoral students.

inositol phosphates; dendrites; molecular dynamics; cerebellar Purkinje cell; biomedical resource;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An intensive three day course to train investigators to apply the Virtual Cell to their research applications has been held annually starting in June 2000. The attendees include PIs, postdoctoral students and graduate students, and the curriculum consists of a general introduction to using the Virtual Cell, followed by intensive, one-on-one sessions developing a Virtual Cell Model and running simulations. The course has been highly successful, leading directly to several long term collaborative projects and several additional continuing users.

To realize the potential of nanomedicine to revolutionize health and medicine, intracellular molecular mechanisms will have to be elucidated. The accelerating progress in cataloguing the critical molecular and structural elements responsible for cell function has led to the hope that cell biological processes can be analyzed and understood in terms of the interactions of these components. Currently, efforts to characterize and analyze cellular mechanisms focus on interactions between molecules that comprise biochemical and gene-regulatory networks. The study of such networks is a central theme of the emerging field known as Systems Biology. However, this focus alone can never provide answers to the key question on the origin and role of spatial organization at supramolecular scales within the context of a cell. This nanoscale bridges molecular level detail, which is derived from biochemistry and molecular structure, and the micro level detail that is derived from studies of cell function and dynamics. The proposed "Center for Cellular Nano-Organization" (CNO) will focus on intracellular dynamic structures at this intermediate scale and explore how these structures are generated and how they mediate key cellular events. This will be achieved through a series of specific collaborative interdisciplinary projects that will explore the assembly and function of prototypical cellular structures, which we call intracellular nanomachines, in carefully selected appropriate cell biological systems. These projects will call upon the expertise and tools available in 3 technology cores: Optics Lab, Modeling Lab and Computation Lab. The latter will also offer web-based services that will assure that the products of the CNO will be available to the other Nanomedicine Centers and disseminated to the greater biomedical research community. We anticipate that these studies will lead to new unifying principles on the initiation, self assembly and function of spatially organized intracellular nanostructures.

computational biology; transcription factor; biomedical resource;

cytoskeletal proteins; computer simulation; cell biology; biomedical resource;

computational biology; computer program /software; biomedical resource;

DESCRIPTION (provided by applicant): Existing proteomics technologies can provide a wealth of information about the biochemistry operating in cells. And systems biology tools are being developed to analyze and model this data. However, they fail to address the fundamental questions of how the spatial organization of molecules in cells is established and how it is utilized to control cell function. To answer these, we will need new tools and new theoretical frameworks that specifically include consideration of cell morphology and dynamic spatial molecular distributions. This proposal aims to establish a Technology Center for Networks and Pathways (TCNP) that will integrate microscope technologies for making quantitative in vivo live cell measurements with new physical formulations and computational tools that will produce spatially realistic quantitative models of intracellular dynamics. The model predictions will then be validated with new measurements as well as novel intracellular manipulation technologies also to be developed in our proposed TCNP. These new technologies will be developed and disseminated by the Center for Cell Analysis and Modeling (CCAM) at the University of Connecticut Health Center (UCHC). The technology will be disseminated throughout the research community via training programs, web-based instructional material, a repository of molecular probes and a database of data and models. The proposed work builds on a firm foundation. CCAM is the home of the Virtual Cell, a computational environment for cell biological modeling, and also hosts a variety of projects in biophotonics and live cell microscopic imaging methods as well as a state-of-the-art user facility for nonlinear, confocal, and widefield microscopy. CCAM is the scientific home of an extraordinary confluence of expertise in physics, chemistry, software engineering and experimental cell biology that is unique for a medical school and is ideal for the concerted multi-pronged effort that is planned for the TCNP.

This award is for support of student participation in the "Third International Symposium on Computational Cell Biology," held March 19-23, 2005 in Lenox, MA.

Computational Cell Biology is a growing interdisciplinary field that responds to the need for computational methods to analyze and organize complex experimental data on the structure and function of the cell. Whereas other meetings in computational biology focus on bioinformatics and structural biology, this is the only meeting geared specifically to the use of computational modeling applications in cell biology and that is primarily targeted to cell biologists. The symposium is being organized by the National Resource for Cell Analysis and Modeling (NRCAM), located in the Center for Biomedical Imaging Technology (CBIT) at the University of Connecticut Health Center. The organizing committee consists of Drs. Leslie M. Loew, Director of NRCAM and CBIT, John Carson, co-Director of NRCAM, and Ann Cowan, Deputy Director of CBIT and head of dissemination and training for NRCAM, who chairs the committee. Previous symposia, held in 2001 and 2003, were highly successful both in their scientific content and in providing a venue for cell biologists to interact with theoreticians and computer scientists. The 2005 Symposium continues to serve as an important focal point for the community of scientists in this rapidly expanding field. The scientific focus of the meeting is on areas of cell biology for which modeling approaches are currently being developed or that are ripe for computational modeling approaches. Topics at the meetings encompass a range of cellular mechanisms including cytoskeletal dynamics, cell cycle control, signal transduction pathways, and regulation of calcium dynamics. The meeting format is an intensive mix of platform sessions, poster sessions and small workshops and tutorials. The venue is a small resort in the Berkshire mountains that is limited to a maximum of 125 on-site participants, a setting and meeting size that is ideal for encouraging individual interactions, and encouraging active participation by graduate and postdoctoral students.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Long term depression of synaptic strength in Purkinje neurons requires repeated inositol-1,4,5-trisphosphate (InsP3)-mediated Ca2+ release, but the InsP3 receptor, while highly abundant, is extraordinarily insensitive to InsP3 in this cell. Synaptic inputs occur at distinct structures, called spines, that are ca. 15m diameter spheres connected to the dendrite via a narrow neck. The ability of the spine to compartmentalize electrical and chemical signals is poorly understood. We are using this project to drive the development of new electrophysiological modeling capabilities of Virtual Cell.

NIH Roadmap Initiative tag; technology /technique development

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Cell crawling is integral to many important biological and biomedical processes, such as wound healing, cancer metastasis, and organism development. A quantitatively predictive model that describes the bulk motion of many cells will have broad impact across many areas of biology and biophysics. This research will combine the general physical features of single cell crawling with a physiologically realistic description of cell-cell adhesion and cell-cell interaction in order to develop a mathematical model for the collective movements of cells in tissue. Specifically, the model will address a number of experimentally-measured behaviors that occur in wound healing assays. For example, recent experiments have shown that there are complex motions and long-range correlations of the movements of cells in epithelial monolayers. In addition, during wound healing assays it is observed that the wound edge undergoes a fingering-like instability, and the average progression of the wound border expands at a supra-linear rate with respect to time. This research will develop and test a continuum model for the collective migration of eukaryotic cells in tissue, with an emphasis toward explaining the dynamics of epithelial cell monolayers during wound closure. Most of the model parameters will be set using existing experimental data; however, some parameters are currently unknown. Therefore, comparison of the model with existing experimental data will allow us to set ranges on these unknown parameters and will suggest new experiments to test the predictions of the model. Analytic and numerical methods will be used to analyze the model for free boundary problems corresponding to two different wound closure experiments, and the model will be used to explore three-dimensional motility in bulk tissue.

To build an organism out of a vast number of cells requires coordination and the ability to move cells from one place to another. Therefore, during development of an organism, it is not surprising that groups of cells often migrate together, as a semi-cohesive unit. Similar migratory behavior is also seen during wound healing and cancer metastasis. This project explores the physics of this motility and the mechanism by which the physical behavior of cells helps maintain the overall coordination of the group. A mathematical model will be developed that takes into account the gross behavior of motile cells, considering the forces generated by an individual cell and the interactions between neighboring cells. This model will be used to describe the behavior of a wounded layer of cells, and the predictions will be tested by comparing them to data from experiments that mimic wounded tissues. The results from this project will provide a deeper understanding of many important biological processes, such as organism development, cancer metastasis, and even the motions of dense communities of swimming bacteria. The strong interdisciplinary nature of the research makes it ideal for training graduate students and postdoctoral researchers to be able to work at the interface between biology, mathematics, and physics.

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. The impact of VCell could be significantly expanded by developing enhanced visualization tools that would vastly reduce the time required to developed a VCell BioModel and to compare simulation results to experimental results. These advances would also facilitate the use of VCell and other spatial simulation technologies by a broader class of both bench and clinical researchers by providing intuitive methods for interacting with mathematical models of biological processes. Two classes of visualization tools are proposed to enhance the VCell modeling environment. Aim 1 integrates enhanced graphical methods for creating and interacting with VCell BioModels. We are integrating powerful graphical methods to enable users to efficiently traverse the reaction network landscape and interact with the system in an intuitive interface that allows facile movement between geometry, reaction pathways, parameter lists, and ultimately simulation results. Aim 2 develops sophisticated three dimensional visualization tools for displaying and comparing simulation results and experimental data sets. Ultimately the tools developed will be linked to provide seamless integration from the original BioModel to the simulation results, providing an intuitive interface by which models and simulation results can be more easily disseminated beyond the modeling community. Enhanced graphics capabilities within the user interface of VCell will help to bring the power of this technology into laboratories and clinics that would not ordinarily have the expertise to utilize advanced modeling technologies.

DESCRIPTION (provided by applicant): This grant application is designed to help the researchers who have been developing emerging community standards in computational systems biology to travel to the R. D. Berlin Center for Cell Analysis and Modeling in Farmington, CT, which will host the Hackathon on Resources in Modeling in Biology (HARMONY) 2013 meeting. The focus is on four core community-accepted languages, standards and ontologies used in biological modeling and data analysis (SBML, BioPAX, SBGN, and SED-ML), whose communities of users and developers have joined forces as part of the Computational Modeling in Biology network (COMBINE). The main purpose of the conference is to improve interoperability between formats and to jump-start efforts to address newly identified unmet needs or critical gaps in coverage. The HARMONY meetings have emerged as an invaluable venue for hashing out concrete high priority issues and devise new solutions for apparent stumbling blocks, with face-to-face meetings and hands-on participation of leaders in the field. The conference will bring together leading scientists and software developers, experts in biological modeling, bioinformatics and computer science, who are actively involved in the development of standards and/or the software tools and repositories that use them. A large part of the meeting will consist of hackathon-style parallel tracks of working groups that are tasked to address concrete problems and to produce deliverables such as draft specifications and/or prototype implementations. We wish to emphasize that most of the leading members of the different groups of the COMBINE network serve as volunteers and do not have dedicated funding for this activity, which is nonetheless crucial to the scientific community. It is importan that as many as possible of these researchers are present at the HARMONY conference. We are striving to maintain the cost of attending to a minimum and we will be able to organize the meeting without charging any registration fee. However, no support is available to directly defray the costs of travel for participants, which often does make up for most of the financial burden to attend meetings that have a global audience. We are therefore requesting funds to partially defray the travel costs for some of the key participants to the conference.

Administration and Management Project Summary The administrative core provides leadership and administrative support for the activities of the Resource. Housed within the Center for Cell Analysis and Modeling (CCAM), an independent research center within the School of Medicine, the Resource represents a multi-disciplinary group of physicists, mathematicians, computer engineers and cell biologists within a strong basic and translational biomedical research environment. Led by Leslie Loew, the resource is organized in a highly integrated team-based approach with a Theory and Numerics team led by Boris Slepchenko, a Software Development team led by Jim Schaff, a Computational Infrastructure team led by Ion Moraru, and a User Interactions team led by Ann Cowan. A strong administrative staff at CCAM, led by Karen Zucker, provides outstanding administrative support. The majority of the resource operation is the development of the software and its associated web presence. Monthly meetings of the entire group serve to ensure that decisions regarding the technology and its dissemination are discussed and prioritized by the entire team. An outstanding External Advisory Committee composed of seven highly recognized experts in a broad range of fields encompassing computational modeling, biophysics, and cell and molecular biology research meet annually at NRCAM to provide advice on new directions for technology development, driving biological projects and outreach strategies. The administration of the School of Medicine continues to provide outstanding support for the Resource in terms of the physical surroundings, targeting faculty recruiting to CCAM, instrumentation to the CCAM microscopy and high performance computing facilities that support modeling applications, and direct support of personnel and service contracts.

TR&D1 Biology to Physics Project Summary This project proposes to develop new network-free spatial modeling software at the mesoscale - occupying the niche between detailed molecular dynamics and cellular reaction-diffusion systems. Specifically, we plan to address spatial scales in the range of 50nm ? 2µm and temporal scales within the range of 1ms to 10s. Examples of systems that would benefit from modeling tools at this ?mesoscale? are receptor signaling platforms and clusters (e.g. the immune synapse or the post-synaptic density), cell adhesion complexes, lipid rafts, chromatin organization, cytoskeletal dynamics, and nucleoprotein phases. Our approach builds on the foundation of the SpringSaLaD software, which uses a Langevin dynamics formalism to model multi-molecular interactions with explicit excluded volumes. It permits spatial simulations of combinatorially complex processes such as clustering and polymerization. The approach is an amalgam of kinetic and molecular modeling, in that it derives probabilities of reactions from both coarse structural features of the molecules and macroscopic biochemical parameters such as on and off rate constants, diffusion coefficients and allosteric state transition rates. Currently SpringSaLaD represents molecules as spherical ?sites? connected by linear linkers, modeled as stiff springs. Molecules can diffuse and react either in a rectangular volume or be anchored to a planar patch of membrane. Our Specific Aims propose to dramatically expand the scope of SpringSaLaD by allowing more realistic representation of structural details and expanding the range of biophysical mechanisms that can be modelled. To better account for the influence of membrane curvature on clustering and the possibility of assemblies that span across thin processes such as filopodia, we will implement methods for Brownian dynamics along curved membranes. We will develop methods to derive the arrangement of spherical sites and linkers from more realistic 3D molecular data, including atomic coordinates. We will develop new optional schemes to better account for the rigidity of molecular structures at this coarse-grained level and, separately, the flexibility of linker domains; the latter will help us represent the influence of intrinsically disordered domains. Finally, we propose to encode the net vectorial force experienced by a site into the probabilities for unbinding (i.e. off rates), binding (on-rates) and the forces at membrane surfaces.

Driving Biomedical Projects Project Summary A series of 18 Driving Biomedical Projects are presented to motivate and test the research and development products of the 4 TR&D projects. They span a broad range of cutting edge science, including cell motility, liquid droplet phases, cell signaling, cytoskeletal dynamics, cardiac physiology, gene regulatory networks and cancer. The Research Strategy section presents details on 9 of these DBPs within the 12 page limit. But additional details can be found in the attached full Table as well as the Letters of Support (located in the ?Overall? section of this grant application). Most of the projects cover multiple TR&Ds, attesting to the breadth of the DBP science as well as the complementarity of the TR&Ds. TR&D1 is driven by 7 DBPs. TR&D2 is driven by 11 DBPs. TR&D3 is driven by 11 DBPs. TR&D4 is driven by 11 DBPs. The list of DBPs includes 5 projects that are continuing from the current project period and 13 new projects. Of the latter, several are transitioning from active Collaborations to DBPs. We expect that 8 of the DBPs will be interacting with NRCAM from the start of the new project period while others will be activated as the new technologies become ready for their input and testing. Each DBP will be assigned a primary contact within the team of NRCAM investigators to act as a liaison to the overall project. Decisions on the entry of new DBPs and the retirement of existing ones will be made by the NRCAM team with the advice of our Scientific Advisory Board.

In this project, the investigators will develop a new experimental and computational platform to answer the longstanding biological question of how cellular receptors generate biochemical signals as they move among various subcellular compartments. By helping to quantitatively resolve certain apparently conflicting observations that have recently been made within a biological system previously thought to be well understood, this project will add fundamentally new understanding of basic mechanisms of cell signaling that may have implications across numerous receptor systems in cell biology. Moreover, due to the broadly important role that receptors, and the biochemical signals they generate, play in virtually every biological process, the new quantitative understanding gained will ultimately impact ongoing efforts to optimally engineer receptor-mediated signaling. This project also includes a broad and detailed set of integrated educational objectives that leverage the specific scientific objectives and activities of this project to reach students at different training stages and from diverse backgrounds. These education and outreach activities involve innovation upon existing programs at the partnering institutions as well as the creation of new outreach efforts that will target larger audiences, such as a summer academy of science for high school students and a collaboration with the Museum of Science and Industry in Tampa, FL to develop a live interaction program on cell signaling.

The biochemical signaling that results upon ligand binding to receptor tyrosine kinases is accompanied by receptor clustering and internalization, or endocytosis. While it has been understood for decades that endocytosis enables cells to degrade or recycle receptors, the question of how endocytosis ultimately impacts downstream signal propagation remains wide open. For example, even for the highly studied epidermal growth factor receptor (EGFR) the literature contains highly cited, yet conflicting reports that conclude that EGFR endocytosis either promotes or impedes downstream signaling through the extracellular regulated kinase (ERK) pathway. In this project, the investigators will explore the hypothesis that the intrinsic complexity of the spatiotemporal signaling regulatory mechanisms creates parameter spaces for both answers to be true depending on the cellular context. The complexity of the receptor trafficking and signaling system cannot be fully dissected, however, without integrating quantitative experimental measurements that faithfully reflect cell biology with spatiotemporal computational models. Accordingly, the overarching goal of this project is to combine novel live-cell imaging of gene-edited cell systems with mechanistic models that capture system complexity with unprecedented detail to resolve the kinds of conflicting reports previously mentioned, as well as more recent surprising observations that certain proteins conventionally thought to remain in complex during signal transduction may become physically separated through trafficking processes. Ultimately, the proposed integration of gene-edited cell systems, quantitative live-cell imaging and biochemical measurements, and a spatiotemporal computational model will lead to an entirely new quantitative platform to answer these kinds of questions for EGFR and other receptor signaling systems.

Project Summary Virtual Cell (VCell) is a problem solving environment built on a central database and disseminated as a client-server application. It is designed to make mathematically sophisticated computational modeling and simulation accessible to cell biologists. The resource is open and free to the cell biology and scientific software developer communities. Currently, there are >6300 users who have built models and run simulations through the VCell servers, all saved in the VCell database. VCell supports a number of key biophysical mechanisms, including reaction kinetics, diffusion, flow, membrane transport, lateral membrane diffusion, electrophysiology and rule-based models of multi-state/multi-molecular interactions. Reaction-diffusion simulations can be based on 1D, 2D or 3D analytical or experimental image-based geometries. Users may choose among multiple available simulation approaches: ordinary differential equations, partial differential equations, stochastic reaction kinetics, network-free simulations, spatial particle- based simulations and spatial hybrid stochastic/deterministic simulations. A newly developed technology now allows for simulations of reaction diffusion equations in geometries with changing shape. The research needed to develop the existing VCell technology was developed over a period of 20 years. This grant application aims to preserve the availability of the large amount of data and unique functionality provided by the VCell platform for the scientific community. It will also provide the time to plan for a careful and deliberate transition to a self- sustaining financial model for the VCell resource.

This project proposes to develop new network-free spatial modeling software at the mesoscale - occupying the niche between detailed molecular dynamics and cellular reaction-diffusion systems. Specifically, we plan to address spatial scales in the range of 50nm ? 2µm and temporal scales within the range of 100µs to 10s. Examples of systems that would benefit from modeling tools at this ?mesoscale? are receptor signaling platforms and clusters (e.g. the immune synapse or the post-synaptic density), cell adhesion complexes, lipid rafts, chromatin organization, cytoskeletal dynamics, and nucleoprotein phases. Our approach builds on the foundation of the SpringSaLaD software, which uses a Langevin dynamics formalism to model multi-molecular interactions with explicit excluded volumes. It permits spatial simulations of combinatorially complex processes such as clustering and polymerization. The approach is an amalgam of kinetic and molecular modeling, in that it derives probabilities of reactions from both coarse structural features of the molecules and macroscopic biochemical parameters such as on and off rate constants, diffusion coefficients and allosteric state transition rates. Currently SpringSaLaD represents molecules as spherical ?sites? connected by linear linkers, modeled as stiff springs. Molecules can diffuse and react either in a rectangular volume or be anchored to a planar patch of membrane. Our Specific Aims propose to dramatically expand the scope of SpringSaLaD by allowing more realistic representation of structural details and expanding the range of biophysical mechanisms that can be modelled. To better account for the influence of membrane curvature on clustering and the possibility of assemblies that span across thin processes such as filopodia or endocytic invaginations, we will implement methods for Brownian dynamics along curved membranes. We will develop methods to derive the arrangement of spherical sites and linkers from more realistic 3D molecular data, including atomic coordinates. We will develop new optional schemes to better account for the rigidity of molecular structures at this coarse-grained level and, separately, the flexibility of linker domains; the latter will help us represent the influence of intrinsically disordered domains. We will develop statistical and analytical methods to analyze simulation results and build lumped models so as to bridge from this mesoscale to the full cell scale. Finally, we propose to support mechanochemistry by accounting for local force experienced by a site and appropriately altering probabilities for unbinding (i.e. off rates), binding (on-rates) and the tension at membrane surfaces. Ultimately, the SpringSaLaD functionality will be incorporated within the Virtual Cell software system.

Project Summary A primary focus of the NIH BRAIN Initiative is to develop new technologies for large scale high resolution imaging of brain activity. Imaging, as opposed to traditional electrode array based measurements, promise much greater spatial resolution and specificity. Excellent fluorescent calcium indicators have been developed to make it possible to image activity with high spatial and temporal resolution. But calcium signaling is secondary to electrical signaling, which is the primary form of information transfer within the brain circuitry. However, current tools for imaging electrical activity have severe limitations, primarily due to limited sensitivity and poor signal to noise. This proposal focusses on the development of new hybrid voltage sensors, hGEVIs, based on genetically encoded fluorescent proteins (FPs) and linked organic fluorescence quenchers. The system is designed so that the fluorophore is localized to the outer surface of the membrane while the tethered quencher translocates across the membrane in response to voltage. Based on our previous experience with an all organic voltage sensor composed of a tethered bichromophoric fluorophore quencher (TBFQ), we know that huge voltage sensitivities can be achieved. Our proposed hGEVIs promise to be bright and produce extraordinary voltage sensitivity. They will have the additional benefit of allowing the sensors to be targeted to specific brain circuits or regions. This new class of voltage indicators could make electrical imaging as widespread and accessible as calcium imaging.

Project Summary This proposal aims to establish a National Resource for Mechanistic Modeling of Cellular Systems to serve the large community of cell and systems biologists. The Resource will encompass COPASI and Virtual Cell (VCell) software platforms, which are arguably the most comprehensive and widely used tools for computational modeling of the biophysical mechanisms controlling cell function. VCell supports a number of key biophysical mechanisms, including reaction kinetics, diffusion, flow, membrane transport, lateral membrane diffusion, electrophysiology and rule-based models of multi-state/multimolecular interactions. Simulations can be based on 0D, 1D, 2D or 3D analytical or experimental image-based geometries. Users may choose among multiple available simulation approaches: ordinary differential equations, partial differential equations, stochastic reaction kinetics, network-free simulations, spatial particle-based simulations and spatial hybrid stochastic/deterministic simulations. COPASI enables the simulation and analysis of complex biochemical reaction networks either deterministically or stochastically. It offers a broad range of analysis tools including parameter estimation/optimization, steady state analysis, stoichiometric analysis, sensitivity analysis and metabolic control analysis. VCell and COPASI each boast thousands of active users. Collectively, their users produce >100 papers per year relying on these software systems. Both VCell and COPASI will be hosted at the University of Connecticut School of Medicine. Thus, the Resource will benefit from: (1) the common institutional organization under which it will operate; (2) a joint website; (3) a common high performance computing facility that will serve the computationally intensive needs of users; (4) coordinated training and outreach to the user community in the form of web-based documentation and tutorials, a yearly Computational Cell Biology Workshop and numerous roadshows at national and international meetings. Additionally, VCell and COPASI will be leveraged by external systems biology software developers as user-friendly platforms for the wide dissemination of their third party algorithms, software and databases. Finally, the Resource will actively engage with the software standards community to assure the reproducibility and reusability of both the software and the models it generates.