Kenneth W. Dunn - US grants

In preparation for a grant to fund a multiphoton microscope at the University of Indiana we would like to use the system at the MR to obtain preliminary data. We will send fixed samples of Iddney tissue which has been stained with rhodamine fluorophores. Dr. Frohlich will observe the samples using the YLF solid-state laser for multiphoton imaging and the ADLAS 532 run solid state laser for confocal imaging. We would like her to collect data sets of this fixed material imaged first by confocal microscopy and then sample the same volume by multiphoton microscopy. Zseries and xzseries data sets will be collected to document the improvement in depth of imaging penetration obtained with multiphoton microscopy compared with confocal microscopy. The data will be transmitted us me via the anonymous FI? site maintained by the MR.

The imaging core is build around a state-of-the-art facility, the expertise of the optical and electron microscopy personnel and the experience of each of the subproject investigators in imaging. The central core facility will constitute a focus of interaction for the program, and will provide for consistent imaging performance and interpretation. The facility is equipped for quantitative confocal and 2-photon microscopy (Bio-Rad MRC-1024), digital deconvolution wide-field microscopy (Applied Precision Deltavision deconvolution software) and electron microscopy (Phillips CM120 TEM). It is the objective of the imaging core facility to apply, develop and combined these technologies to provide a rational, integrated approach to three-dimensional microscopic imaging of polarized epithelial cells. The specific mission of the core facility will be to, 1. provide quantitative imaging to support each of the 4 program subprojects, 2. implement digital image deconvolution techniques to support a 4 way strategy of high resolution three-dimensional optical imaging employing confocal microscopy, 2-photon microscopy and image deconvolution of wilde-field and confocal images and, 3. develop methods of three-dimensional imaging of living polarized epithelial cells. With the completion of these aims, the imaging core will have been developed into a unique resource for the study of renal cell biology.

Endocytic membrane transport is critical to many facets of renal function. Vectorial transport of ions, solutes and proteins is regulated by the polarized expression of transporters and receptors, which is determined by rates of endocytic insertion and retrieval. The plasma membrane expression of transporters is frequently regulated by signaling receptors, whose expression is likewise regulated by endocytosis. As many diseases, such as insulin-dependent diabetes, cystic fibrosis and Liddle's syndrome are associated with disruptions of endocytic membrane transport, it is clearly important to understand the molecular mechanisms regulating endocytosis. Combined molecular and cell biological studies of fibroblasts have elucidate the molecular regulation of many endocytic transport steps. However, comparable studies have not been conducted in polarized epithelial and it is clear that the studies of fibroblasts do not apply to epithelia, whose endocytic pathways are regulated by epithelial-specific proteins, and by ubiquitous proteins that acquire new functions within the more complex membrane pathways of epithelia. To better understand molecular regulations of epithelial endocytosis we propose to address the epithelial function of proteins known to participate in membrane recycling, both those common to epithelia and fibroblasts, Rab4a, Rab11a and Rme-1, as well as those specific to epithelial cells, Rab17 and Rab25. We will also identify proteins that regulate polar sorting in endosomes by characterizing how the protein constitution of endosomes is altered when polar sorting is disrupted. Endosome protein analysis will be assayed using subcellular fractionation, 2-dimensional gel electrophoresis and mass spectroscopy. The cellular effects of transfected proteins will be evaluated through the combined use of quantitative confocal microscopy of polarized cells expressing GFP chimeras of mutant and wildtype proteins and biochemical analysis of stable cell lines expressing transfected proteins. The dissection of the molecular mechanisms of endocytosis provided here will illuminate the processes that regulate membrane recycling, transcytosis, transport to the trans-Golgi network and plasma membrane polarity. In so doing, they will provide the framework for understanding diseases, and developing therapeutics for transporter disorders, immune disorders, toxin-mediated pathologies and polarity disorders, such as polycystic kidney disease.

Funding from Indiana University, the NIH and the Lilly Endowment have given Indiana University a world-class center for biomedical imaging. The Indiana Center for Biological Microscopy is one of a handful in the world equipped for low-light level microscopy, confocal microscopy, UV confocal microscopy, 2-photon microscopy, digital deconvolution microscopy, live cell microscopy and the latest systems for digital image analysis and visualization. The facility represents a strong institutional commitment to optical imaging technology development. In addition to providing state-of-the-art support for Cancer Center members, the core is also actively involved in research into biological imaging, resulting in the development and dissemination of new methods of microscopy and digital image analysis software. The products of these activities are disseminated through a program of education, including seminars, courses and individual training.

Funding from Indiana University, the NIH and the Lilly Endowment have given Indiana University a worldclass center for biomedical imaging. The Indiana Center for Biological Microscopy (ICBM) is one of a handful in the world equipped for low-light level microscopy, confocal microscopy, UV confocal microscopy, 2-photon microscopy, digital deconvolution microscopy, live cell microscopy and the latest systems for digital image analysis and visualization. The facility represents a strong institutional commitment to optical imaging technology development. Since 1998, Indiana University has committed more than $5M to this facility. In 2003 the Imaging Center moved into new laboratory space, a move that nearly doubled the space, and provided more efficient space utilization and better environmental control. In addition to providing state-of-the-art support for Cancer Center members, the core is also actively involved in research into biological imaging, resulting in the development and dissemination of new methods of microscopy and digital image analysis software. The products of these activities are disseminated through a program of education, including seminars, courses and individual training.

[unreadable] DESCRIPTION (provided by applicant): This request is for funds to purchase an Olympus Fluoview 1000MPE confocal/multiphoton fluorescence microscope system. This instrument will be housed, managed and maintained by the Indiana Center for Biological Microscopy (ICBM), a core facility of the Indiana University School of Medicine and the Indiana University George M. O'Brien Center for Advanced Renal Microscopic Analysis. Confocal and multiphoton microscopy are essential services of the center, which operates four confocal microscopes, two of which are equipped for multiphoton fluorescence microscopy. An eleven-year old BioRad MRC1024MP system accounts for a major fraction of current microscope utilization, but, due to its age, will soon lose service contract support. We seek to replace this dated and increasingly unreliable system with a new Olympus Fluoview system. Replacing the BioRad system with a new Olympus system will, 1. Provide users with a reliable instrument that will be supported by an ongoing manufacturer's service agreement, 2. Provide users with a modern system with enhanced performance and capabilities. The requested system is absolutely essential to the ICBM, which supports the research of over 68 NIH- funded laboratories, and is particularly crucial to the group of major users listed in this proposal, whose research has come to depend upon the current BioRad system. In addition to supporting Indiana University researchers, this system is also the primary microscope of our NIH-funded O'Brien Center, supporting the research of renal investigators throughout the world. The loss of this system will profoundly compromise the ability of the ICBM to function as a university core facility, and as a center of renal microscopy. Acquiring the new Olympus system will ensure the continued productive use of confocal/multiphoton microscopy at the ICBM Relevance - The proposed instrument is a crucial tool in the research of numerous biomedical investigators studying the cellular basis of disease. In particular, studies of living animals conducted with this instrument are uniquely powerful for elucidating disease processes and therapies in the context of the whole organism, making them better predictors of clinical outcomes. [unreadable] [unreadable] [unreadable]

The Digital Image Analysis core will complement the Intravital Multi-photon Microscopy Core by providing digital image processing services for O'Brien investigators to support data exploration and quantification. The Core will provide analysis services, will train investigators in the use of image analysis software and will develop new tools to enhance and extend the visualization and quantification of deep tissue microscopy images. In particular the Digital Image Analysis core will develop and distribute user-friendly software that will provide effective, efficient tools for quantifying 2-dimensional and 3-dimensional fluorescence microscopy images. These services will be made available remotely through the dissemination of free, flexible image analysis software and an interactive televisualization system. The Digital Image Analysis Core will provide researchers with powerful new tools enabling them to conduct novel studies addressing fundamental issues of renal physiology, cell biology and pathophysiology.

Fluorescent protein biosensors have grown into one of the most powerful tools for the study of cell biology. When combined with intravital microscopy, these biosensors have the potential to sensitively interrogate molecular, physiological and physical events occurring within living cells In the most relevant, in vivo setting. However, the utility of fluorescent protein biosensors for in vivo study has been limited by the difficulty of in vivo gene delivery, largely restricting their application to studies of cells in, or derived from transgenic animals, in order to realize the potential of fluorescent biosensors for studying cell biology in vivo, flexible methods of in vivo gene delivery must be developed. The goal of the Probe Delivery Core is to develop and characterize simple, inexpensive and effective methods for expressing fluorescent biosensors In the rodent kidney and to provide these methods to end-user investigators in the form of fully characterized probes and detailed protocols for the use of these probes and for the development of new probes. These probes will provide renal researchers with powerful new tools that will enable them to conduct novel intravital microscopy studies addressing fundamental issues of renal physiology, cell biology and pathophysiology.

DESCRIPTION (provided by applicant): Pharmacological and toxicological processes occur across a wide range of spatial and temporal scales and include multiple organ systems. A Systems Biology in silico toxicological model must include submodels that cover the multiple scales and the multiple tissues relevant to human medicine and toxicology. We will develop a liver centered mechanism based multiscale in silico simulation framework for xenobiotic toxicity and metabolism that incorporates four key biological scales: Population genetic and exposure variation scale Physiologically Based Pharmacokinetic (PBPK) whole body scale Tissue level and multicellular scale Subcellular signaling and metabolic pathways scales The multiscale in silico simulation will be centered on the liver, a critical organ in many toxicological, pharmacological, normal and disease processes. For our initial simulations of toxic challenge to the liver we will build a mechanism based in silico simulation of Acetaminophen (APAP) toxicity. APAP is a widely used over-the-counter pain reliever and fever reducer. An acute overdose of APAP is a leading cause of liver failure in the western world. APAP overdose leads to centrilobular liver necrosis that can progress to liver failure and in some cases patient death. Our multiscale in silico simulation will link existing open source modeling tools for the various spatiotemporal scales into an aggregate in silico model. This approach allows us to leverage existing tools, modeling modalities and models at the individual biological scales. Furthermore, this approach facilitates swapping models at individual scales without extensive modification of the sub-models at the other scales and allows us to leverage existing model development tools and resources. The complete multiscale in silico model will provide a mechanism based framework that incorporates effects at the various scales and will also provide a framework to predict changes in clinically used serum markers of liver function and failure. Our in silico simulation will be calibrated using microscopic imaging in the liver of a living mouse, mouse liver immune-histology, along with standard histology and serology in animal studies of APAP toxicity. The proposed in silico model is a first step in toxicity prediction 1. 2. 3. 4. simulatio that ultimately will lead to improved techniques for prediction toxicity of therapeutic agents and environmental toxins while simultaneously reducing the need for animal toxicity studies.

ABSTRACT (MICROSCOPY CORE) The overall goal of the Microscopy Core is to provide IDRC investigators with effective access to advanced methods of light microscopy, particularly the powerful new techniques of intravital microscopy developed during the previous grant period. The Microscopy Core utilizes the extensive facilities of the Indiana Center for Biological Microscopy (ICBM), the School of Medicine's 20 year-old core microscopy facility. The Core is equipped with three point-scanning confocal/multiphoton excitation microscopes, as well as a spinning-disk confocal microscope and multiple digital workstations supporting quantitative two- and three-dimensional image analysis. The Core is fully equipped to support intravital microscopy, providing investigators with space in a surgical suite with all necessary supplies for surgery, anesthesia, and animal monitoring. The Microscopy Core benefits not only from the extensive facilities of the ICBM, but also the reliable performance of systems that are fastidiously maintained by ICBM staff and continuously supported with manufacturers' service contracts. Specifically, the Core will apply and adapt methods of intravital microscopy that the ICBM has developed over the past 12 years to studies of the pathophysiology of diabetes and will develop and implement novel assays of protein and cell function based upon fluorescent protein biosensors, applying a combined approach of spectral and time-resolved fluorescence quantification. The Microscopy Core is Directed by Dr. K. Dunn, with Dr. R. Day serving as Associate Director. The Core leverages the expertise of a staff scientist and technician, and interfaces closely with the Islet & Physiology Core of the IDRC to ensure that tissue resources and surgical expertise are tightly integrated. The Aims of the Microscopy Core include: (1) Implementation of methods of quantitative intravital microscopy. (2) Continued development of intravital microscopy methods that will be applied to biological systems of interest to IDRC investigators, including approaches for studies of muscle and neurons in the brain. (3) Implementation of fluorescent protein biosensors for intravital and in vitro studies, including the optimization of biosensors for confocal and multiphoton microscopy, the design of viral vectors, validation of biosensor performance, optimized methods of image collection and quantitative analysis. (4) Training in the methods of microscopy and image analysis developed by the core for investigators whose needs exceed the capacity of the core. (5) Technical support for laboratories lacking microscopy expertise to utilize additional challenging techniques; e.g., fluorescent biosensor-based in vitro studies, fluorescence lifetime microscopy).

Summary (Digital Analysis and Development) Over the past 15 years, advances in molecular biology, genetic engineering and microscopy technology have provided biomedical researchers with the capability to use fluorescence microscopy to capture the dynamics of multiple processes occurring in living animals or to characterize the structure and constitution of tissue volumes approaching centimeter scale. The unprecedented size and complexity of this these data has extraordinary potential for enriching our understanding of the mechanisms underlying physiology and disease. However, realizing this potential requires image processing software and hardware powerful enough to address the size and complexity of the data, while being simple enough to be used by mainstream biologists. Since 2002, the IU O'Brien Center has pioneered intravital and 3-dimensional imaging of renal tissues, with the Digital Analysis and Development Core (DAD core) providing consultation, training and software development to facilitate the exploration and quantitative analysis of microscopy data. The ongoing goal of the DAD core is to develop image processing solutions that will help biologists explore and quantify complex image data. During the next grant period, this goal will be addressed via three specific aims: (1) Implement software tools previously developed by the core into an intuitive and efficient software interface. (2) Develop the Distributed and Networked Analysis of Volumetric Image Data (DINAVID) high-performance image analysis system that will support storage, management, visualization, quantitative analysis, and exploration of large (multiple gigabyte) image volumes collected by the 3D tissue cytometry core, and (2) support real-time by remote investigators and (3) Develop new tools for automated image segmentation based upon machine learning.

Project summary/Abstract This request is for funds to purchase a Leica SP8 MPE confocal/multiphoton fluorescence excitation microscope system. This system will be housed, managed and maintained by the Indiana Center for Biological Microscopy, a core facility of the Indiana University School of Medicine, the Indiana University NIDDK P50 George M. O'Brien Center for Advanced Renal Microscopic Analysis, the NIDDK-P30 Indiana Diabetes Research Center and the NIDDK-P30 Indiana Cooperative Center of Excellence in Hematology. The proposed Leica system is sought to replace two aging confocal/multiphoton microscope systems, one of which has outlived its eligibility for service contract support, and the other of which will be ineligible for support as of March, 2019. The microscopy capabilities of these two systems are crucial to the Center grants listed above as well as to a number of other NIH-funded projects. This proposal is predicated upon the need to avoid a dangerous dependence on these unsupported systems for the successful completion of these projects. In addition to replacing the Olympus systems with a more reliable system with an extended life- expectancy, the new system will also provide users with a more modern instrument with numerous advantages in both performance and functionality that will enhance the research of all users.