Marcel P. Bruchez - US grants

This proposal describes the development of semi-conductor nanocrystals (quantum dots) as probes for the specific detection of multiple markers of breast cell transformation simultaneously. Quantum dots are novel luminescent probes that are photo-stable, easily multiplexed and can be sensitively detected with simple, inexpensive optical systems. In phase I of the project we will use conjugated quantum dots to detect three selected breast cancer markers in sections of well-characterized breast cancer cell lines to demonstrate their specificity in a simple model system. In phase II we will multiplex this system, incorporate a fourth marker and develop it for use on tissues and tissue micro-arrays. We will detect the expression of three protein markers (p53, estrogen receptor and progesterone receptor). Initially we will characterize the assay specificity and performance of the probes individually. We will use this model to compare the stability and sensitivity of quantum dots with conventional fluorescent dyes. It is anticipated that this technology will allow the use of these probes to characterize breast tumor biopsies and ultimately quantum dots could be used to more confidently phenotype any disease for which multiple markers exist. PROPOSED COMMERCIAL APPLICATIONS: This research could directly lead to a novel product that allows more sensitive, quicker and more informative diagnostic and prognostic evaluation of breast cancer tissue sections. The technology is also directly applicable to many different kinds of tumor analysis and could be used on tissue microarrays to vastly increase the throughput and aid the archiving of tissue section analysis.

We aim to develop new probes and methods that will allow direct measurements of the spatio- temporal behavior of proteins in living cells. Using multifunctional dendrimers decorated with two dyes, a fluorescent donor array and a fluorogenic quencher dye, we will develop very high brightness probes that are activated by small fusion protein tags. Because the binding of these modules produces an intermittent signal, we will integrate these probes into a new superresolution imaging method that enables molecular scale localization and tracking of all tagged proteins in a sample region by stochastic activation at equilibrium. We will validate these new probes and methods in studies of [unreadable]2 adrenergic receptor and cytoskeletal dynamics. Collectively, this program will develop, validate, and deliver new imaging tools and methods to track positions and dynamics of tagged proteins at nanometer length scales. Collectively, these methods are anticipated to deliver at least an order of magnitude improvement in the single molecule brightness, the localization accuracy, and the timescale of dynamic single molecule measurements in living cells.

DESCRIPTION (provided by applicant): We will investigate the factors regulating the subcellular distribution of the high conductance, calcium- and voltage-gated BK channel, a critical ion channel that regulates neuronal firing output in health and disease. Despite its powerful role in modulating excitability, experimental evidence indicates that it is sparsely distributed at the plasma membrane, a phenomenon that is regulated via interactions with the brain-specific accessory subunit, ?4. Conventional methods to study membrane protein localization have relied heavily upon overexpression of tagged proteins, a method that can significantly alter protein distribution by changing the stoichiometry of the target with its regulatory factors. To accurately determine how BK channels are distributed across the cell, it is important to be able to determine the location of individual molecules at endogenous expression levels to preserve critical concentration-dependent interactions with regulatory partners. We have developed a novel protein/dye tag with high-fluorescence emission that enables single-molecule detection, for both high- and low-abundance proteins. To preserve normal channel expression levels, we will generate a transgenic mouse where this tag has been inserted into the endogenous BK channel gene. The localization of this channel in primary neurons derived from these animals will be evaluated, and its accessory subunit and activity-regulated surface distribution will be determined.

DESCRIPTION (provided by applicant): The regulation of the endothelial barrier in blood vessels is a coordinated signaling process that controls the exchange of oxygen and nutrients with the surrounding tissues. Dysregulation of barrier integrity or function is implicated in a range of pathologies, including metabolic disorders such as diabetes (affecting 8.3% of the US population) and cardiovascular disorders such as atherosclerosis (affecting 25% of the US population). Regulation of the barrier is controlled by small gaseous reactive signaling molecules (e.g. nitric oxide and superoxide) that have proven challenging to detect and quantify with adequate selectivity and sensitivity in cells and more importantly in complex tissues and living animals. Imaging has provided significant insight into regulation of the barrier and related physiologic changes that correlate with disease and disease treatment. Yet imaging of signaling molecules associated with the barrier continues to pose significant challenges because the currently available fluorescent biosensors are not sufficiently specific or sensitive to directly report the concentrations and locations of the analytes. Both dye based fluorescent probes and fluorescent protein sensors suffer from limitations that prevent simultaneous correlative measurements of molecular signaling and vascular physiology. In this proposal, we develop a new class of fluorescent molecular biosensor dyes that combine the advantages of indicator dyes with the specificity of genetic encoding. By using tissue specific expression and genetically encoded subcellular targeting, these new biosensors will allow detection of Ca(II), reactive oxygen species (ROS), and reactive nitrogen species (RNS) in specific cells, at specific subcellular locations. These novel targeted fluorescent biosensors are constructed by linking together a sensitive optical sensor of Ca, ROS, or RNS with a fluorescent signaling moiety (FRET acceptor) that is activated upon binding to a genetically encoded receptor, called a fluorogen activating protein (FAP). FAP-bound sensor is able to report (fluorescence signal) the physiology of the sensing at the site of interest. Any biosensors that are not bound to the FAP target are incapable of producing a fluorescence signal and there is no background or non-specific fluorescence to complicate images or analysis of the data. The targeted biosensor dyes will be optimized to work in both cultured endothelial cells and living zebrafish. Transgenic zebrafish will be generated that express the FAP at subcellular locations in specific cells using tissue specific Cre-recombinase expression. We will use these sensors in zebrafish to assess the correlation between Ca(II), ROS and NO signaling, blood flow and barrier function. This project is a close collaboration of three Principal Investigators with distinct expertise at Carnege Mellon University and University of Pittsburgh. Dr. Bruchez is an expert on the development of multichromophore structures for biological detection, and designed the hybrid indicators for biosensing using the fluorogen activating proteins; Dr. St. Croix is an expert on endothelial cell biology RNS/ROS signaling and regulation of endothelial function and more specifically the imaging of endothelium in vitro and in vivo. Dr. Waggoner is an expert in the design of environmentally sensitive dyes, and original developer of the fluorogen activating protein technology.

? DESCRIPTION (provided by applicant): Neurons in the cerebral cortex communicate with each other using highly specified, hierarchical rules of connectivity. There are more than 30 cell types in the neocortex, and these cell types can be differentiated by their developmental lineage, projection target, or expression of marker genes. Previous studies have attempted to reveal the logic of neural circuits by low-throughput anatomical or electrophysiological methods. Here we propose to develop and employ a novel trans-synaptic fluorescent complex formation strategy to chemically tag synapses defined by pre- and post- synaptic cell identity. Cell contacts made between genetically specified pre- and post- synaptic neurons will bring together a fluorescence-activating protein and one of a pair of covalently anchored fluorogenic dyes to trigger a 20,000-fold increase in fluorescence, easily detectable over background signal. The outstanding signal-to-noise and spectral properties of the dye will enable quantitative and in vivo analysis of cell-type specific synapses in the mammalian neocortex. We will use sequential labeling in different colors to differentially label newly formed synapses, allowing single endpoin measurement of synaptic density changes in response to experience. Applying these tools in the context of seizure models will reveal the cellular and molecular mechanism underlying changes in inhibition in cortex that result in increased seizure risk. The long- term goal of this proposal is to develop chemical biology tools for a complete index of cell-type specific synaptic contacts in order to establish how these contacts change in health and disease states.

? DESCRIPTION (provided by applicant): A key player in mast cell activation is the high affinity IgE receptor, FceRI. While much is known about the chain of events initiated by crosslinking of FceRI through multivalent antigen, there is a fundamental gap in our understanding of how protein dynamics facilitate signaling. Our long term goal is to understand how the dynamic and stochastic behavior of protein-protein interactions influences signal propagation. The objective of this proposal is to directly quantify protein interactions and changes in the surrounding membrane environment during early events in FceRI signaling. Our central hypothesis is that the spatiotemporal coordination of FceRI signaling complex formation modulates the strength and duration of the cellular response. The rationale for the proposed research is that understanding the role of protein-protein interaction dynamics in signaling is the next step in understanding how the cell shapes the strength and quality of an immune response. The goal of this proposal is to develop novel FRET measurements based on fluorogen activating peptide (FAP) technology that will provide improved signal detection for measuring protein-protein and protein-lipid interactions in living cells. This new methodology will complement our ongoing projects by allowing us to address additional questions about FceRI signaling dynamics that are simply unaddressable with conventional methods. The approach is innovative because we will develop methodology for imaging biochemical events at the molecular level that will allow us to obtain dynamic information about signaling events that cannot be determined using traditional biochemistry techniques. The proposed research is significant because the quantitative information that we propose to obtain has not been directly measured before and will bring new perspectives to the cell biology community. Furthermore the FAP-based FRET methods will be broadly applicable to other cell signaling pathways. The information we gain about FceRI signaling will help to fill the gaps in our knowledge of how FceRI initiates signaling. Ultimately, we expect that this information will open new avenues for drug design that target protein interactions and localization.

PROJECT SUMMARY Advancements in the field of microscopy and imaging have pushed the boundaries of what was once thought possible in many fields of research. New techniques coupled with the application of new technologies allows researchers to probe further and with greater accuracy to answer increasingly complex questions. While these new techniques allow for far greater specificity of observation and increased sensitivity in regard to both resolution and frequency, the amount of data generated is increasing to a point where conventional systems are unable to manage it. At the current time, there is no practical way to analyze, mine, share or interact with large (100+TB) brain image datasets. The development of a national, scalable archival solution for such datasets is a pressing problem extremely important and central to the NIH mission as in the future there will be a continuous and sustained growth in data scale. To address this issue, this proposal establishes the BRAIN Imaging Archive (or more simply ?the Archive?) data service in Pittsburgh PA as a collaboration of the Pittsburgh Supercomputing Center (PSC), the University of Pittsburgh (PITT), and Carnegie Mellon University (CMU). The Archive encompasses the deposition of datasets, the integration of datasets into a searchable web- accessible system, the redistribution of datasets, and a computational enclave to allow researchers to process datasets in-place and share restricted and pre-release datasets. The Archive will, for the first time, provide researchers with a practical way to analyze, mine, share or interact with large (100+TB) image datasets by creating a unique public resource for the BRAIN research community.

PROJECT DESCRIPTION Synapses are formed, broken and reformed dynamically both during development, normal function and in response to activity. Although this general principle is well-established, the way in which this is manifested in specific subtypes of neurons across a complex network, and how altered patterns of synaptic input will determine network function, have not been quantitatively investigated. Here we propose to develop molecular genetic tools for defining synaptic organization and connectivity in the mouse brain using fluorogen activating proteins (FAPs), a robust and modular system that enables multiplexed fluorescence identification of synapses and cell-specific connectivity. Our preliminary data indicate that we can target FAPs to synapses for quantitative analysis, as well as import 3D fluorescence image data for automated synapse detection using the image processing platform Imaris. Here we will create and validate pre- and postsynaptic targeting of fluorescent and FAP proteins respectively, acheiveing trans-synaptic FRET signal with high signal-to-background sensitized emission, allowing selective detection of synaptic connections formed between two genetically selected cell populations. These constructs, and the associated imaging and analysis approach, establish a pipeline for high- throughput data acquisition and analysis for assignment of cell-type specific contacts. As a test- bed for this technology, we will employ it to determine the synaptic input map for an important subset of cortical interneurons, somatostatin-expressing GABAergic cells, in the mouse neocortex.

Despite the predominance of monoclonal antibody based technologies, the means by which monoclonal antibodies are generated and disseminated, together with the size and structure of IgGs, impose fundamental limitations on their usefulness, especially for research purposes. Poor specificity and documentation has led to a crisis in experimental reproducibility, leading to an annual waste of ~$350 million in US research expenditures. Rectifying this by the systematic generation of sequenced, validated monoclonals would cost an estimated $50,000 per antibody and would fail for many targets. Limitations of monoclonals will be addressed by developing a yeast `secrete and capture' co-display system for the high throughput isolation and improvement of recombinant nonimmune Nanobodies (NBs), small protein affinity reagents derived from camelid antibody VHH domains. Fluorogen-based FACS technology that quantifies displayed NBs and captured target protein domains (TPDs) will be integrated with next generation sequencing (NGS) that identifies the associated complex. Integration will greatly expand the repertoire of biological targets for which cognate NBs may be isolated, and facilitate the creation of focused NB toolkits. Current nonimmune scaffold screens use purified target protein to isolate candidate binders that are physically cloned and individually evaluated. This resource-intensive approach will be replaced by the following: (1) A FACS reporter assay that quantitatively reports the interaction of co-expressed NB and TPDs in terms of specificity, affinity and kinetics, thus avoiding the use of purified protein. The assay is based on fusing surface-displayed NBs and secreted TPDs to spectrally distinct fluorogen activating proteins (FAPs) that fluoresce when binding non-fluorescent dyes (fluorogens); fluorogens may flexibly linked by PEG as a `tie-dye'. A cleavable tie-dye is used to stabilize and report on a cell surface complex of secreted TPD and cognate displayed NB; upon cleavage, kinetic analysis of TPD-FAP release from the cell surface allows one to estimate NB/TPD affinity. (2) A method that physically fuses the encoded genotypes of co-expressed NB and TPD, enabling NGS analysis to resolve FACS assays of complex populations into the binding phenotypes of individual clones, thus eliminating the need for physical cloning. After mass mating of yeast libraries respectively encoding NBs and secreted TPDs on separate plasmids, CRISPR will be used to force bulk recombination of sequences encoding the NB and a barcode that identifies the associated TPD into a single NGS decodable read frame. (3) Multiplexed screens in the form of bioinformatics derived TPD query sets to directly obtain groups of related reagents, thus minimizing the need to serially evaluate individual clones. Queries will be used to isolate NBs that probe biological functionalities that were previously very difficult to approach; our test cases will be: (i) neuroligin splice isoforms; (ii) neuroligin/neurexin complexes; and (iii) bacterial surface protein ectodomains.