1992 — 1993 |
Nonet, Michael L |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Analysis of Synapse Formation and Function in C Elegans @ University of California Berkeley
neurotransmitters; developmental neurobiology; neurogenesis; synaptogenesis; gene expression; genetic mapping; electron microscopy; Caenorhabditis elegans; mutant;
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0.933 |
1995 — 2007 |
Nonet, Michael L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms Controlling Synaptic Function
DESCRIPTION (provided by applicant): The primary means by which most neurons communicate with their target cells is through the regulated release of neurotransmitter from synapses. Modulation of the release properties of these synapses has long been postulated, and in certain circumstances been directly demonstrated, to be a critical component of the cellular mechanisms that underlie learning and memory. A molecular understanding of the mechanisms that mediate this release process is crucial to developing a comprehensive understanding of brain function both in health and in disease. Over a decade of molecular studies of the presynaptic terminal has led to the identification of over one hundred gene products that are implicated in the release process at the presynaptic nerve terminal. Yet, the role of many of these proteins in regulating release still is unknown. Homologs of most of the molecules implicated in this process are present in the nematode Caenorhabditis elegans. Included among these conserved molecules are about a dozen proteins implicated in the function of rab3, a small synaptic vesicle-associated GTPase of the ras superfamily. Analysis of mutants lacking rab3 components in C. elegans and mouse have demonstrated a role for this pathway both in regulating basal transmitter release and in presynaptic-forms of synaptic plasticity. This proposal aims to utilize molecular genetic tools available for the study of C. elegans to further dissect the role of rab3 pathway constituents in synaptic function. Nematode mutants lacking four rab3 components have been previously isolated and characterized. Analysis of these mutants has revealed that some of these molecules play central roles in neuronal function, while others appear to be largely dispensable. Building upon this foundation of mutants previously isolated in C. elegans, we propose to isolate additional mutants lacking rab3 pathway components and to characterize the neuronal defects of animals lacking these molecules. Secondly, we propose several lines of experimentation aimed at dissecting the molecular mechanisms that govern how neurons localize a subset of these pathway components specifically at the nerve terminal. The ultimate aim is to integrate the role of the rab3 regulatory machinery seamlessly into a detailed description of the basic mechanisms that underlie the release of neurotransmitter from synaptic terminals.
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1 |
2000 — 2018 |
Nonet, Michael L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Molecular Genetic Analysis of Developing Synapses
DESCRIPTION (provided by applicant): The primary means by which nerve cells communicate is via the release of neurotransmitter at chemical synapses. The function of the brain and the ability of the brain to store and process information depends on synaptic connections forming precisely and reliably. This proposal is directed towards developing a molecular understanding the mechanisms that regulated this process of synaptogenesis. We propose to use a combination of genetics, cell biology, molecular biology and live imaging to identify and characterize the role of molecular components of the signaling pathways that coordinate synaptic development by studying the specific assembly of a set of nerve-nerve synapses. In previous work, we described the order of cellular events in nascent synapse formation by visualizing the recruitment of fluorescent-tagged components to newly forming synapses. Furthermore, we isolated mutants that disrupt the formation of these synapses. Cloning of a subset of these identified an F-box protein that selectively targets proteins for ubiquitin-mediated degradation, several regulators of the cytoskeleton, a transcriptional regulatory protein and a novel conserved protein. Using a variety of molecular, genetic and protein interaction studies we now propose to determine at a mechanistic level how these proteins function to regulate synapse assembly. In addition, we will use genetic approaches to isolate and characterize additional genes that disrupt signaling between mechanosensory neurons and their synaptic partners in C. elegans to extend our molecular understanding of the process. Together these approaches will help define mechanisms that cells use to identify and communicate with one another during the process of synapse formation and synaptic maintenance. While synaptogenesis is undoubtedly less complex in C. elegans than in vertebrates, it is already clear that similar pathways operate in both systems. Thus, analysis of the molecules participating in the process in C. elegans should help define a set of general and likely conserved principles that are common to synaptogenesis mechanisms in general.
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2005 — 2007 |
Nonet, Michael L |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Monoclonal Antibody Resources For C. Elegans
DESCRIPTION (provided by applicant): Antibodies (Abs) are widely used to detect specific cellular components both in situ and in biochemical experiments aimed at defining protein-protein interactions, protein processing, and reversible protein modifications such as phosphorylation. Polyclonal Abs are relatively simple to make and are widely used in molecular and cellular studies. However, such reagents are limited in quantity and thus rarely made widely available. Monoclonal Abs are more complicated to make, but hybridomas secreting the monoclonal antibody can be passaged ad infinitum permitting the identical reagent to be used by many different research group over long periods of time. Polyclonal and monoclonal Abs are available commercially that are directed against a huge array of vertebrate cellular components. By contrast, few such commercial reagents are available for work with model organisms such as C. elegans probably because the market for these reagents is too small to make them commercially viable. I propose to develop a 'tool kit' consisting of monoclonal Abs directed against a series of proteins that label discrete cellular components and subcellular compartments of C. elegans. The compartments include organelles common to all cells (e.g. endoplasmic reticulum, the Golgi, endosomes), organelles of specialized cells (e.g. synaptic vesicles), subcellular structures common to all cells (e.g. nuclear membrane, centrosomes and caveoli) and components that label domains in the developing embryo, germline, and nervous system. The cellular components include macromolecular complexes (e.g. splicesomes, proteosomes, replication origin complexes) and common cellular proteins (e.g clathrin, kinesin, dynein and intermediate filaments). The Abs will be thoroughly characterized and made publically available through the Developmental Studies Hybridoma Bank. The tool kit will be extremely useful for C. elegans researchers. It will provide a set of standardized renewable reference antibody tools available to the entire community. Secondly, it will greatly facilitate localizing other cellular components by providing a large set of cellular markers that can be used coordinately with rabbit polyclonal antibodies for co-localization studies. Finally, it will stimulate the use of biochemical approaches, such as subcellular fractionation and immunoprecipitations, by providing means to detect specific cellular compartments in extracts and the presence of specific components in immunoprecipitates.
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2006 — 2007 |
Nonet, Michael L |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Synaptic-Gfp Tags For Zebrafish
[unreadable] DESCRIPTION (provided by applicant): GFP-tags have been extremely useful in the genetic dissection of molecular pathways that regulate the formation of synapses in model organisms such as C. elegans. Similarly, many such fusion proteins are now being used to examine synaptic development in cultured mammalian neurons. However, such fusions remain largely unutilized in Zebrafish even though the fish is uniquely suited for forward genetic dissection of synaptic development because of its fast development, transparent body during larval development, and the ease of genetic manipulation. With the genome sequence of Zebrafish nearing completion, such tools are ripe for development in the Zebrafish. As a first step in developing a research program in Zebrafish aimed at dissecting the molecular mechanisms that regulate the specificity of synapse target selection, I propose to create a set of GFP-tagged synaptic proteins to facilitate the analysis of synapse formation in vivo in Zebrafish embryos and larvae. Using a combination of modern recombinant DNA technologies (the GAL4/ DAS system, Gateway cloning technology, transposon vectors, and bacterial artificial chromosomes), I propose to create vectors for expression of Zebrafish synaptic protein-GFP fusions and transgenic Zebrafish animals expressing these synaptic protein-GFP constructs. We will concentrate on 'moving' constructs that we have shown in C. elegans to express robustly and precisely and label either synaptic vesicle populations or the active zone domain of the synapse. Specifically, we have recently created a novel fusion to a C. elegans synaptic vesicle protein that allows for 10-fold more sensitive detection of synapses in live worms. Furthermore, this C. elegans fusion works across species as the worm protein fusion localizes robustly to the neuromuscular junction when expressed in Drosophila. Preliminary studies indicate that the equivalent Zebrafish fusion forms puncta when expressed in neurons in a transient expression system suggesting it localizes to synapses. We propose to develop this fusion in Zebrafish to label presynaptic specializations. Similarly, we propose to test whether several recently developed C. elegans active zone tags will permit the visualization of Zebrafish active zones when the analogous Zebrafish protein fusions are expressed in Zebrafish neurons. [unreadable] [unreadable]
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2008 — 2009 |
Nonet, Michael L |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Transgenic Tools For Nematodes
DESCRIPTION (provided by applicant): Modern biology research relies extremely heavily on transgenic tools. This is true of virtually all model systems that are being used to explore questions as diverse as the cell biology of mouse brain development to the highly specialized antigenic variation-based immunological defenses of human pathogens like trypanosomes. In the last five years, rapid advances in transposon and integrase molecular engineering and recombinant DNA technology have led to the development of sophisticated transposon and integrase based transgenic techniques for many model systems. These tools include efficient transposons for zebrafish, efficient site- specific integration systems for Drosophila as well as a new generation of genome engineering tools for mice. These advances have not been applied to all common model systems. In particular, few improvements have been made in transgenic methodologies for the C. elegans model system. Development of these tools is critical to leveraging the substantial genetic, molecular, and intellectual resources that have already been committed to develop this promising model system. This grant proposes to develop molecular genetic resources by testing the feasibility of using two mobile genetic elements for creating transgenic nematodes. The utility of these tools will include the improved ability to create mutations to study gene function, to create gene reporters for cell biological analysis, and to create transgenic animals for the study of basic cellular processes that underlie the cause of many human diseases. Public Health Relevance: This grant proposes to develop transgenic tools for a model system used in the study of basic cellular processes that underlie human disease. The contributions of this model system to understanding disease include the discoveries of several biological processes each of which was recently recognized by a Nobel prize. The first discovery is that of programmed cell death which plays critical roles in cellular responses to stroke. The second is the discovery of the process of RNA interference that is widely recognized as very promising methodology to treat human diseases such as cancer that result from the mis-expression of genes. Basic research such as the work proposed herein is essential for long-term progress in public health.
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1 |
2012 — 2013 |
Nonet, Michael L |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Quantifying Cellular Complex Composition in Vivo
DESCRIPTION (provided by applicant): Modern molecular biological approaches have provided enormous insight into a vast array of cell biological mechanisms from polarized cell division to neuronal cell migration, autophagy, neuronal exocytosis and countless others. While many molecular players participating in these processes have been elucidated defining the more mechanistic and structural aspects of these processes has been more difficult. Importantly, these mechanistic insights provide the cellular basis for our understanding of disease and serve as the primary guides for developing therapy. One systemic problem in developing mechanistic insight has been the difficulty in obtaining quantitative data about the organization and stoichiometry of components in many cellular complexes. For example, over a dozen proteins have been documented to localize to the cytomatrix of synaptic active zones, a structural presynaptic feature essential for vesicular neurotransmitter release site from neurons in the brain. Furthermore, proteomic analysis of synaptic active zones suggests that the number of distinct proteins associated with presynaptic release sites is greater than one hundred. Defining the structure and organization of such complex subcellular assemblies is a daunting challenge, but it is essential if we wish to understand the basic cellular process of neurotransmitter release in the brain at a mechanistic level. Here we propose to develop methodology for assessing quantitatively the composition of various components of subcellular assemblies in vivo. Multiple methods for performing such quantitative measurements have previously been described, but none are easy to implement for the analysis of structures in complex multicellular organisms. Herein we propose to create in vivo internal calibration standards that can be used in combination with fluorescent protein fusions to quantify levels of specific proteins present at specific sites in vivo. Specifically, we propose to use GFP-LacI bound to LacO sites integrated in known copy number to specific sites on chromosomes as a calibration curve to quantify the numbers of molecules found in subcellular assemblies in vivo. We propose to develop the system in yeast, transfer the technology to C. elegans, and then apply the technology to characterize the relative and absolute stoichiometry of a half dozen proteins found at presynaptic active zones. The system we propose to develop should be applicable to any molecular genetic model system that can be manipulated using transgenic techniques including mouse and zebrafish, as well as human cell culture. PUBLIC HEALTH RELEVANCE: This grant proposes to develop methodology and tools for a model system that will enable better quantification of components of subcellular assemblies in vivo. Model systems are used in the study of basic cellular processes that underlie human disease. Understanding of cellular mechanisms is a fundamental requirement for designing therapy. The contributions of this model system to understanding disease include the discoveries of several biological processes each of which was recently recognized by a Nobel prize. The first discovery is that of programmed cell death which plays critical roles in cellular responses to stroke. The second is the discovery of the process of RNA interference that is widely recognized as very promising methodology to treat human diseases such as cancer that result from the mis-expression of genes.
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1 |
2014 — 2015 |
Nonet, Michael L |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Genetically Encoded Sparse Labeling and Expression For in Vivo Studies
DESCRIPTION (provided by applicant): Sparse labeling of experimental tissue has been a powerful approach for cell biological analysis from the time of Ramon y Cajal, who used Golgi staining to describe in great detail the structure of neuronal tissue. To this date such labeling i critical for the analysis of neurons in whole tissue as the density and complexity of axonal and dendritic processes make tracing individual cells virtually impossible in ubiquitously labeled samples. Sparse labeling techniques also play critical roles in examining the behavior of cells during migration, in determining lineage relationships of cells, and assessing the cell autonomy of gene function. A variety of approaches have been used to sparsely label samples both in fixed and living tissue including Golgi staining, single cell dye injections, transient expression f DNA constructs, viral infection and cell transplantation. In vertebrate systems, several transgenic approaches to sparsely label cells have also been developed including use of promoters that display extensive variegation in expression, drug inducible activation of promoters and stimulation of mitotic interchromosomal recombination. However, none of these approaches for obtaining sparse labeling are easily applied for large-scale analysis in vertebrates (genetic screens or drugs screens). We propose to develop a robust, reliable method to create transgenic animals that will sparsely label a cell population and be readily reproduced from generation to generation. Conceptually, the idea is to use a newly described CRISPR/Cas9 site-specific nuclease to drive non-homologous end joining DNA repair in select cells to modify a non-expressed GFP reporter into one that expresses. If successful the approach should be widely applicable for the analysis of neuronal structure (and other cell types also) in mouse or zebrafish models of human neuronal diseases.
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1 |
2021 |
Nonet, Michael L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Novel Transgenesis and Expression Technology For Nematodes
Recombinant DNA technology plays an integral role in virtually every research program using the C. elegans model to dissect conserved biological mechanisms that mediate many aspects of human biology in health and disease. While the development of CRISPR technology has revolutionized the ability of scientists to make small modification of the genome, creating transgenic animals that contain large multi-kilobase inserts remains laborious. These types of transgenic animals are required for many critical aspects of dissecting cellular mechanisms including to express genes in specific cell types, to tag and visualize sub-cellular components, to monitor concentrations of signaling molecules using genetically encoded sensors, and to perturb cellular functions using RNA interference and selective protein degradation technologies. I have recently developed a novel recombinase-mediated cassette exchange approach for C. elegans that increases the frequency of transgenesis about five-fold over current techniques. Furthermore, I used this novel technology to develop four bipartite reporter systems for use in nematodes to facilitate robust expression of transgenic tools. While the novel approach is a significant improvement over current approaches, it remains greater than an order of magnitude less efficient than CRISPR technology. Insights made during the development of the technique point to critical limitations that this grant aims to overcome to further increase the efficiency of the approach. Furthermore, the new approach comes with significant limitations due to the use of Flp and Cre recombinases. This grant also proposes further technological development of the approach to overcome these limitations. Successful implementation of the proposed work would have extreme impact on the C. elegans research community by greatly facilitating transgene development removing this common bottleneck for many research programs.
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