Barry Ganetzky - US grants

Segregation distorter (SD) is a naturally occurring meiotic drive system on the second chromosome of Drosophila melanogaster. Heterozygous SD/SD+ males transmit the SD chromosome in vast excess over the normal homolog. The basis of the distorted transmission involves the induced dysfunction of the spermatids the receive the SD+ chromosome but the underlying molecular mechanism is unknown. The SD system comprises at least three distinct loci: Sd, E(SD) and Rsp. Our long term goals have been to characterize these loci with the aim of elucidating the structure, function, and origin of SD chromosomes. Molecular analysis of Sd indicates that it is associated with a tandem duplication of a normal genomic segment. The tandem duplication encodes an Sd-specific 4.2 kb transcript. In addition, the duplication specifies a family of 2 kb transcripts some or all of which are also present in Sd+. Sequence analysis of Sd and Sd+ cDNAs reveals that most members of the 2 kb family share the same ORF. cDNAs corresponding to the 4.2 kb transcript have not yet been identified. Our data suggest that the neomorphic phenotype of Sd results from expression of a novel fusion protein or from the ectopic or overexpression of a normal (Sd+) protein. To elucidate the molecular nature of Sd in greater detail we propose to: isolate and characterize cDNAs corresponding to the 4.2 kb transcript; use anchored PCR techniques to characterize the complete set of transcripts in the 2 kb family and to compare this array in SD and SD+ with emphasis on identifying any transcripts that may be Sd-specific or expressed preferentially in testes; raise antisera against the common ORF for Western blot and immunolocalization analysis of Sd and Sd+ polypeptides;a nd isolate and characterize Sd+ null mutations. Insight into the segregation- distortion system should enhance our understanding of a fundamental genetic mechanism, meiotic drive. But it is also clear that such an understanding could be of great practical use in designing gene vectors which have an insertion advantage.

psychophysiology; behavioral genetics; neurophysiology; genetic regulation; nervous system; neural transmission; alleles; genetic mapping; electrical potential; genetic manipulation; molecular cloning; gene expression; synapses; gene mutation; genetic recombination; gene complementation;

The electrically excitable cell membrane of neurons and muscle fibers these cells to receive, process, and transmit information within the nervous system. Electrical excitability is conferred by transmembrane proteins called channels that play the key roles in action potential propagation and synaptic transmission by mediating fluxes of specific ions across the membrane. Despite important recent advances, many unanswered questions remain concerning the structure, function, and regulation of ion channels. The long term goal of this work is to elucidate the molecular mechanisms that underlie membrane excitability by isolating and characterizing mutations in genes that encode ion channels or that otherwise affect their function. Experiments described in this proposal focus on the three mutations: eag (ether a go-go), slo (slowpoke), and nap (no action potential). We have shown that each of these mutations profoundly disrupts membrane excitability owing to specific defects: eag diminishes the function of one class of potassium channels; slo entirely abolishes the function of another class of potassium channels; nap reduces the number and/or function of sodium channels. However, the molecular lesions responsible for these defects are not yet understood. The mutations could alter genes encoding structural components of channels or cause perturbations in one of the cellular mechanisms thought to regulate channel activity or synthesis. Genetic, electrophysiological, and molecular experiments are proposed to elucidate the underlying defects in these mutants. We will complete the cloning of eag and nap, map the molecular limits of these loci, identify their transcripts, isolate and sequences cDNA clones and use this information to characterize the proteins encoded by these genes. We will perform a detailed cytogenetic analysis of slo to isolate new alleles and to assign slo a precise cytological location. Based on the information and new mutants thus generated the molecular analysis of slo will be initiated by chromosome walking or transposon tagging. The new slo alleles will be characterized electrophysiologically to determine whether they display the array of phenotypes expected for defects in a structural gene. Finally, we will identify mutations at new loci by continuing our screens for mutations that (1) suppress or enhance existing membrane excitability mutations or (2) display any locomotor defects such as paralysis or uncoordination. These studies will provide new information on the structure and function of ion channels and on the mechanisms that regulate their synthesis, assembly, membrane insertion, nd modulation. Since various human disorder are known to be associated with perturbations in the function of ion channels, the information we obtain in Drosophila should have broad biological and medical significance.

Information is encoded and transmitted in the nervous system in the form of electrical impulses or action potentials. Neurons are characterized by an electrically excitable membrane that enables them to receive, process and relay these impulses. Transmembrane proteins, called ion channels, form voltage-sensitive, ion-specific pores in these cells and carry out the key functions in nerve signalling by mediating the fluxes of particular ions across the membrane. Sodium channels in particular perform the central role in the generation and propagation of action potentials. Despite important recent advances, many unanswered questions remain concerning the structure, function and regulation of sodium channels. The long term goal of the work proposed here is to elucidate the function and regulation of sodium channels in Drosophila by use of a combined genetic and molecular approach. We will concentrate on the para locus, mutations of which cause lethality or temperature-sensitive paralysis correlated with a block in nerve conduction. We have cloned the para locus, determined the sequence of the encoded protein and demonstrated that para is a sodium channel structural gene. The existence of other putative sodium channel loci in Drosophila besides para suggests that, as in mammals, these genes comprise a small family whose members are differentially utilized and may subserve physiologically distinct functions. To understand the function and regulation of para in the Drosophila nervous system we now propose to characterize its expression and the distribution of its gene product and to examine the perturbations caused by mutations at para and two other loci (nap and tip-E) that apparently interfere with expression or function or para. To accomplish these goals we will use NOrthern blot and nuclease S1 experiments to determine the abundance and developmental profile of para transcripts in wild type. The normal spatial distribution of para transcripts will be characterized by tissue in situ hybridization. para- specific antibodies will be raised against synthetic peptides or para-lacZ fusion proteins and used for immunolocalization of the paraprotein in the nervous system and to identify it on Western blots. To elucidate the phenotypic effects of para, nap and tip-E mutations, similar experiments will be carried out on these mutants to determine their effect on the structure, abundance or distribution of the para transcript or protein. Finally, we will develop germline transformation for para by use of a hybrid genomic/cDNA fusion gene to pave the way for future experiments that will utilize site-directed mutagenesis for more detailed studies of para function and regulation in vivo. Because para is the only sodium channel structural gene in any organism that has been mutated in situ, it provides a unique opportunity to obtain novel insights into to molecular mechanisms of sodium channel function, expression and regulation in vivo. Since a number of human neurogenetic disease are known to be associated with perturbations in the function of ion channels, the information we obtain may have significant implications for the understanding and treatment of these disorders.

Our long term aim is to elucidate the molecular mechanisms of neural signaling by characterization of mutations in Drosophila that affect ion channels. We discovered a new, evolutionarily conserved family of voltage-activated K+ channels consisting of three subtypes (Eag, Elk, and Erg) encoded by the genes eag, elk, and sei. A primary goal of the present application is to characterize the physiological properties of these channels and to elucidate their in vivo functions, using a combination of genetic, molecular, and electrophysiological techniques. We will relate the physiological roles of these channels with the molecular structure and function of the corresponding polypeptides by in vivo electrophysiological studies of eag and sei mutants, immunohistochemical analyses of the distribution of the channels, and biophysical characterization of wildtype and mutant channels expressed in Xenopus oocytes. We will extend these analyses to Elk channels as well by isolation and characterization of elk mutations. New genes that regulate the expression, localization, or function of Erg channels will be sought by screening for enhancers of sei. Such mutations can pave the way for molecular dissection of these regulatory mechanisms, as our work on Hk has shown. We discovered that the Hk locus encodes a K+ channel Beta subunit that coassembles with, and affects the functional properties of, pore-forming subunits. We also found that Hk is a member of a family of NADPH-utilizing oxidoreductases. To further elucidate the role of Beta subunits in regulating the diversity and function of K+ channels in vivo, we will perform molecular and electrophysiological experiments to describe the expression, localization, and properties of Hk splice variants. The possibility that Hk retains an essential enzymatic activity will be tested by characterizing the electrophysiological effects of site-directed mutations in vivo and in Xenopus oocytes. Because Drosophila is the only organism in which in vivo studies of K+ channel mutations can be readily combined with in vitro manipulations and functional analyses in heterologous systems, these studies will yield novel insights on K+ channel structure, function, expression, regulation, diversity, and evolution. Since K+ channels are essential for neural function in all higher organisms and mutations of K+ channels (including members of the Eag family) cause genetic disease in humans, results from our studies should continue to have broad biological and medical significance.

DESCRIPTION: Na channels perform the central role in the generation and propagation of action potentials, which are the primary form of electrical signalling in the nervous system. The long term objective of the proposed work is to elucidate the molecular mechanisms that underlie neural signalling. Here, the investigator focuses on analysis of the structure, function, and regulation of two Na channel structural genes in Drosophila, called para and Dsc. These Na channel genes comprise a small family whose members appear to be differentially utilized and could subserve physiologically distinct functions. The goals are to understand the respective functions of these two Na channel genes in the Drosophila nervous system, the basis of their differential expression, and the phenotypic consequences of mutations. The investigator will approach these goals using a combination of genetic, molecular, and electrophysiological techniques. To characterize Dsc, cDNAs representing the entire open reading frame will be isolated and sequenced; null mutations will be generated by imprecise excision of P element insertions within the gene; and behavioral and electrophysiological phenotypes of these mutants alone and in various double mutant combinations will be characterized. To characterize further the spatial and developmental expression of para and Dsc and to elucidate some of the regulatory mechanisms governing their expression, the investigator will: raise para- and Dsc-specific antisera for immunolocalization of the corresponding channel polypeptides; examine the cellular distribution of the large array of para splice forms using exon- specific probes for in situ hybridization, splice-dependent reporter constructs, or single-cell PCR analysis; and identify the upstream transcriptional regulatory sequences of para for use in germline transformation experiments. To explore structure-function relationships of para and Dsc, the investigator will determine the exact lesion in a large collection of in vivo-generated para mutations using a sensitive SSCP technique in combination with sequence analysis. Electrophysiological recording of Na currents in cultured Drosophila neurons and in heterologous expression systems will be used to compare the properties of channels encoded by wild-type and mutant alleles of para and Dsc as well as by the different splice variants of para. Finally, the investigator proposes to use a PCR-based strategy to search for additional Na channel genes in Drosophila that will be targeted for mutagenesis and phenotypic analysis. Because para and Dsc are the only neuronal Na channel genes in any organism that have been mutated in situ, these genes provide unique opportunities to obtain novel insights into the molecular mechanisms of Na channel expression, function, and regulation, in vivo. A number of human hereditary neuromuscular diseases are known to be associated with perturbation in the structure or function of ion channels, including Na channels. Therefore, the results obtained from these studies could have direct significance for the understanding and possible treatment of these disorders.

Segregation Distorter (SD) is a naturally occurring meiotic drive system on the second chromosome of Drosophila melanogaster with the property that heterozygous SD/SD+ males transmit the SD chromosome to their offspring in vast excess over the normal homolog. The basis of this distorted transmission is the dysfunction of spermatids that receive the SD+ chromosome. Dysfunction of these spermatids is associated with a failure of chromatin condensation. The SD system comprises a collection of genetic elements that act in concert to cause distortion. These loci include: Sd, E(SD), M(SD), St(SD) and Rsp.Sd is the gene primarily responsible for distortion; E(SD), M(SD) and St(SD) are strong upwards modifiers of distortion; and Rsp is the target site at which Sd and the modifiers act. SD chromosomes carry an insensitiveRsp allele whereas SD+ homologs whose transmission is affected carry a sensitive Rsp. The sd locus has been demonstrated to encode a truncated version of the nuclear transport protein, RanGAP. However, the mechanism by which this altered protein causes selective sperm dysfunction is unknown. It is hypothesized that the mutant protein has an aberrant location inside the nucleus rather than outside which could disrupt nuclear import and export leading ultimately to failure of chromatin condensation and sperm dysfunction. To explore this model the developmental changes that occur in nuclear morphology during spermatogenesis in normal and distorting males will be determined, the subcellular distribution and localization of wildtype and mutant RanGAP during spermatogenesis in distorting and non-distorting backgrounds will be characterized and the nuclear vs cytoplasmic distribution of GFP reporter constructs containing nuclear import and export signals will be observed in order to test the hypothesis that nuclear transport is defective. M(SD) and Su(SD) will be studied. Identification of the proteins encoded by these genes and a determination of their cytological location will be the initial steps in an attempt to understand their suppressor effects. Identification of the Sd gene product as a mutant RanGAP raises questions about the role in distortion of other components of the Ran system and thus, the consequences on distortion of overexpressing wildtype or dominant negative forms of Ran and RanGEF in the male germline will be explored.

Analysis of SD is significant because it represents a striking exception to the fundamental principles of genetic transmission and population genetics and may therefore provide insights into the mechanisms that normally ensure the fidelity of these processes, the perturbations that can affect these mechanisms, and the consequence of such perturbations. Furthermore, SD appears to involve disruption of basic biological processes such as spermatogenesis and the regulation of chromatin structure and function and can thus be informative about their genetic control.

DESCRIPTION (provided by applicant): Our long-term aim is to elucidate molecular mechanisms of neural signaling using a genetic approach in Drosophila. Here we focus on synaptic development and plasticity of the larval neuromuscular junction (NMJ). We have discovered several mutations among our collection of ts-paralytics, including nwk and 9-76, that cause striking defects in NMJ development and arborization, providing us with novel starting points to dissect regulatory mechanisms that control synaptic growth and plasticity. Our goals are to determine the in vivo functions of these genes using genetic, molecular, biochemical, and confocal microscopic techniques to analyze how defects in particular proteins result in the observed synaptic phenotypes. nwk causes NMJ overgrowth with an increase in bouton number and hyperbranching. It encodes an SH3-domain containing adaptor protein conserved from yeast to humans that binds directly to WASP, a regulator of actin assembly. It localizes to presynaptic periactive zones, a region specialized for endocytosis and regulation of synaptic growth. We have found that Nwk interacts genetically and biochemically with known endocytic proteins. Conversely, NMJ overgrowth in Nwk is suppressed by decreasing TGFp signaling. We hypothesize that Nwk normally functions to integrate actin assembly with endocytosis to downregulate TGFp signaling to sculpt synaptic growth. Genetic epistasis, yeast two-hybrid, and immunocytochemical experiments are proposed to test this hypothesis. 9-76 also causes NMJ overgrowth and hyperbranching. We found that it corresponds with the clumsy locus, which encodes a kainate-type glutamate receptor (GluR). Rescue experiments show that clumsy* is required presynaptically rather than postsynaptically for normal NMJ growth. Strong presynaptic overexpression of c/umsy+ mimics the 9-76 mutant phenotype and our results suggest that 9-76 encodes a mutant subunit that is overactive. We hypothesize that the Clumsy GluR is a component of a presynaptic signaling mechanism through which boutons self-monitor glutamate release enabling NMJ growth regulation in response to synaptic activity. We will test this hypothesis and dissect the affected mechanism by immunolocalization, structure-function, overexpression, and genetic epistasis experiments. Additional novel mechanisms regulating synaptic growth will be studied by phenotypic and molecular analysis of new mutants we have discovered that cause NMJ overgrowth or undergrowth. Synaptic growth and plasticity are of fundamental importance to neural communication, learning, and memory and are disrupted in many human neurological diseases. In particular, mutations of human homologs of Nwk have been associated with severe mental retardation. Consequently, our proposed analysis of nwk and other mutations affecting synaptic growth should have broad biological and medical significance by contributing important new information that will advance our understanding of the underlying molecules and mechanisms that regulate synaptic growth and plasticity.

[unreadable] DESCRIPTION (provided by applicant): Funds are requested to purchase a Zeiss LSM 510 laser-scanning confocal microscope for integration into the Genetics Training Program (GTP) Imaging Facility. The University of Wisconsin GTP encompasses more than 75 faculty trainers in 10 departments and 3 colleges. It is recognized as one of the premier programs worldwide for research and training in Genetics. To maintain and advance our leadership in this field and to continue to train the most promising scientists, it is essential to have access to the state-of-the-art instruments and facilities on which our research depends. A confocal microscope has become one of the most essential research tools in modern biology, regardless of the particular experimental organism or particular problem under investigation. However, we currently lack consistent, affordable access to a confocal microscope. To fill this need, we are applying here for funds to purchase a confocal microscope. Eight primary users who will manage and subsidize maintenance of the microscope are submitting this application on behalf of the entire GTP. With reliable access to a confocal microscope, GTP scientists will be able to (i) simultaneously observe the cellular and sub-cellular localization of three different molecules (ii) follow the in vivo expression and localization dynamics of multiple proteins; (iii) visualize thin optical sections of thick biological specimens; and (iv) obtain high-resolution 3-D reconstructions of their specimens. The addition of a confocal microscope to our imaging facility will have a major impact on the research programs of each of the primary users. These programs range from studies of yeast to mice, and address fundamental questions in biology from the regulation of gene expression to the molecular mechanisms of cell division, from embryonic and organ development to neural signaling and memory formation. Relevance: The ongoing studies of the primary users are yielding important insights into the biological processes underlying human disorders including cancer, birth defects, mental retardation, deafness, blindness, and neurodegeneration, among others. We also will attract new users from within the GTP, so an accessible confocal will broaden and strengthen the research programs of laboratories beyond those of the initial primary users. All of the GTP faculty are engaged in research of significant relevance to human health; most have current funding through NIH. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our long-term aim is to elucidate molecular mechanisms of neural signaling in Drosophila. Here we focus on analysis of mutants with defects in maintenance of neuronal viability. We have discovered several mutations among our collection, including wstd and comt, that exhibit shortened lifespan and age-dependent, progressive neurodegeneration providing us with novel starting points to dissect neuroprotective mechanisms. Our goals are to determine the in vivo roles of the affected proteins using genetic, molecular, biochemical, and histological techniques to analyze how defects in these proteins result in the observed phenotypes. wstd encodes the glycolytic enzyme, triose phosphate isomerase (Tpi) responsible for the interconversion of DHAP (dihydroxyacetone phosphate) and GAP (glyceraldehyde 3-phosphate), only the latter of which is able to continue through glycolysis. Mutations of Tpi in humans result in Triosephosphate isomerase deficiency, characterized by early death and neurodegeneration but the underlying mechanism has remained unclear. We hypothesize that the enzymatic block in Tpi-deficient flies and humans leads to excess accumulation of methylglyoxal (MG), which reacts with target proteins to generate advanced glycation end products (AGEs) causing loss of protein activity, cross-linking, aggregation, and ultimately neuronal death. We propose genetic and biochemical experiments to test and refine this hypothesis. comt, which encodes NSF-1 (N-ethyl- maleimide sensitive fusion protein), exhibits a deficit in lysosomes and accumulation of ubiquitinated protein complexes in parallel with neurodegeneration. We hypothesize that comt is deficient in autophagy. Experiments are proposed to test this hypothesis and to dissect the step(s) in the process that are impaired. Additional mechanisms of neuroprotection will be investigated by phenotypic and molecular analysis of other mutants in our collection that exhibit neurodegeneration. Neuroprotective mechanisms are essential for proper neural communication and their disruption leads to some of the most devastating human neurological disorders. Detailed understanding of these mechanisms is thus of fundamental biological as well as medical importance. Drosophila has already proven to be a potent experimental system for elucidating these mechanisms. Moreover, both wstd and comt have direct links with human neurodegenerative disorders. Consequently, our proposed analyses should have broad biological and medical significance by contributing important new information that will advance our understanding of the underlying molecules and mechanisms that maintain neuronal viability and integrity.

DESCRIPTION (provided by applicant): For decades, the Drosophila larval neuromuscular junction (NMJ) has been a powerful model system for genetic and molecular dissection of synaptic growth, structure, and function. More recently the peripheral nervous system of third instar larvae has been employed to study acute neuronal responses to axon damage and disease. However, due to the short time interval between the third larval instar and pupariation, the system is not well suited to study processes that extend over a longer time period. Recent studies demonstrate that third instar larvae mount a rapid initial response to axon damage and display tantalizing beginnings of axonal regrowth. However, the onset of metamorphosis with replacement of most larval tissues precludes more complete analysis of the response to injury - including involvement of glia and possible axonal repair - over time. Similarly, the window of observation in experiments probing mechanisms that maintain NMJ structure and function over time, or how these are compromised with age or by disease, is significantly limited by the onset of pupariation. The goal of this application is to characterize and demonstrate the utility of an experimental system we are developing that overcomes these time constraints while preserving the features of the larval NMJ that makes it such a powerful model. We exploit genetic variants in which larvae develop normally but subsequently remain in the third instar for up to 10 days (4 times longer than normal), during which time they continue to grow before finally undergoing metamorphosis and eclosion. On the basis of our preliminary results, we are confident that the expanded third instar lifespan provides a novel and powerful opportunity for experiments that probe time-dependent neurobiological processes. To establish the validity and utility of this Extended Larval Life-span (ELL) model, we propose experiments that aim to answer the following questions: (1) Is NMJ growth normal in ELL larvae during development? Does the NMJ continue to grow along with the increase in larval size during ELL? Do the key signaling pathways known to regulate NMJ growth during normal development continue to function during ELL? (2) Does the NMJ remain structurally and functionally intact throughout ELL? (3) Can we prove the utility of the ELL system as an experimental tool by employing it to expand our understanding of the injury response in larval motor axons and peripheral nerve glia over an extended time frame? We believe that this novel experimental system has enormous potential to greatly expand the power of the larval NMJ as a model system and enable us to make unique inroads in studies of axonal regeneration and synaptic maintenance, both of which are highly relevant for understanding and treatment of a number of human neurological disorders.