Craig Montell - US grants

The long-term objective of the proposed research is to use a molecular genetic approach to study vision in the fruitfly, Drosophila melanogaster. The approach takes advantage of the combination of molecular, biochemical, genetic and physiological techniques available to Drosophila, to identify and characterize molecules important in visual physiology that have not been previously identified. The goal of the current proposal is to understand the functions of a gene, ninaC, which is required for formation of the photoreceptor cell cytoskeleton. This gene encodes two highly related proteins with linked domains homologous to protein kinases and the myosin heavy chain. Protein kinases play a role regulating many cell processes including signal transduction and cell growth. Myosins are proteins which convert the chemical energy in ATP into mechanical force used in a variety of cell movements. Two approaches to the study of ninaC are proposed. The first involves construction directed mutations to determine the functions of the putative kinase and myosin domains in vivo. The altered genes will be introduced into the genome of ninaC null mutant using P-element germline transformation. The second is to test the hypothesis that the ninaC proteins are genuine protein kinases joined to the myosin heavy chain. This will be investigated by assaying for the biochemical activities characteristic of protein kinases and the myosin heavy chain. The specific aims are to: 1) determine whether the ninaC proteins have myosin activity, 2) determine whether the ninaC proteins have protein kinase activity, 3) construct and analyze the effect of a mutation which should eliminate the kinase activity, 4) construct and analyze the effects of mutations which should affect just the myosin activity, and 5) analyze the effects of mutations which should result in expression of just one or the other ninaC protein. The results from the experiments proposed here should contribute not only to understanding visual physiology but also to the structure/function relationships of myosins in general and are part of the long-term goal to identify and characterize new components important in vision in the fruitfly. Homologs of many of these molecules may be found in other organisms including humans and may lead to a better understanding of human and may lead to a better understanding of human retinal diseases caused by defects in these molecules.

Dr. Craig Montell was awarded the Prestigious Presidential Young Investigator award. A well-trained investigator and academician, he appears to be well on his way to achieving an illustrious career in the Biological Sciences. Dr. Montell will continue his indepth investigations and characterizations of a family of genes, and the functions of the proteins they encode, associated with the phototransduction of light stimuli into electrical stimuli within the visual system. His molecular biological model is in the Drosophila and utilizes sophisticated methodologies of germ-line gene transfer. Additionally, Dr. Montell has characterized a number of Drosophila mutants that will be exploited to understand the molecular basis for differing spectral sensitivities of rhodopsin cell type pairs affiliated with the visual transduction process. The potential for Dr. Montell to provide outstanding contributions to the field of molecular neurobiology is considerable. This award will assist in the professional development of a young, enthusiastic, and highly motivated scientist.

The long-term objective of the proposed research is to understand the molecular mechanisms underlying vision in the fruitfly, Drosophila melanogaster. The goal of the current proposal is to understand the functions of a gene, ninaC, which is required for signal transduction in photoreceptor cells (phototransduction) and to prevent retinal degeneration. The gene encodes two highly related proteins, p132 and p174, with linked domains homologous to protein kinases and the head region of myosin heavy chains. The two NINAC isoforms are the major calmodulin. The approach to studying ninaC takes advantage of a combination of molecular, biochemical, genetic, electrophysiological, histological and germline transformation techniques. To study the functions of ninaC we plan to: 1) test the hypothesis that the calmodulin bound to the two different calmodulin binding sites has different functions, 2) identify the rhabdomere localization signal (rhabdomeres are the photoreceptor cell microvillar structures), 3) test the kinase regulates subcellular localization or calmodulin binding, 4) test the proposal that the PEST sequence targets NINAC for degradation, 5) test the hypothesis that the NINAC proteins are bona fide actin-based motors by characterizing the enzymatic properties and testing for in vitro motility, 6) test the hypothesis that pp28 is a target for the NINAC kinase activity, and 7) identify and characterize other Drosophila retinal calmodulin binding proteins. The results of the proposed experiments should contribute not only to understanding visual physiology and the functions of protein-calmodulin complexes, but perhaps also to a more general understanding of the roles of unconventional myosin and proteins kinases. Retinal degeneration is a major health problem; however, in most cases the causes of retinal degeneration are not understood. The degeneration might be due to a requirement for p174 to link the actin filaments in the rhabdomere to the membrane. A number of proteins, such as dystrophia, bridge actin filaments with the plasma membrane and are required to maintain the integrity of the cell. Thus, a description of the mechanism underlying the degeneration of fly photoreceptor cells might also contribute to understanding the bases of diseases which lead to the degeneration of other cell types as well.

Three ABI 377 automated DNA sequencers are requested to fulfill the needs for DNA sequencing and genescan analyses at the basic sciences of the Johns Hopkins School of Medicine. The approximately 100 basic science laboratories at this institution are pursuing a wide range of research topics which depend heavily on these techniques. The range of ongoing projects span from developmental, cell and molecular biology, biochemistry and neurobiology. The proposed instruments would be managed by our non-profit DNA core facility, experienced in a variety of DNA analysis services, and would be available to the entire basic science community at cost. The Dean of the School of Medicine has committed co-funding for this project.

The long-term objective of the proposed research is to understand the molecular mechanisms underlying vision in the fruitfly, Drosophila melanogaster. The goal of the current proposal is to understand the mechanisms regulating TRP, a novel plasma membrane Ca2+ channel specifically expressed in Drosophila photoreceptor cells. Changes in the concentration of intracellular Ca2+ are crucial for signal transduction in virtually every cell. During the last couple of years, it has been found that one of the most effective and prevalent Ca2+ influx pathways, referred to as capacitative Ca2+ entry, occurs via Ca2+ selective ion channels in the plasma membrane that are activated following depletion of intracellular Ca2+ stores. The mechanisms regulating these ion channels are poorly understood and the molecular identification of the proteins responsible for these plasma membrane Ca2+ conductances have been elusive. Based on a variety of electrophysiological, pharmacological and molecular analyses, it appears that TRP is the archetypical member of the family of capacitative Ca2+ entry channels. The approach to characterizing TRP takes advantage of a combination of molecular, biochemical, electrophysiological, pharmacological, genetic and germline transformation techniques. To study the mechanisms regulating TRP we plan to: 1) test the hypothesis that TRP function is regulated by interaction with calmodulin, 2) test the hypothesis that TRP localization to the base of the rhabdomeres is mediated by interaction with alpha-actinin, through the TRP ankyrin repeat, 3) test the hypothesis that TRP is regulated by interaction with GTP, 4) test whether the PEST signal is important for TRP function, 5) characterize the effects of systematic deletions in TRP on subunit assembly, activation and inactivation, and 6) characterize TRP expressed in the neural cell line, NG115-401L. The results of the proposed experiments should contribute not only to understanding visual physiology but to the widespread phenomenon of capacitative Ca2+ entry which has been proposed to be important in such diverse cellular processes as fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signaling. Several studies indicate that Ca2+ influx is important in memory involving NMDA receptors and other studies suggest that blockage of Ca2+ influx is important in controlling certain types of epileptic seizures.

DESCRIPTION (Adapted from applicant's abstract): The goal of this proposal is to understand molecular mechanisms regulating TRP, the archetypal member of a new class of proteins referred to as store-operated channels (SOC). SOCs are activated by Ca2+ release from internal stores and appear to be responsible for a prevalent Ca2+ influx pathway. Vertebrate SOCs were recently identified, but mechanisms that regulate SOC's in general are poorly understood. Three general strategies to study SOCs are to be used. The first will employ in vitro binding to characterize the nature of the protein interactions in the TRP signaling complex. The second is to probe TRP function in vivo using the Drosophila visual system. A series of site-specific mutations will be made in TRP and the effects of the mutations will be assessed following germ-line transformation of the mutant genes. The third strategy is an electrophysiological analysis of members of the TRPC family expressed in cultured cells. The specific aims of the proposal focus on several hypotheses: (1) that INAD binds directly to multiple proteins required in vision to facilitate feedback regulation; (2) that CaM functions in feedback regulation of TRP; (3) that TRP is regulated by interaction with GTP; (4) that the vertebrate TRPC proteins form heteromultimers; and (5) that TRPR1 and TRPR2 define a new subfamily of TRP-like channels.

The long-term goal of our research is to understand the molecular mechanisms regulating the G-protein coupled signaling cascade critical for vision in the fruitfly, Drosophila melanogaster. The rapid activation and feedback regulation of many signaling cascades may be tightly coupled. Recently, many of the components required for Drosophila phototransduction have been found to be linked into a supramolecular signaling complex (signalplex). The coordinator is INAD, a protein with five tandem protein interaction motifs, referred to as PDZ domains. The identifications of the signalplex permits a reevaluation of the modes by which fly vision is regulated. The goal of the current project is to investigate the role of the INAD signalplex in activation and termination the Drosophila photoresponse. Several strategies to study the function of the INAD signalplex are proposed. The first will employ in vitro techniques to characterize the interactions of INAD with various target proteins as well as sites important for homomultimerization and studies to probe the role of the signalplex in vivo. This will be accomplished by constructing site- specific mutations that disrupt specific protein interactions and introducing the altered genes into the fly using P-element mediated germ-line transformation. The effects of these alterations will be assessed using a combination of electrophysiological, immunocytochemical and biochemical techniques. The specific aims are to test the hypotheses that: 1) association of signaling proteins with INAD is critical for activation and termination, 2) interaction of calmodulin with the signalplex functions in termination of the photoresponse, 3) the association of signaling proteins with INAD is regulated dynamically by PKC, and 4) homomultimerization of INAD is required in vivo for termination and/or activation of the photoresponse. The fundamental importance of activating and switching signals off with normal kinetics is illustrated by the profound retinal degeneration resulting from defects in activation and termination of phototransduction in Drosophila photoreceptor cells. Mutations in many signaling proteins central to Drosophila phototransduction result in retinal degeneration. In most cases, homologs of these Drosophila proteins are known to be expressed in the vertebrate retina. A long range goal of the current research will be to ascertain whether similar retinal dystrophies result from mutations in these vertebrate homologs.

Autosomal dominant polycystic kidney disease is one of the most common inherited diseases in humans. Recently, the two genes responsible for nearly all cases of this disease have bene isolated, PKD1 and PKD2. PKD1 is predicted to contain multiple transmembrane domains and has motifs suggesting that it interacts with the extracellular matrix. PKD2 is predicted to have six transmembrane domains, a topology that is reminiscent of the superfamily of voltage-gated and second messenger-gated ion channels. Moreover, PKD2 has modest, but significant homology to several types of ion channels. The predicted structure of PKD2 has led to the suggestion it may be a cation channel. Nevertheless, the functions of PKD1 and PKD2 remain obscure. To characterize the functions of PKD2, both in vivo and in vitro, this proposal focuses on model experimental organism, the fruitfly Drosophila melanogaster. An opportunity to use this model organism has emerged through the identification of a Drosophila homolog of PKD2 (Dpkd2). The proposed research takes multi-disciplinary approach to the characterization of dPKD2 using a combination of genetic electrophysiological, cell biological, biochemical and molecular approaches. The first aim of the proposed research is concerned with completing a molecular analysis of dPKD2, including characterization of the spatial distribution of Dpkd2. A second and major aim is to define the role of dPKD2 in vivo. A third goal is to test the hypothesis that dPKD2 is a cation influx channel. The modes of activation, regulation and the biophysical properties of the putative channel will be explored. A fourth aim is to characterize the topology and multimerization of Dpkd2. While PKD2 has been proposed to consist of six transmembrane domains, there is currently no experimental evidence to support this model. Finally, the fifth specific aim will be devoted to testing the hypothesis that dPKD2 may also form functional heteromultimeric channels with a related channel, TRP, in vitro and in vivo. Human PKD2 has recently been shown to bind to TRPC in vitro. However, such interactions have not been described in vitro and the functions of the PKD2/TRP association are not known. The long-term goal of the proposed research is to understand the normal roles of PKD2 and the basis for the disease state at the molecular level.

DESCRIPTION (provided by applicant): The Graduate Program in Biochemistry, Cellular and Molecular Biology (BCMB) is an interdisciplinary training program comprised of 93 faculty members in the Departments of Biological Chemistry, Molecular Biology and Genetics, Biophysics and Biophysical Chemistry, Pharmacology and Molecular Sciences, Cell Biology and Anatomy, Physiology and Neuroscience. The program trains young scientists in these disciplines and provides them with a breadth of knowledge and understanding so that they can initiate independent and fruitful research careers. Applicants apply to a single admissions committee and follow a single curriculum. They take five "core" courses: Molecular Biology, Physical Biochemistry, Genetics, Bio-organic Chemistry, and Biochemistry and Cell Biology. The students also participate in several small group discussion courses, carry out three rotations in different laboratories, and then choose a thesis advisor. During the subsequent years they take several electives and attend journal clubs and departmental seminars presented by visiting scientists. Upon completion of their training, the students present their thesis research in a public seminar. The entire training program takes between 4 and 6 years. The 23-year-old program has been very successful. It continues to recruit high quality applicants. The successful candidates are outstanding science majors from top-ranked undergraduate schools. The "steady-state" level of predoctoral students is 170, with an average of approximately 25 new students admitted each year. The graduates of the program hold research and teaching positions at all levels in academia, the government and industry. The training facilities are the classrooms and laboratories of the seven participating departments. The program fosters an extraordinary level of collaboration and interaction among the faculty. The students are an essential part of the scientific community and they appreciate not only the enormous variety of exciting research opportunities, but also the fact that the Johns Hopkins University School of Medicine is a very pleasant place to work.

The long-term goal of the proposed research is to understand the molecular mechanisms underlying visual transduction in the fruitfly, Drosophila melanogaster. Drosophila phototransduction is the fastest known G-protein coupled signaling cascade and it culminates in sodium and calcium influx, via the TRP, TRPL and TRPgamma cation channels. During the last few years, a large family of mammalian TRP-related proteins has been identified, which play critical roles in processes ranging from sensory physiology to vasorelaxation and male fertility. As is the case for the Drosophila TRPs, many of the mammalian TRP channels are activated through signaling pathways that are coupled to phospholipase C. However, the molecular mechanisms linking phospholipase C to the activity of the TRP channels are enigmatic. While the activation mechanisms of many mammalian TRP-related proteins have been addressed, a key limitation of most of these studies is that the analyses have focused exclusively on proteins overexpressed in heterologous systems. The goal of the current proposal is to characterize the mechanisms underlying the regulation of TRP channels using a multidisciplinary approach. Experiments are outlined to study the Drosophila TRP channels in parallel in vitro and in vivo systems, using a combination of molecular, genetic, biochemical, electrophysiological, calcium-imaging and cell biological approaches. The specific aims of the proposal are to test the following hypotheses: 1) TRP is regulated by PKC and PIP3; 2) TRP is regulated through direct interaction with a member of newly described class of membrane-associated proteins expressed exclusively in excitable cells; 3) TRPgamma is required for the visual response; and 4) TRP is a tetrameric channel with six transmembrane segments. The final specific aim (5) is devoted to characterizing a new activation mechanism underlying a mammalian TRP channel, TRPM1, and to initiate characterization of TRPM3. TRP channels appear to be important for human health as mutations in TRP-related channels cause a neurodegenerative disease, mucolipidosis, and polycystic kidney disease. Moreover, defects in TRP channels have been associated with changes in growth control and one TRP-related protein, TRPM1, may be a tumor suppressor.

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand the molecular mechanisms underlying phototransduction in the frutifly, Drosophila melanogaster. Most of the proteins that function in Drosophila phototransduction are coupled into a supramolecular signaling complex, referred to as the signalplex. The central protein in the signalplex is a PDZ protein referred to as INAD. The signalplex represents the first example of a G-protein coupled signaling cascade that is linked in a large macromolecular assembly. However, recent evidence indicates that similar G-protein coupled signaling complexes exist in mammalian cells. Nevertheless, characterization of the composition, assembly and functions of these complexes in native tissues has been limited. The Drosophila visual system offers a tractable in vivo model for characterizing a G-protein coupled signaling complex, using a combination of genetics, biochemistry, molecular biology, cell biology and electrophysiology. The first specific aim of the proposed research is to characterize novel molecular mechanisms regulating a new member of the signalplex, Arrestin2. Arrestin2 is of particular interest since recent studies indicate that, in addition to functioning in inactivation of the receptor, abnormally stable rhodopsin/arrestin complexes contribute to one form of retinal degeneration. We propose to investigate the role of the INAD/ Arrestin2 interaction and to test the hypothesis that arrestins are regulated by phosphoinositides. One INAD binding protein is protein kinase C (PKC) and interaction of PKC with INAD could provide a mechanism for rapid light-dependent regulation of PKC substrates, which also associate with the signalplex. However, the substrates and mechanisms regulated by light-dependent PKC phosphorylation are poorly understood. In the second specific aim, we propose to test the hypothesis that the G-alpha-q, which transiently interacts with the signalplex, is phosphorylated by PKC and this modification regulates its GTPase activity. The goal of the third specific aim is to test the hypothesis that PKC phosphorylation of INAD regulates dynamic INAD/target protein interactions. The fourth specific aim is concerned with characterizing the mechanisms regulating the localization of the signalplex. Several of the proteins that are the focus of the proposed research are related to proteins that function in mammalian phototransduction and, if mutated, result in retinal degeneration. Therefore, the proposed studies should contribute to a general understanding of the mechanisms regulating phototransduction, as well as provide new insights into the mechanisms underlying certain forms of retinal degeneration.

[unreadable] DESCRIPTION (provided by applicant): Funds are requested to cover partial support for the 2004 FASEB Conference on Calcium and Cell Function, to be held in Snowmass, Colorado. Calcium is a critical second messenger, which is essential for processes ranging from T cell development to fertility, neurotransmitter release, sensory transduction and cell migration. Furthermore, defects in calcium signaling are associated with a host of diseases affecting excitable and non-excitable cells. The goal of this conference, which takes place every two years, is to provide a forum for scientists from a diversity of fields to present and discuss the latest and most exciting developments in the calcium field. We have aimed to attract many scientists to this meeting who have not participated in previous conferences on calcium signaling, despite the relevance of there work to the topic of the meeting. In particular, many scientists who work on sensory signaling, neuronal development and neuronal physiology have not attended past meetings, due to a former emphasis on non-excitable cells. We expect the mix of participants, combined with the informal setting and relatively modest size of the meeting (maximum of 165 participants), to generate lively discussions and the establishment of new interactions among scientists who ordinarily do not attend the same conferences. The meeting will include nine sessions with oral presentations, two keynote addresses and four poster sessions covering topics ranging from calcium regulation of gene expression to sensory signaling, calcium regulation of development and regulation of vesicular and protein trafficking by calcium. The topics to be covered at the meeting are of fundamental importance and are relevant to understanding the bases of a diseases such as neurodegeneration, communicative disorders, aging, cancer, kidney and cardiovascular disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The taste modality is critical not only to aid in the identification of food, but also to avoid consuming noxious chemicals. The long-term goal of the proposed research is to understand the molecular mechanisms underlying sweet and bitter taste transduction in the fruitfly, Drosophila melanogaster. The fruitfly has proven to be a valuable model organism for characterizing other sensory modalities, such as vision and olfaction;however, taste transduction has received less scrutiny. Recent studies indicate that the mammalian and Drosophila taste transduction pathways are initiated by G- protein coupled receptors. However, the mechanisms operating downstream of the Drosophila receptors are largely unknown. In mammals, there is evidence that taste transduction operates through a phospholipase C mediated signaling pathway that culminates with the activation of the TRPM5 channel. Nevertheless, many questions remain including the mechanisms of adaptation, the role of cAMP signaling in taste transduction, the mechanisms that regulate the gustatory channel in vivo and the proteins regulating the gustatory receptors. The goal,of the proposed research is to apply a combination of genetic, electrophysiological, cell and biochemical approaches in Drosophila to define the biochemical basis for taste transduction initiated by either attractive or aversive compounds. The specific aims of this proposal are to test the hypotheses that: 1) the PI signaling proteins involved in Drosophila vision also function in taste, 2) cAMP signaling functions in Drosophila sweet and bitter transduction, 3) Gr66a is the caffeine receptor and misexpression of this receptor can elicit an attractive behavioral response to caffeine, and 4) the TRPA2 gustatory channel is regulated by calmodulin and interactions with the scaffold protein Homer. Given the apparent parallels between mammalian and Drosophila taste transduction, these studies should provide important new insights into the mechanisms of gustatory transduction that apply to human taste receptor cells.

The long-term goal of our research is to understand the mechanisms underlying phototransduction in the fruitfly, Drosophila melanogaster. Drosophila phototransduction functions through a phospholipase C (PLC)- dependent signaling system, and culminates in Ca2+ and Na+ influx, via TRP channels. It is now clear that there exists a large family of mammalian TRP channels, many of which participate in a diversity of sensory signaling processes. The over-riding theme of this proposal is that phosphoinositide (PI) signaling is critical for regulatory events in Drosophila photoreceptor cells, which are more complex and varied than the established concept of gating the TRP channels through hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Experiments in this proposal are designed to challenge the current view that the only effector pathway for light-activated rhodopsin is a heterotrimeric G-protein/PLC pathway. Rather, we propose to test the idea that light activated rhodopsin also couples to a small GTPase, which in turn stimulates a phospholipase D (PLD), and the small GTPase and PLD are required for the light-dependent translocation of the rhodopsin regulatory protein, arrestin. We also propose to test the idea that PIs have an additional role in vivo, namely to regulate the TRP channel by preventing its interaction with inhibitory calcium/calmodulin. As part of a long-term goal to define the proteins that participate in PI signaling in photoreceptor cells, we propose to address the functional requirement for a type of PI-transfer protein that is conserved from flies to humans, but has not been characterized in any organism. To address these questions, we propose to employ a combination of genetics, electrophysiology, cell biology, germline transformation and biochemical approaches. Finally, to generalize from our work on fly vision to mammalian phototransduction, experiments are proposed to provide genetic evidence for a requirement for PI signaling in the intrinsically photosensitive retinal ganglion cells (ipRGCs) of the mouse, which function in the pupillary light reflex and several accessory light-driven behaviors. To study the regulation of TRP channels and phototransduction by PIs, we propose to test the hypotheses that: 1) light-dependent movement of Arrestin functions through a small GTPase and PLD-dependent pathway, 2) Drosophila TRP undergoes dual regulation by PIP3 and Ca2+/calmodulin in vivo, 3) a new PI-transfer protein is required for the Drosophila photoresponse, and 4) a mouse PI-transfer protein is required for function of the ipRGCs. During the last few years, four human diseases have been shown to be due to mutations in TRP channels, including the most common disease due to mutation in a single gene, autosomal dominant polycystic kidney disease, and mucolipidosis type IV, which causes severe neurodegeneration, mental retardation and retinal degeneration. Given that the molecular mechanisms underlying the activation of these human TRPs are poorly understood, Drosophila TRP provides an in vivo model for defining the mechanisms regulating TRP channels.

DESCRIPTION (provided by applicant): The goal of our proposed research is to understand the molecular mechanisms underlying thermotaxis in the fruitfly, Drosophila melanogaster. Thermotaxis, which is the movement towards a preferred temperature, has been studied in a wide range of vertebrate and invertebrate organisms. However, only a few of the genes and proteins required for temperature discrimination are known. There are two rationales for the proposed research. First, thermotaxis in insects has potential medical relevance, as the host-seeking behaviors of disease spreading vectors, such as the malaria-spreading insect, Anopheles gambiae, appear to involve temperature sensation. Thus, identification of the proteins essential for this behavior may lead to approaches to interfere with it. Drosophila homologs of TRP channels, which are thermosensors in mammals, also function in thermotaxis. Since several thermoTRPs in mammals are also regulated by aversive chemicals, we propose that Drosophila thermoTRPs may be targets for insect repellents. The discovery of the molecular targets for repellents has medical implications, given that mosquito-borne disease is a worldwide health problem. Second, the observations that TRPs are themosensors in flies and mammals raise the possibility that other proteins that function in thermosensation may be shared. Thus, identification of genes and proteins that function in Drosophila thermotaxis may provide new insights into mammalian thermosensation. To characterize thermotaxis and thermoTRPs, we propose to use a multidisciplinary approach, using a combination of genetics, biochemistry, cell biology, molecular biology and electrophysiology. The specific aims of the current proposal are to: 1) test the hypothesis that a TRP channel (Painless) functions in thermotaxis in adult flies, 2) test the hypothesis that thermoTRPs are targets for insect repellents, 3) test the hypothesis that the TRPV channels (Nanchung and Inactive) operate in combination for larval thermotaxis, and 4) test the hypothesis that rhodopsins function in a thermotaxis signaling pathway. This last aim is concerned with testing the proposal that rhodopsins are direct thermosensors, which may account for the long-known phenomenon that dark-noise and spontaneous activation of rhodopsin is temperature sensitive. A long-term goal of the proposed research is to apply the insights on Drosophila thermoTRPs to identify improved insect repellents and to test the efficacies of drugs that inhibit thermally-driven thermotaxis behaviors that could be applied to medically important Diptera. PUBLIC HEALTH RELEVANCE: The proposed research is concerned with identifying the genes and proteins that are important for thermotaxis and the responses to insect repellents, in the fruitfly. A long-term goal of the proposed research is to apply the insights on fruitfly to identify improved insect repellents and to test the efficacies of drugs that inhibit thermally-driven thermotaxis behaviors that could be applied to medically important insects, such as those that spread malaria and West Nile Virus.

DESCRIPTION (provided by applicant): The goal of the proposed research is to more fully exploit the fruitfly, Drosophila melanogaster, as an animal model for retinal degeneration. The Drosophila visual system has proven to be a very powerful genetic model to identify genes required for phototransduction and for photoreceptor cell survival. Over the last funding period, we set out to increase the pace of gene discovery by combining a comprehensive microarray analysis for "eye-enriched" genes with new large-scale screens for mutations that cause defects in visual transduction. It now appears that most of the genes essential for phototransduction are in hand. Mutations in virtually all of these result in retinal degeneration. The goal of the research proposed here focuses on this medically important phenomenon. We propose to develop additional fly models for retinal degeneration, with a new emphasis on those genes that function in processes other than phototransduction. This is an important transition in our research, since the genes that cause retinal dystrophies in humans are not restricted to those that contribute to phototransduction, but function in many additional processes, such as protein transport, cytoskeletal function, lysosomal function and sphingolipid metabolism. Moreover, many of the retinal degeneration diseases are syndromic and cause clinical manifestations outside the visual system. Multiple retinal diseases also result from defects in the production and regeneration of the chromophore in the retinal pigment epithelium. Currently, flies have not been exploited as an animal model for diseases originating in the retinal pigment epithelium or for syndromic diseases. The four specific aims of the current research are to fill these voids by developing new retinal degeneration models in Drosophila. Moreover, based on our preliminary results, we propose that studying seemingly diverse models for retinal degeneration in parallel will promote the discovery of common underlying mechanisms, raising the possibility of identifying common therapies. To accomplish our goals, we propose to employ a multidisciplinary approach using a combination of genetic, cell biological, germline transformation, electrophysiological, biochemical and microarray techniques, which we have employed over the past 19 years to dissect the molecular mechanisms of phototransduction. PUBLIC HEALTH RELEVANCE: The proposed research is to uses the fruitfly as an animal model to identify and characterize new genes and proteins associated with retinal degeneration. The long-term goal of the proposed research is to identify new therapies to reduce the severities of human retinal degenerations.

DESCRIPTION (provided by applicant): Funds are requested to cover partial support for a conference on TRP Channels, From Sensory Signaling to Human Disease, to be held at the Karolinska Institute, Stockholm, Sweden (Sept. 26-27, 2009). TRPs are global sensory detectors, enabling us to sense tastants, mechanical stimuli, and environmental chemicals, changes in temperature and in some animals, light and pheromones. Defects in TRP channels underlie many diseases, including the childhood neurodegenerative disorder, MLIV. Excessive activities of TRP channels cause certain forms of chronic pain, and are associated with several cardiovascular diseases, and the ischemic cell death following stroke. In addition to diseases affecting the central and peripheral nervous systems, defects in TRP channels affect other tissues and cause autosomal dominant polycystic kidney disease and other diseases. However, the therapies for diseases due to perturbation of TRP activity are very limited and many questions remain concerning the modes of activation of these channels. The goal of this conference, which is the only meeting focusing on TRP channels in 2009, is to provide a forum for scientists from a diversity of fields to present and discuss the latest exciting developments in the field. We have attracted both established and young scientists to this conference who do not ordinarily attend the same meeting and have limited interactions. This includes scientists working on many systems and model organisms, ranging from worms to flies, mice and humans. In addition, the invited scientists have a broad diversity of technical expertise, such as biophysics, cell biology, genetics and biochemistry. We expect the informal setting and relatively modest size of the meeting (~150 participants) to promote lively discussions and new interactions among established scientists and younger, emerging scientists. The meeting will include five seminar sessions, and a poster session covering topics ranging from temperature sensation and pain pathways to taste, vision, neurodegeneration, cardiac and kidney disease and diabetes, drug addiction and synaptic transmission. One of the major challenges, which will be discussed, is the development of therapeutic strategies for treating human diseases due to defects in TRP channels.

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to clarify the molecular mechanisms underlying the detection and discrimination of chemicals through contact chemosensation in the fruit fly, Drosophila melanogaster. Contact chemosensation allows flies to distinguish sweet from bitter molecules, as well as nonvolatile pheromones. Insect gustatory organs express a diversity of candidate molecular detectors. These include gustatory receptors (GRs), TRP channels, ionotropic receptors (IRs) and odorant binding proteins (OBPs), the latter of which promote the detection of chemicals by receptor proteins. However, the functions of most of these candidate gustatory receptors and binding proteins are unknown, or are understood poorly. We propose experiments to dissect the mechanisms underlying contact chemosensation in flies using a multidisciplinary approach that includes electrophysiology, behavior, genetics, and cell biological approaches. During the last few years, the concept that GRs are required broadly for sensing sugars and bitter-tasting compounds has been confirmed. However, the biochemical functions of GRs are unclear. Aim 1 is to test the hypothesis that GRs are tastant-activated cation channels. Aim 2 addresses one of the longstanding questions in taste sensation- the nature of sour receptors. These receptors are thought to be cation channels, and many candidates have been suggested. We propose experiments to dissect whether an IR is required for the responses to most sour tastants, which cells require the IR, and investigate the contributions of two other IRs that are expressed primarily in gustatory receptor neurons. Aim 3 is devoted to characterizing the roles for odorant binding proteins (OBPs) in contact chemosensation. OBPs are known primarily for promoting the detection of certain olfactory stimuli. We found that some OBPs are highly enriched in taste organs up to 800- fold over other tissues. Experiments are proposed to test the hypothesis that gustatory OBPs promote behaviors mediated by nonvolatile pheromones. Finally, the goal of our last aim is to dissect the molecular, cellular and biological basis for selective taste plasticity. We present preliminary data indicating that flies exposed to camphor reduce their repulsion specifically to camphor and not other bitter tastants, and this taste plasticity results from a reduction in the level of a TRP channel. Aim 4 is to distinguish between different possible models underlying this taste plasticity, and to address the biological basis for adaptation to some, but not all, unappealing tastants. An additional long-term goal of this research is to apply the findings to the control of insect pests that spread disease.

DESCRIPTION (provided by applicant): The goal of the proposed research is to more fully exploit the fruitfly, Drosophila melanogaster, as an animal model for retinal degeneration. The Drosophila visual system has proven to be a very powerful genetic model to identify genes required for phototransduction and for photoreceptor cell survival. Over the last funding period, we set out to increase the pace of gene discovery by combining a comprehensive microarray analysis for eye-enriched genes with new large-scale screens for mutations that cause defects in visual transduction. It now appears that most of the genes essential for phototransduction are in hand. Mutations in virtually all of these result in retinal degeneration. The goal of the research proposed here focuses on this medically important phenomenon. We propose to develop additional fly models for retinal degeneration, with a new emphasis on those genes that function in processes other than phototransduction. This is an important transition in our research, since the genes that cause retinal dystrophies in humans are not restricted to those that contribute to phototransduction, but function in many additional processes, such as protein transport, cytoskeletal function, lysosomal function and sphingolipid metabolism. Moreover, many of the retinal degeneration diseases are syndromic and cause clinical manifestations outside the visual system. Multiple retinal diseases also result from defects in the production and regeneration of the chromophore in the retinal pigment epithelium. Currently, flies have not been exploited as an animal model for diseases originating in the retinal pigment epithelium or for syndromic diseases. The four specific aims of the current research are to fill these voids by developing new retinal degeneration models in Drosophila. Moreover, based on our preliminary results, we propose that studying seemingly diverse models for retinal degeneration in parallel will promote the discovery of common underlying mechanisms, raising the possibility of identifying common therapies. To accomplish our goals, we propose to employ a multidisciplinary approach using a combination of genetic, cell biological, germline transformation, electrophysiological, biochemical and microarray techniques, which we have employed over the past 19 years to dissect the molecular mechanisms of phototransduction.

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to use the fruit fly, Drosophila melanogaster, as an animal model to unravel the mechanisms through which insects respond to sensory cues, ranging from changes in temperature to insect repellents. These questions are of potential relevance to the control of insect pests, since mosquitoes that spread diseases are attracted to humans through thermosensory, visual and chemical cues. Aversive temperatures and chemical repellents deter insects. Therefore, understanding the mechanisms underlying avoidance behavior may provide important insights into insect pest control. A key group of receptor proteins that sense environmental stimuli are Transient Receptor Potential (TRP) cation channels. Among the 13 Drosophila members, TRPA1 is of particular note as it is a detector for a wide array of noxious sensory inputs, including slightly warm or hot temperatures, insect repellents, and excessive light. Here, we propose to dissect the molecular, cellular and behavioral mechanisms through which TRPA1 allows larvae and adult flies to elude aversive stimuli. To accomplish our goals, we propose to employ a multidisciplinary approach, using a combination of molecular genetics, biochemistry, cell biology, electrophysiology and behavioral approaches. Aim 1 will test the hypothesis that bright light, which larvae avoid, activates TRPA1 through a novel mechanism of light detection that is independent of a cell surface protein. In Aim 2 we propose to test the hypothesis that TRPA1 functions as a detector that allow flies to use the daily changes in temperature to set circadian cycles of locomotor activity. The experiments proposed in Aim 3 will test hypotheses concerning the function and mechanism by which TRPA1 is activated via a thermosensory signaling cascade. This cascade represents a new mode of activation of TRP channels in contrast to the well-known direct activation of thermoTRPs by changes in temperature. Aim 4 is an outgrowth of the observation that TRPA1 is required for flies to avoid the insect repellent, citronellal, and this response occurs through both direct and indirect mechanisms in both mosquitoes and fruit flies. In the first part of this aim we will test the hypothesis that an additional TRPA channel functions in the aversive responses to other insect repellents (geraniol and camphor). The second part of aim 4 tests the hypothesis that G-protein coupled receptors detect repellents directly, and initiate signaling cascades that lead to indirect activation of TRP channels. The proposed studies ultimately could lead to the identification of a new generation of safer and more effective repellents to control insect-borne disease by identifying and characterizing molecular targets for such compounds.

Abstract The goal of this project is to address the major unanswered questions concerning the mechanisms through which the TRP channels are activated, regulated and trafficked in Drosophila photoreceptor cells. These questions are of significance in part due to the striking similarities between the phototransduction cascades in Drosophila photoreceptor cells, and in mammalian intrinsically photosensitive retinal ganglion cells (ipRGCs). Both are initiated by similar visual pigments, which activate Gq and phospholipase C? (PLC), thereby leading to the opening of TRPC channels. It is long established that PLC activity is crucial for gating the TRP and TRPL channels in Drosophila photoreceptor cells. However, the link between stimulation of PLC and activation of these channels remains controversial. Aim 1 is devoted to addressing this question. We have identified a candidate agonist that increases in wild-type photoreceptor cells in a light-dependent manner, but not in a mutant devoid of PLC activity. We propose experiments to test whether this lipid represents the physiologically relevant agonist for the TRPC channels in photoreceptor cells. To provide further insights into the mechanisms activating and regulating TRP, we performed a proteomics analysis to identify the repertoire of proteins that associate with TRP in vivo. Aim 2 focuses on one such protein, which we propose has dual roles in phototransduction. In addition to its classical function in phototransduction, we outline experiments to discriminate between whether the direct interaction with TRP promotes activation, or serves to suppress dark noise in the photoreceptor cells. Aim 3 addresses the enigmatic mechanisms through which TRP traffics through the secretory pathway and inserts in the plasma membrane. Using a biochemical approach, we identified a candidate chaperone, which we propose is a critical component necessary for promoting TRP transport. While TRPL can be functionally expressed in heterologous cells, the classical TRP gets retained in the secretory pathway. As such, in vitro electrophysiological studies of TRP have been challenging. We propose that this new chaperone may solve this problem. Aim 4 is devoted to testing whether two small single transmembrane proteins that interact with TRP represent elusive TRP ? subunits. Many ion channels, such as voltage-gated cation channels, intimately associate with ? subunits. These proteins bear similar overall sizes and topologies with the candidate TRP regulatory subunits that are the focus here. We propose to test whether these proteins have two roles. The first is to enable TRP channels to traffic to the specialized portion of the photoreceptor cells where phototransduction takes place. The second is to shape the gating properties of the channel. To accomplish our goals, we propose a multidisciplinary approach, including electrophysiology, molecular genetics, biochemistry and cell biology. We propose that these studies will provide the framework for addressing similar questions relevant to the TRPC6 and TRPC7 channels in ipRGCs, which are essential for multiple behaviors important for human health, including normal circadian rhythms and sleep patterns.

? DESCRIPTION (provided by applicant): The most devastating infectious diseases worldwide are the insect-borne diseases, malaria and Dengue fever. Half the world's population lives in areas at risk for these diseases. According to WHO and the CDC, one billion people came down with malaria or Dengue last year. Despite intensive efforts to control the mosquitoes that spread malaria and Dengue, they remain enormous health problems. These diseases affect >100 countries, and kill >1,000,000 people each year. Dengue is of particular concern, as its incidence has increased 10-fold over the last 50 years. Now, the Dengue flavivirus infects upwards of 500,000,000 people annually. Clearly, current approaches to control malaria and, especially Dengue, have been inadequate. The latest approach to dealing with the spread of Dengue, is to release transgenic male mosquitoes bearing dominant mutations that, upon mating, render indigenous females either sterile or unable to reproduce the flavivirus. However, the #1 obstacle to the success of these release strategies is that the transgenic males do not compete adequately with native males, greatly limiting the feasibility of this otherwise promising approach. We discovered mutations in three Drosophila signaling proteins that greatly increase male sex drive, and this allows the male flies to outcompete wild-type males in mating. Since flies and mosquitoes are both Diptera, and the genes we discovered in flies are conserved in mosquitoes, we propose that these findings will allow us to launch a revolutionary new approach in mosquitoes, aimed at increasing the sex drive of transgenic Dengue mosquitoes-Aedes aeypti. We propose to create mutations in the three corresponding Aedes genes, which we posit will greatly increase the fitness of these mosquitoes in outcompeting indigenous males. As a first test of this model, we will perform competition tests with wild-type males in laboratory cages, and then in field cage studies in outdoor field enclosures. We

Abstract This project has the long-term potential to exploit the differences in taste mechanisms between insects and mammals to develop new strategies to control insect disease vectors that infect hundreds of millions of people annually. In addition, this research explores unexpected similarities between insect and mammalian taste that are evolutionarily conserved. This project will exploit the vast array of tools provided by the fruit fly, Drosophila melanogaster, to address fundamental, longstanding questions in the field of contact chemosensation. The aims will employ an usually diverse combination of approaches, including electrophysiology, Ca2+ imaging, behavior, molecular genetics and cell biological approaches. Aim 1 tests the idea that there exists a signaling pathway that functions in the detection of low levels of bitter compounds, and which bears previously unrecognized similarity to mammalian sweet, bitter and umami taste transduction. The capacity to taste aversive compounds is essential for survival, and high levels of many noxious chemicals are detected through so-called ?gustatory receptors,? which are insect ligand-gated channels distinct from mammalian taste receptors. In addition, TRP channels are also employed to sense bitter compounds. Aim 1 will test the idea that a G-protein coupled receptor (GPCR) couples to a TRP channel in bitter responsive gustatory receptor neurons, and enables flies to sense noxious chemicals at low levels. Thus, despite the striking differences in some mechanisms of taste reception between insects and humans, aim 1 is to define a GPCR/TRP dependent taste transduction pathway in an insect. However, the specific GPCRs that appear to initiate taste transduction in the fly are highly unexpected. Aim 2 concerns a well-known and highly conserved behavior in which animals are attracted to low salt foods and reject foods with high salt. The first aim leverages our recent discovery as to how low and high Na+ taste perceptions are differentially encoded in gustatory receptor cells, and thereby induce distinct behavioral responses. An ionotropic receptor (IR76b) is the low salt sensor, but the high salt receptor remains unknown. Aim 2 is to define the high Na+ receptors, and address whether they form a multi- subunit cation channel. Another behaviorally conserved, but poorly characterized gustatory modality is Ca2+ taste. Aim 3 is to reveal the enigmatic Ca2+ receptors that endow animals with the ability to taste Ca2+. Finally, aim 4 is to dissect the molecular and cellular mechanism through which food texture affects taste preferences. This would break important new ground since the molecular basis for food texture detection in animals is as yet completely unexplored. Aim 4 will test the idea that the hardness of food is detected through the Drosophila homolog of ?transmembrane channel-like? (TMC) proteins, which in mammals are implicated as subunits of a channel complex in auditory hair cells. In summary, in those cases in which insect and human receptors are distinct, the differences can be exploited to develop strategies to control disease vectors. Conversely, focusing on flies also offers to reveal evolutionarily conserved gustatory detection mechanisms.

Abstract The goal of the proposed research is to decipher unconventional roles for opsins. This project builds on our recent discoveries that opsins are not just light sensors, and offers to transform our understanding of the sensory roles of these receptors, which were discovered nearly 150 years ago. Rather than functioning exclusively in photosensation, we suggest that opsins are broadly required sensory receptors, potentially as wide-ranging as TRP channels. Opsins are expressed in a diverse array of cell types and tissues outside of the visual system. Currently, the roles of these extra-ocular opsins are largely unknown. The goal of the present proposal is to exploit state-of-the-art tools available for use with the fruit fly, Drosophila melanogaster, to identify a range of roles for opsins. The proposed research brings to bear a wide set of in vivo and in vitro electrophysiological techniques, in vivo Ca2+-imaging, behavioral assays, cell biology and molecular genetics to address a variety of unconventional roles for opsins. The first aim is to clarify a role for an opsin in the central brain. Aim 1 will test the idea that a previously uncharacterized Drosophila opsin, Rh7, functions as a photosensor in circadian pacemaker neurons in the brain, and is required for normal circadian photoentrainment. This aim will address the hypothesis that Rh7 promotes entrainment to low levels of light by coupling to the same type of phototransduction cascade that operates in known photoreceptor cells. Aim 2 will test the iconoclastic hypothesis that Rh7 also has a light-independent function in taste. We propose to identify the signaling proteins that couple to Rh7 in gustatory receptor neurons, which may be the same as those that function in phototransduction. We posit that the primordial function of opsins was in chemosensation. In addition to gustation, another ancient chemical sense is olfaction. Aim 3 will investigate a potential role for another opsin in olfaction. Aim 4 investigates the functions of opsins in thermosensation. Experiments are outlined to test whether opsin-dependent signaling cascades are required in larval temperature discrimination to promote thermal adaptation, in addition to amplifying small temperature differences. Based on recent preliminary data, aim 4 will address the question as to whether opsins also enable adult flies to select their preferred temperature in the comfortable range. Mammalian opsins are expressed in a broad range of extra- retinal tissues. The proposed experiments will provide a deeper understanding of the biological functions of extra-ocular opsins, and form the conceptual basis for investigating unconventional roles for mammalian opsins. Because G-protein coupled receptors similar to opsins are the most successful drug targets, non-visual opsins may provide effective new possibilities for drug development. Currently, drugs are in development to treat retinal degenerations that result from dominant mutations affecting rhodopsin. Understanding the diversity of roles for opsins is also an important step towards selecting drugs that have minimal effects on non-visual opsins, so as to reduce unintended side effects of these therapies.

Abstract The goal of the proposed project is to build on our recent discovery of non-classical roles for opsins in diverse sensory contexts, and specifically to unravel their contributions to the senses of taste and smell. It turns out that opsins, which have been studied for >100 years as light sensors, are expressed outside of the visual system and function in a variety of sensory modalities. Rather than serving exclusively in photosensation, we are finding that opsins are broadly required sensory receptors, potentially playing roles as far-ranging as TRP channels. In mammals, opsins are expressed in a large variety of extra-retinal cell types and organs. However, the extra-ocular roles of these opsins are essentially unexplored. We propose to bring to bear a wide combination of tools available for use in Drosophila to elucidate extra-retinal roles for opsins. The project will employ an extensive suite of in vivo approaches including electrophysiology and Ca2+ imaging, behavioral assays, cell biology and state-of-the art molecular genetic approaches, to study unconventional roles we found for opsins in gustation and olfaction. In addition to the six rhodopsins that are required in the visual system (Rh1-Rh6), flies encode a seventh family member (Rh7). Aim 1 will clarify a light-independent role for Rh7 in the response of gustatory receptor neurons (GRNs) to an aversive tastant. We will test the proposal that Rh7 couples to a signaling cascade that endows GRNs with high sensitivity to the aversive chemical. Aim 2 will investigate a potential role for an opsin in olfaction. Conceptually similar to our recent discovery that opsins expressed in thermosensory neurons allow flies to sense small increases in temperature in the comfortable range, we propose to test that an opsin acts in olfactory receptor neurons to detect minute concentrations of an aversive odorant through an amplification cascade. Aim 3 focuses on illuminating the potential roles of two opsins in the discrimination of foods on the basis of texture. The physical features of food include the viscosity of liquid foods, and the hardness of solid foods. However, the mechanisms underlying food texture sensation are poorly understood. We will investigate the contributions of two opsins to sensing the physical properties of foods. Temperature also influences food palatability. Aim 4 will address the concept that the temperature- dependent change in appeal of sugar-containing foods requires a member of the opsin family. In summary, the proposed project will deepen our understanding of light-independent roles of opsins in gustation and olfaction, and shape the proposal that opsins are broad, polymodal sensors. In view of the wide extra-ocular expression of mammalian opsins, we suggest that this project will provide the conceptual framework for exploring similar roles for opsins in mammals. Finally, taste and smell are critical for insect disease vectors to identify and bite human hosts, and thereby spread diseases that afflict over a billion people annually. Therefore, unraveling highly sensitive receptors for gustation and olfaction in insects has important potential for developing new strategies for interfering with the recognition and attraction of humans by insect disease vectors.

Abstract The goal of the proposed research is to decipher unconventional roles for opsins. This project builds on our recent discoveries that opsins are not just light sensors, and offers to transform our understanding of the sensory roles of these receptors, which were discovered nearly 150 years ago. Rather than functioning exclusively in photosensation, we suggest that opsins are broadly required sensory receptors, potentially as wide-ranging as TRP channels. Opsins are expressed in a diverse array of cell types and tissues outside of the visual system. Currently, the roles of these extra-ocular opsins are largely unknown. The goal of the present proposal is to exploit state-of-the-art tools available for use with the fruit fly, Drosophila melanogaster, to identify a range of roles for opsins. The proposed research brings to bear a wide set of in vivo and in vitro electrophysiological techniques, in vivo Ca2+-imaging, behavioral assays, cell biology and molecular genetics to address a variety of unconventional roles for opsins. The first aim is to clarify a role for an opsin in the central brain. Aim 1 will test the idea that a previously uncharacterized Drosophila opsin, Rh7, functions as a photosensor in circadian pacemaker neurons in the brain, and is required for normal circadian photoentrainment. This aim will address the hypothesis that Rh7 promotes entrainment to low levels of light by coupling to the same type of phototransduction cascade that operates in known photoreceptor cells. Aim 2 will test the iconoclastic hypothesis that Rh7 also has a light-independent function in taste. We propose to identify the signaling proteins that couple to Rh7 in gustatory receptor neurons, which may be the same as those that function in phototransduction. We posit that the primordial function of opsins was in chemosensation. In addition to gustation, another ancient chemical sense is olfaction. Aim 3 will investigate a potential role for another opsin in olfaction. Aim 4 investigates the functions of opsins in thermosensation. Experiments are outlined to test whether opsin-dependent signaling cascades are required in larval temperature discrimination to promote thermal adaptation, in addition to amplifying small temperature differences. Based on recent preliminary data, aim 4 will address the question as to whether opsins also enable adult flies to select their preferred temperature in the comfortable range. Mammalian opsins are expressed in a broad range of extra- retinal tissues. The proposed experiments will provide a deeper understanding of the biological functions of extra-ocular opsins, and form the conceptual basis for investigating unconventional roles for mammalian opsins. Because G-protein coupled receptors similar to opsins are the most successful drug targets, non-visual opsins may provide effective new possibilities for drug development. Currently, drugs are in development to treat retinal degenerations that result from dominant mutations affecting rhodopsin. Understanding the diversity of roles for opsins is also an important step towards selecting drugs that have minimal effects on non-visual opsins, so as to reduce unintended side effects of these therapies.

Abstract The goal of the proposed research is to decipher a diversity of unexplored behavioral roles for rhodopsins in Drosophila melanogaster, and in the major disease vector, Aedes aegypti. The proposed studies build on our recent discovery that a fly rhodopsin (Rh7) acts as a light sensor in a small set of neurons in the brain, where it contributes to circadian photoentrainment. This represents the first function for a rhodopsin in the central brain, despite decades-old reports that rhodopsins are expressed in the brains of many animals, including humans. We propose to bring to bear a wide set of tools to reveal the rhodopsins and signaling proteins employed in the fly brain and eye for circadian photoentrainment and for sleep. We will then leverage our extensive background in flies and our recent expansion into mosquitoes to reveal the mosquito rhodopsins required for circadian photoentrainment and for detecting humans under different light conditions. This goal is important, since mosquitoes rely heavily on vision to identify humans. Yet, there are no molecular genetic studies focusing on vision or rhodopsins in any insect vector. The project?s success will be made possible by our development and application of an extensive suite of in vivo approaches including electrophysiology, behavioral assays, cell biology, molecular genetic approaches, and expertise in creating gene knockouts in Aedes. We propose to capitalize on a transformative technical innovation we have developed to accelerate molecular genetics in mosquitoes. Aim 1 will test the hypothesis that Rh7 in the fly brain functions in circadian photoentrainment through a signaling cascade distinct from the one used in the compound eye. The experiments will also test the idea that Rh7 confers greater light sensitivity to pacemaker neurons than is possible through Cryptochrome, the other light sensor in the brain. Aim 2 focuses on illuminating the roles of different rhodopsins in the Drosophila eye and brain on sleep. We will address the concept that two rhodopsins in a small subset of photoreceptor cells in the compound eye are required for normal nighttime sleep, while Rh7 in the brain affects daytime sleep. In Aim 3, we will decipher roles for rhodopsins in Aedes that are required for increased visual attraction to humans, following exposure to CO2. We will clarify which rhodopsins are most important for host recognition at different intensities of light. Finally, we will unravel roles for two rhodopsins expressed in the Aedes brain. In summary, the proposed project will deepen our understanding of visual and non-visual rhodopsins that function in circadian photoentrainment and sleep. These are important goals, since our understanding of the mechanisms underlying sleep is rudimentary, and many diseases are exacerbated by impairments in this evolutionarily conserved behavior. Finally, given the importance of mosquito vision for identifying humans, we propose that unraveling the rhodopsins that contribute to human recognition offers to lead to creative CRISPR/Cas9-based approaches to control insect vectors and reduce disease.

Mosquitoes such as Aedes aegypti are disease vectors that rely on their keen senses to locate human hosts, and for finding conspecifics, so that they can mate. However, we have only a rudimentary understanding of the receptors that control these critical behaviors. The goal of the proposed research is to address this gap. To find human hosts, female Ae. aegypti integrate information from diverse stimuli, including CO2, visual cues, organic molecules, and skin temperature. We have discovered another cue. Aim 1 builds on our preliminary data that Ae. aegypti use infrared (IR) radiation as an additional host stimulus. We outline experiments to reveal the IR-sensing neurons, and the receptor that detects IR. To pursue this aim, we devised a highly effective behavioral assay for monitoring IR attraction, and a new molecular genetic approach to bypass complications in combining multiple genetic elements. Aim 2 takes advantage of a mutation that we recently created in a TRP channel, which renders males and females deaf. We will test the roles this channel and hearing in swarm formation, mating and in finding human hosts. Our proposed experiments will interrogate the iconoclastic idea that mosquitoes are attracted to human speech. This would be a particularly specific cue to help them recognize and zero in on people. In Aim 3, we propose a new strategy to overcome a major impediment limiting the efficacy of the sterile insect technique (SIT). SIT is a promising strategy to suppress Ae. Aegypti. It involves inundating a local population with sterile males, which render females sterile upon mating. However, an impediment to lasting suppression of mosquito populations is that wild-type males outcompete the sterile males. We propose two complementary approaches to significantly elevate male mating success. This work has the potential to transform SIT, and thereby effectively suppress Ae. aegypti. Aim 4 concerns another key sensory issue?identification of the receptors for repellents. We recently discovered that rhodopsins function as multi-modal sensory receptors, challenging 100 years of dogma that they detect only light. Here, we propose to test the hypothesis that an opsin in Ae. aegypti functions as a highly sensitive receptor for naturally-occurring insect repellents. If confirmed by our proposed experiments, this would demonstrate that opsins comprise a new, unsuspected class of olfactory receptor. To accomplish our goals, we will bring to bear an extensive repertoire of state-of-art approaches, some of which we have developed specifically to pursue this project. These include new molecular genetic tools, a suite of behavioral assays, original video tracking software, and several types of electrophysiological recordings. In summary, the unifying theme in this project is the identification of elusive receptors and mechanisms through which mosquitoes sense human hosts and conspecific mates. The insights gleaned from this work have exciting potential to lead to innovative strategies to control Ae. aegypti, and reduce insect-borne disease.