Theodore G. Wensel, PhD - US grants

The interactions of photoreceptor membranes with the signal-coupling protein transducin are being studied in order to gain a deeper understanding of the roles of membranes and proteins bound to them in intracellular signal transduction. Visual signal transduction has in common with many other intracellular communication processes that it is mediated by peripheral proteins at the surface of membranes, as well as integral membrane proteins. In vision, the GTP-binding protein transducin shuttles between photoexcited rhodopsin (the primary signal receptor, an integral membrane protein) and cGMP phosphodiesterase (the target effector enzyme, a peripheral membrane protein). Transducin can be solubilized under mild conditions, but it requires the disk membrane to carry out its functions. An understanding of the underlying principles controlling membrane-association in the normal functioning of cellular regulators like transducin and other g-proteins should facilitate an understanding of the pathological conditions that arise when this normal regulation becomes disrupted by disease. A major emphasis is the characterization of a recently discovered subpopulation of transducin (Tm) that is much more potent than the major population in stimulating cyclic-GMS phosphodiesterase, and is much more tightly bound in the GTP form. This coexistence of membrane-bound activity, and to purify and characterize the protein(s) responsible for this activity. In addition, the membrane interactions of the major soluble population of transducin (Tm) will be studied in depth. These experiments will employ membrane binding assays, phosphodiesterase activation measurements and fluorescent probes to determine equilibrium and kinetic properties of Ts interactions with disk membranes and other proteins bound to them. Analysis of these interactions is essential in order to understand the function of this protein, and to compare its functional properties to those of Tm. This understanding should provide insight into the physiological modes of action of these proteins, and into the mechanisms by which G-proteins transduce signals in other cell types as well.

structural biology; orphan disease /drug; human tissue; eye; enzymes; spectrometry; biomedical resource;

DESCRIPTION (Adapted from applicant's abstract): This project seeks to uncover the molecular mechanisms of RGS-mediated GTPase regulation in photoreceptor cells of the retina. The expression patterns for different RGS family members will be determined at the mRNA and protein levels, and correlated with patterns of G protein expression and cellular function. Much of the effort will be focused on RGS9, recently discovered to be a major phototransduction GAP, localized to rod and cone outer segments. Cloning and characterization of the mouse and human genes for RGS9 will facilitate determining the functional consequences of deficiencies of RGS9 in vivo and the biochemical bases for those effects will be determined. The relationships between structure and function of RGS9 will be systematically explored by comparisons within the RGS family by analysis of chimeric proteins, and by site-specific collaborative multidisciplinary efforts to explore the role of RGS9 in vision using techniques of molecular genetics, structural biophysics, electrophysiology, and enzymology. Regulation of RGS function by protein-protein interactions, calcium ions, and phosphorylation will also be explored. These studies will add an important piece currently missing from the phototransduction puzzle, and may provide important information on retinal disease resulting from defects in phototransduction components.

electron microscopy; histology; vision; biomedical facility; bioimaging /biomedical imaging;

DESCRIPTION (provided by applicant): RGS proteins are a family of regulators of G proteins, the mediators of signal transduction in retinal photoreceptor cells, and in most signal transduction pathways in mammals. One member of this family, RGS9-1, plays a key role as a regulator of photoresponse timing, and serves as a model for understanding the function of this large family. The goal of this project is to understand the role of RGS proteins, particularly those of the RGS9 subfamily, in regulating the timing and sensitivity of intracellular signaling cascades in the vertebrate retina. The focus will be on the roles of these proteins in the kinetics of recovery in rods and cones, on the molecular mechanisms through which these roles are played, and on determining the mechanisms by which accelerated recovery is achieved in cones, relative to recovery in rods. There are four specific aims: 1. To elucidate the molecular mechanisms of GTPase regulation in phototransduction, by combining structural and functional studies of purified GTPase accelerating proteins (RGS9-1 and related RGS proteins, Gbeta5L, and engineered forms and fragments of them) with physiological and biophysical studies of mice and frogs genetically altered to express these proteins. 2. To assess the role of GTPase acceleration relative to other recovery reactions in the fast recovery of cone photoresponses, and in the "dominant time constant" of phototransduction, by quantifying the amounts of RGS9-1, Gbeta5L, and other phototransduction components in cones, and varying RGS9-1 and Gbeta5L levels in rods and cones. 3. To determine the mechanisms for modulation of RGS9-1 function by interactions with other photoreceptor proteins and lipids. 4. To determine the roles of RGS11 and RGS7 in vision by characterizing their multiple splice variants, identifying the retinal cells in which they are expressed, and identifying proteins with which they interact. Understanding the roles of RGS proteins in G protein signaling is necessary to understand how these pathways, which serve as targets for most drugs, normally function, how they are disrupted in disease states and how they are impacted by therapies. Understanding the role RGS of proteins in vision will help us understand normal vision, and provide insight into retinal diseases and potential therapies.

DESCRIPTION (provided by applicant): The objective of this project is to delineate the role of specific RGS proteins in regulating the timing and sensitivity of G-protein-mediated signal transduction pathways in the mammalian striatum. RGS (Regulators of G-protein Signaling) proteins accelerate the recovery, and in some cases, the activation phases of neuronal responses to stimulation of G-protein-coupled receptors. In the striatum, these include, among others, receptors for dopamine, histamine, serotonin, cannabinoids, and opioids. These receptors and the neurons that use them for signaling play important roles in drug abuse and addiction as well as in human neurological diseases such as Parkinson's and Huntington's diseases. The focus will be on RGS9-2, an RGS protein with highly specific localization in the striatum, whose expression is dramatically reduced by amphetamine treatment. The receptors, G-proteins, and effectors with which it co-localizes will be determined in order to identify the signaling pathways in which it likely functions. Localization within the striatum of another neuronal RGS protein with similar domain structure to RGS9-1, RGS7, will also be examined. The role of RGS9-2 in specific pathways will be tested by assays of signal transduction in wild type and RGS9-knockout mice. Mice with and without the RGS9 gene will also be tested for compensatory changes in other RGS proteins and other signaling proteins, including Gbeta5S, which forms a tight complex with RGS9-2. These studies should reveal the role of RGS9-1 and RGS7 in striatal signal transduction, and should provide insight into the normal functioning of these important signaling pathways and into the mechanisms of their disruption or exploitation in various disease states and in response to drug abuse.

DESCRIPTION (provided by applicant): The Houston Area Molecular Biophysics Program (HAMBP) is a Ph.D. research training program in molecular biophysics involving 43 faculty mentors in six educational/research institutions in the Houston- Galveston area: Baylor College of Medicine, Rice University, University of Houston, University of Texas Health Science Center at Houston, University of Texas M.D. Anderson Cancer Center, and University of Texas Medical Branch at Galveston. Because of the large number of faculty, and the large and diverse applicant pool represented by this group of institutions, we propose to maintain our number of funded trainees at 9. Students pursue a rigorous course of study in the classroom and in the laboratory. Program activities required of all trainees include didactic courses in Molecular Biophysics and responsible conduct of research, a weekly seminar, monthly trainee meetings, an annual research symposium and progress review, and attendance and presentation at two annual research conferences. They receive a strong foundation in the fundamentals-of Molecular Biophysics, with exposure to a broad range of topics, and pursue cutting-edge thesis research in world-class biophysics laboratories. Research areas include x-ray crystallography, macromolecular electron microscopy, fluorescence, magnetic resonance and other types of macromolecular spectroscopy, thermodynamics, kinetics, molecular dynamics, theoretical biophysics, protein folding, nucleic acid structure, molecular recognition, and others. Biological structures studied range in size from peptides and lipid mediators to viruses, transcription complexes, and muscle filaments. Supported trainees have included 5 underrepresented minorities, and have had average GRE scores of 78%tile (Q), 82%tile (A), 74%tile(V), with average undergraduate GPAs of 3.50/4.0. The average time-to-degree of the 29 students supported in the last 10 years who have completed their Ph.D. is 5.27 years. The average number of publications per trainee is 3.8 for graduated trainees, including many high-profile papers and journal covers. Alumni have secured excellent positions upon completion of training, and continue to perform research at a high level. The Program is administered by a Steering Committee of representatives from all partner institutions, which closely monitors the progress of trainees, and make decisions about admission of applicant students and mentors. HAMBP is one of several highly successful cooperative training and research ventures of the partner institutions, which together represent a larger pool of research resources and trainees than any single institution in the U.S. RELEVANCE: Training in Molecular Biophysics enables research careers leading to fundamental understanding of human disease and development of innovative approaches to diagnostics, therapeutics, and disease prevention. Biophysics provides tools used by all specialties in biomedical research and in medical practice

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have used cyo-EM and single particle analysis to determine structures of ion channels of the TRP family. Those studied included TRPV1, the receptor for painful heat that also senses the active ingredient in hot chili peppers, capsaicin, TRPV2, another heat sensor, and TRPY1, an ion channel of the yeast vacuole that releases calcium into the cytoplasm in response to osmotic stress. We have also used cryo-EM to characterize vesicles into which TRP channels been reconstituted, which allowed us to quantify the activity of the reconstituted channels. The TRP family of non-selective cation channels represent one of the most important classes of ion channels for human physiology, and constitutes a major class of drug targets. Our work represents the first reliable three-dimensional structural information available for any full-length TRP chanels, and also the first quantitative assays of purified and reconstituted TRP channels.

bioimaging /biomedical imaging; three dimensional imaging /topography

ABSTRACT NOT PROVIDED

[unreadable] DESCRIPTION (provided by applicant): The goal of this project is to understand the roles of RGS proteins of the R7 subfamily in regulating the timing and sensitivity of intracellular signaling cascades in the vertebrate retina, and in influencing the course of retinal degeneration. RGS proteins are a family of Regulators of G Protein Signaling, the most common type of signaling pathway in the mammalian retina and throughout the body. G protein signaling pathways are the targets of most drugs. RGS proteins are uniquely poised to control the timing and amplitude of cellular responses to extracellular signals. Of particular importance in the retina and elsewhere in the central nervous system is the R7 subfamily. The critical role of one R7 subfamily member, RGS9-1, in determining the kinetics of photoresponse recovery in rod and cone photoreceptor cells is well established. There are four specific aims: 1. Structures and dynamics of complexes that carry out RGS protein function will be determined using multi-resolution biophysical techniques. 2. The role of RGS7 and its complexes in responses of ON bipolar to mGluR6 activation will be determined using biochemical and proteomic techniques along with collaborative electrophysiological approaches. 3. The role of RGS11 in retinal development and function will be explored using genetically engineered mice, immunolocalization, and proteomics. 4. The ability of increases in the concentration and activity of the RGS9-1-GP5-R9AP complex to prolong the survival of rods in retinal degenerative disorders will be tested. Understanding the roles of RGS proteins in G protein signaling is necessary to understand how these pathways, which serve as targets for most drugs, normally function, how they are disrupted in disease states and how they are impacted by therapies. Understanding the role RGS of proteins in vision will help us understand normal vision, and provide insight into retinal diseases and potential therapies. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Fundamental Issues in Vision Research Course contributes specifically and responds directly to the recommendation of the National Plan of the National Eye Institute to "train high-caliber predoctoral trainees to study the visual system and sight-threatening diseases and disorders of vision." In addition, the course responds to the plan's assessment for the continued need to train vision scientists in basic science areas of immunology, molecular biology, genetics, and cell biology. The course is held biennially in August, the most recent session in 2004. The specific aims are to introduce the students to the full range of the study of vision and of visual disorders at the molecular and cellular level and to foster an experience of bonding and collaboration. In order to gain an opportunity to consider careers of investigation in the vision research fields, the students encounter 25-30 of the current leaders in the field, who introduce the major problems in vision research. Parallels between the visual system and studies in other systems are emphasized whenever possible. Accordingly, a very diverse faculty of both basic scientists and clinicians is chosen to introduce the students to the subjects in a variety of formats. The resultant interaction between the faculty and students introduces the students to a new community of researchers and to the questions that they are asking. Each student brings the intensity of their own perspectives to the class and enhances the quality of discussion, benefiting from the input of their classmates from other fields and from their exposure to the broad faculty interests. Each class becomes a social cohort with close friendships forged from a mutual intense learning experience together. Through this experience, the students will continue to feed each other insights and contacts, just as their more senior colleagues have done for decades. Through this exposure to the field of vision research and its investigators, the students are trained and nurtured for a future in the study of the visual system and its disorders. [unreadable] [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal is to obtain three-dimensional images of photoreceptor cells and their internal structures from wildtype retinas and from retinas of mice with mutations corresponding to human retinal disease. Understanding these structures will help us to understand the normal structure of healthy photoreceptors, and the structural correlates of disease pathogenesis. The most interesting structures will be the disk membranes, flattened membrane sacks which fill up the photoreceptor outer segments. These are packed with rhodopsin at high density and the organization of rhodopsin in these membranes is a subject of considerable controversy. Disorganization of these membranes is observed in a number of mouse models of human retinal degeneration. We hope to see the structural organization of these membranes and their connections to cytokeletal elements and the plasma membrane. These have been previously observed only in samples that have been sectioned and negatively stained, or subjected to freeze-etch procedures. Cryo-electron tomography offers the opportunity to observe these in intact cells (at least on the margins of the cell) with no manipulations other than rapid freezing. Also of interest will be the connecting cilium, a bundle of microtubules that connects the inner and outer segments and serves to transport outer segment components to their proper location. Defects in genes involved in the structure and function of this cilium and the trans-cilium transport also lead to retinal degeneration.

DESCRIPTION (provided by applicant): This application is for partial support of the 14th biennial Federation of American Societies of Experimental Biology (FASEB) summer conference on "The Biology and Chemistry of Vision" to be held on June 15-20, 2011 in Carefree, Arizona. Since its inception in 1985, this has been one of the premier and most successful meetings focused on photoreceptor biology. The goals of the 2011 are to provide a uniquely intimate and interactive forum for the integration across disciplines and career stages of research efforts and ideas focused on photoreceptors, their downstream and upstream cellular partners, and the diseases that affect them. The conference is planned to have 37 platform speakers plus a keynote speaker. Of these, 26 are identified speakers who will be new this time (have not spoken in last two conferences), 3 are identified junior investigators, and four will be junior investigators, selected as the meeting approaches, to highlight exciting new results from young researchers. There will also be two innovative" data blitz" sessions, first instituted in the 2009 meeting, with short talks selected from submitted poster abstracts, which will provide speaking opportunities for another 10 junior investigators, students, or postdoctoral fellows. Another innovation will be a "Meet the Experts Session" designed to maximize interactions between senior and junior participants. Organizing will include efforts to optimize the inclusion of women, members of underrepresented groups, and those with disabilities. The lion's share of the requested funding will go to support participation by junior investigators and trainees. PUBLIC HEALTH RELEVANCE: Blindness and visual deficits represent major problems for health and quality of life of the population. Major blinding diseases of the retina, such as retinal degeneration and diabetic retinopathy, are increasing in incidence as the population ages. This conference plays a vital role in integrating the research efforts of leading laboratories focused on the biology and diseases of the retina, so that state-of-the art approaches can be applied to this daunting problem in human health and well-being. Many new ideas for research directions and collaborations are likely to emerge from this conference.

DESCRIPTION (provided by applicant): Protein-protein and protein-small molecule interactions at membrane surfaces underly most signal transduction pathways and many regulatory networks. The interfacial nature of these interactions presents many challenges to understanding them in terms of molecular structures and mechanisms. One of the most informative model systems for developing this understanding is the phototransduction pathway of retinal rod outer segments, in which a light signal, photon capture by the G-protein coupled receptor (GPCR), rhodopsin, is converted into a highly amplified electrical signal, hyperpolarization of the transmembrane voltage. This proposal aims to continue work aimed at understanding the structural and mechanistic bases of two key steps: activation of the heterotrimeric G protein, transducin, by photoactivated rhodopsin (R*), through accelerated exchange of GTP for GDP on the G protein ¿ subunit (G¿), and activation of a cGMP-specific phosphodiesterase, PD6, by G¿-GTP. These reactions happen on a sub-second time scale at the surface of disk membranes, and depend critically on the environment provided by the membrane lipids, so that they must be studied in that context. The proposed work is technically innovative in that cutting-edge approaches will be developed and used to determine structures that have proven intractable to conventional crystallographic approaches, and to determine the kinetics of individual steps in the G protein activation reaction. It is conceptually innovative in that it will determine for the first time the rate-limiting step in activation of a G protein, and he first structures of a G protein regulated effectors in the presence and absence of the activated G protein. There are two specific aims: 1. to refine the structures of holo-PDE6 and its complexes with transducin and a prenyl-binding protein using cryo-electron microcopy, cryo-electron tomography, and electron crystallography, combined with fitting of high resolution structures of fragments. 2. To determine the rate- limiting step in G protein activation. The startling finding i the previous funding period that the surface density of the phototransduction G protein, transducin, can be reduced by two-thirds without effect on phototransduction kinetics, eliminates binding of G protein to photoactivated rhodopsin, R*, as a candidate for the rate-limiting step in G protein activation, and leaves open the question of which step is limiting. We will use innovative approaches including flash-activation coupled with a scintillation proximity assay and time resolved fluorescence to determine the kinetics of each sub-reaction in the activation process.

? DESCRIPTION (provided by applicant): Phosphoinositides are low-abundance membrane phospholipids which act as important recognition sites for a host of membrane associated proteins involved in intracellular signal transduction cascades, and in intracellular membrane biogenesis, homeostasis, and trafficking. Previously, their low abundance has precluded quantification of their levels in retinal photoreceptor neurons, but we have recently developed methods that allow accurate measurements of unprecedented sensitivity. These measurements have revealed striking light-induced increases in levels of PI(4,5)P2, its precursor, PI(4)P, and PI(3)P, without measurable increases in PI(3,4,5)P3. These results contrast with previous reports of light driven decreases in PI (4, 5) P2, and light activation of a PI-3 kinase isoform tht produces PI (3, 4, and 5) P3, and they reveal previously unknown pathways of light responses in photoreceptors. Retinal degeneration induced by a rod-specific knockout of the type III PI-3 kinase which produces PI (3) P underscores the physiological importance of phosphoinositide synthesis. We will use a range of techniques to manipulate gene expression or activity of enzymes of lipid metabolism in photoreceptors, along with multi-scale analysis of retinal structure, electrophysiological and behavioral measures of retinal function, and our innovative methods for quantifying and localizing specific phosphoinositides, to determine the molecular mechanisms for regulation of photoreceptor phosphoinositide levels and their roles in retinal function, health, and disease.

The goal of this Supplement request is to acquire a Typhoon 5 to enable the experiments planned for project R01-EY026545, Cilium-Associated Structures in Photoreceptors, as well as those for R01-EY031949 and other NEI-funded projects at Baylor College of Medicine. An instrument of this kind is essential for rigorous quantification of proteins, nucleic acids, lipids and other small molecules separated by gel electrophoresis or thin-layer chromatography. It will eliminate the need for outmoded x-ray film-based detection and allow routine rapid quantification of immunoblots, a workhorse technique for our NEI projects. A condensed summary of the parent grant follows. The goal of this project is to develop a thorough understanding of the structural and molecular basis of p function of the rod sensory cilium, and to understand the molecular mechanisms of rod cell death in ciliopathies. There are three Specific Aims: 1. Use cryo-electron tomography (cryo-ET) and recent developments in sub-tomogram averaging to determine the three-dimensional structure to nanometer resolution of repeating structures of the rod cell connecting cilium and basal body, including microtubule doublets and triplets, microtubule inner proteins, ?Y-shaped links?, transition fibers and appendages. Our goal is to apply recent developments in hardware and software to rod cells in both wild type retinas and in animal models of retinal degeneration. 2. Use superresolution fluorescence to test hypotheses about trafficking of specific proteins and about the roles of IFT (intraflagellar transport) particles and the BBSome (a coat-forming protein complex implicated in the blinding ciliopathy, Bardet-Biedl syndrome) in ciliary trafficking in rods. Two- color superresolution fluorescence and quantitative interaction analysis will be used to assess putative interactions between IFT proteins or BBS proteins and outer segment membrane proteins, as well as well as proteins normally excluded from the outer segment which mis-accumulate there in BBS-deficient mice. These experiments will test the hypothesis that specific membrane proteins are actively trafficked through the connecting cilium membrane through their association with IFT particles, whereas others are transported via alternative routes and excluded proteins are actively removed by the BBSome. 3. Use mouse models to test the hypotheses that CEP290 is a major component of the ?Y-shaped links? extending from the ciliary axoneme to the membrane, using superresolution fluorescence, conventional TEM, and cryo-electron tomography with timed gene disruption or gene restoration at different developmental stages to distinguish initiating as opposed to secondary events in the development of the pathophysiology of ciliopathies associated with this protein

Abstract Signaling across biological membranes is critical to living cells and involves membrane- embedded receptors, which transduce extracellular stimuli into cytoplasmic responses through long-range allosteric communication. G protein-coupled receptors (GPCRs) constitute the largest family among these receptors. They are encoded by more than eight hundred genes in humans and are involved in a large diversity of critical functions but also diseases, making GPCRs the target of more than 40 % of current marketed drugs. Although a wealth of genomic and functional data is available on these receptors, the lack of high-resolution structural and mechanistic information hinders the development of specific therapies to modulate their function. The long-term goal of the proposed research is to develop novel integrated computational-experimental approaches and use these methods to uncover the sequence, structure and energetic relationships that govern GPCR interactions with extracellular ligands, intracellular proteins and GPCR signaling functions. We will address this problem using structure modeling, computational protein design, statistical analysis and experimental approaches. Specifically, we will develop novel computational techniques to model how a large diversity of regulatory molecules (ranging from solvent to lipids and peptides) binds to GPCRs even when no structural information is available on the molecules. We will also combine computational design and experimental approaches to uncover the allosteric determinants underlying GPCR signal transductions and use this knowledge to design GPCRs with reprogrammed signaling activities. Lastly, we will engineer novel GPCR/G protein signaling pairs to study the binding specificity determinants between receptors and effectors and to generate signaling switches that may prove useful for improving immune cell engineering in future immunotherapeutic applications. These studies, by advancing capabilities for predicting GPCR structures and their interactions with a wide diversity of molecules and for designing receptors modulating intracellular signaling pathways, will have high biomedical significance.

CORE C: RESEARCH EXPERIENCE AND TRAINING COORDINATION CORE PROJECT SUMMARY The Research Experience and Training Coordination Core (RETCC) will harness the resources of both Baylor College of Medicine and Rice University, both outstanding research institutions that have a solid history of training successful scientists and engineers. These institutions have a long history of academic cooperation, along with the other Texas Medical Center institutions, including the University of Texas Medical Branch at Galveston (UTMB) and M.D. Anderson Cancer Center, the Baylor-Rice Superfund Research Program (SRP) will provide a unique opportunity to create a new and highly innovative training program within the largest medical center in the world focused specifically on scientific and medical research related to environmental impacts of Superfund sites.. The purpose of the RETCC core is to create a cross-disciplinary training environment and mentorship program to help develop next-generation scientists and engineers to tackle complex environmental health and biomedical science challenges relevant to Superfund-oriented research. This RETCC will leverage the emerging resources in the Texas Medical Center relating to environmental health and science, including the newly established T32 Training in Precision Environmental Health Sciences (TPEHS), a joint initiative between Baylor College of Medicine and University of Texas Health Science Center ? Houston School of Public Health. This SRP will focus on detecting and assessing the effect of maternal polycyclic aromatic hydrocarbon exposure on fetal and childhood health and health impacts of potential remediation strategies. The Research Projects encompassed in this SRP will expose trainees to innovative approaches, state-of-the-art technologies, and established investigators/mentors to accomplish hypothesis-driven research and importantly, use their findings to bring about lasting change to communities and Superfund stakeholders.

The goal is to understand regulation of phosphoinositide synthesis, degradation and localization in the retina and retinal pigmented epithelium (RPE), and to understand the role of phosphoinositides in retinal signaling, development, health and disease. By illuminating molecular details of such processes as membrane trafficking, autophagy, phagocytosis, endocytosis and exocytosis, this understanding can help us better understand how these processes are disrupted in retinal disease and how therapeutic interventions could make use of them to preserve vision. The specific aims are: 1. Determine the relationship between each step in the phototransduction cascade and regulation of PI(3)P PI(4)P, and PI(4,5)P2 regulation in rods. Light, independently of time-of-day, drives massive increases in the levels of inner segment phosphoinositides. Preliminary results suggest the signal driving this increase is downstream of the phototransduction cascade, and our published results demonstrate a critical role for the Class III PI-3-kinase, Vps34. It remains unclear how light leads to upregulation of the activity of Vps34 or what drives PI(4,5)P2 increases. The following hypotheses will be tested: A. Transducin activation by photoexcited rhodopsin is essential for the light-driven increases. B. PDE6 activity is essential for light-driven increases. C. The cyclic nucleotide-gated channel is essential for the increases. 2. Determine the role of PI(4)P-5 kinase activity in PIP2 (phosphatidylinositol- (4,5)bisphosphate) regulation in the outer retina and the functional role there of PI(4,5)P2. We will generate mice with inducible rod-cell- or RPE-cell-specific knockouts of the principal enzyme responsible for synthesizing PI(4,5)P2 in neurons, PIP-5-kinase?, and determine the phenotype with respect to phosphoinositide levels, cell morphology and survival, protein trafficking, endocytosis, phagocytosis and autophagy. These experiments will test the following hypotheses: A. PI(4,5)P2 is primarily synthesized in rods and RPE by the action of the PIP-5-kinase? isoform using ATP and PI(4)P as substrates; B. PI(4,5)P2 synthesis is essential for a range of membrane trafficking functions and cell viability. If needed, we will also test the global knockouts of the ? and ? isoforms, which are viable, as well as double and triple. 3. Determine the distinct phosphoinositide-regulated mechanisms of LC3 recruitment and lysosome fusion in autophagy and phagocytosis. We have found deficiencies in the standard models of LC3 recruitment to autophagosomes and phagosomes, so we will combine in vivo experiments with experiments with RPE cell lines to determine which proteins are critical for LC3 recruitment in RPE cells and what sequence of events leads to this key event in both pathways. We will also identify the PI(3)P binding proteins that are essential for LC3 recruitment to phagosomes and those that are essential for lysosome fusion of both autophagosomes and phagosomes.