Maureen Powers - US grants

Several series of experiments are proposed in a continuing effort to understand the neural mechanisms that mediate visual detection. The different series represent three different empirical approaches to the problem of luminance detection at threshold. In Series 1, neurophysiological recordings from single ganglion cell axons in the optic tract of the goldfish will be compared mathematically with the animal's psychophysical frequency-of-seeing curve, in an attempt to provide the first direct (within species) evidence for the nature of the transforms between the behavior of ganglion cells and the probability of producing a particular psychophysical response. Some experiments in this series will involve testing fish of different sizes; because of the differential rates of addition of rods and cones to this animal's retina throughout life, the hypothesis that relative rod- and cone-mediated sensitivity might shift with age is tenable, and the outcome of this investigation may provide new information about the determinants of visual sensitivity via both scotopic and photopic pathways. Experiments in Series 2 are intended to provide the first complete description of cyclic fluctuations in visual sensitivity in two vertebrate species, goldfish and human; preliminary evidence suggests that such changes do exist in both species. I propose to record absolute psychophysical scotopic and photopic detection threshold in both species at 3 hr intervals throughout the 24 hr day, during all seasons of the year. Additional experiments are planned to determine whether the rhythm is circadian and whether it originates within or beyond the eye. Series 3 will provide a first inquiry into the neural mechanisms that might mediate the daily changes in visual sensitivity described in Series 2. I propose to examine the responses of goldfish retinal ganglion cells to determine whether some aspect of their pattern of discharge changes systematically with time of day, and if so, whether the removal of the pineal gland affects that rhythm. The successful completion of these three series of experiments will help to increase our knowledge about the relation between the interactions among nerve cells in vertebrate species and the ability to detect light.

This is a request for a Core Grant for Vision Research at Vanderbilt University. Support is requested for 4 service modules that are designed to facilitate the research of participating investigators and to enhance their scientific interactions and collaboration by providing services that are centrally located and directed by members of the Core. Because of the availability of space in the new Psychology Building (to be completed spring, 1989) and of the concentration of participating investigators in that department, we propose that 3 of the 4 modules should be located there, as well as the administrative headquarters. Support for the following service modules is requested: Shops Module, computer Module, Illustration/Photography Module and Animal Care Module. The Shops Module will provide professional and technical consultation with Core vision researchers in the design and fabrication of specialized optical, mechanical and electronic instruments needed for individual and collaborative research projects. The Shops Module will be located in the Engineering Building approximately 2 city blocks from Psychology in the direction of the Medical School. The Computer Module will provide consultation and programming services for the development and implementation of new stimulus configurations, and for data collection and analysis. It will be located on the 5th floor of the new Psychology building, near the Perception laboratories. The Illustration/Photography Module will provide services appropriate for developing figures for public presentation, whether in journals, slides or posters; this Module will also utilize the Computer Module facilities. It will be located on the first floor of the new Psychology Building, in the Neuroscience laboratories. The Animal Care Module, also located on the first floor, will provide specialized care for breeding colonies, surgical preparation and assistance and other services not provided by the Division of Animal Care but which we have found are essential for the maintenance of healthy animals for vision research.

The focus of this project is on understanding the relations between visual function and structural changes in the retina. Extensive structural changes occur naturally in teleost retina. These changes, together with the wide range of techniques that are available in the PI's laboratory to evaluate their effects on vision, will permit refined insights into structure-function relations in the vertebrate retina. The effect on visual function of three general classes of structural change will be studied: changes that occur during development in young animals, changes that are induced by light in adult animals, and changes that occur under the control of circadian clocks. The techniques to be employed include electrophysiological recording (ERG's and retinal ganglion cells), pharmacological manipulations, and retinal morphometry. The project aims to address the following specific questions: 1.How do developmental changes in photoreceptor density influence visual sensitivity? 2.Do retinomotor movements influence visual sensitivity? 3.Where do circadian rhythms in visual sensitivity originate? The significance of the research lies in the explicit acknowledgment that the retina is structurally dynamic, and that retinal dynamics must therefore be considered in the development of current theories of structure-function relations in retina. Studying a naturally dynamic system--the teleost fish retina--will allow new perspectives on the relations between structure and function at the first stages of visual processing.

vision; biomedical facility;

vision; computer center; biomedical facility;

animal care; vision; biomedical facility;

A fundamental question in cell biology is the mechanism by which one structure, the nuclear pore complex, controls all trafficking between the nucleus and the cytoplasm of the eukaryotic cell. Despite this vitally important function, much of the composition of the pores of higher eukaryotes remains unknown, and the molecular mechanism by which the pore engages and translocates substrates is still not understood. Significant progress has been made recently in understanding interactions between pore components and soluble transport factors during import into the nucleus. In contrast, progress is just beginning in the study of factors required for RNA export The Xenopus laevis model provides a uniquely powerful system in which to study RNA export. Using this system, I have shown that the nuclear pore protein, Nup98, is an essential component of multiple RNA export pathways. Additionally, Nup98 specifically interacts with a second protein, Gle2; genetic evidence has linked mutations in the homologs of each of these proteins to defects in polyA+ mRNA export in yeast. The central hypothesis behind this proposal is that the Nup98/Gle2 complex is a key mediator of nuclear export, providing a primary binding site to link export substrates to the transport machinery of the pore. The goals address the specific associations formed by this one subcomplex of the nuclear pore; however it is expected that understanding of this critical interaction will advance our knowledge of the mechanism of nuclear export and provide a framework for characterizing interactions between other export factors and the nuclear pore. The long term goal of this work is a full understanding of how export from the nucleus is conducted through precise cooperation between soluble proteins and the nuclear pore complex.

The broad goal of this proposal is to elucidate the structural details of the numerous protein-RNA interactions that are essential in the recognition and modification of eukaryotic messenger RNA. The nascent eukaryotic transcript is subjected to numerous modifications before the journey out of the nucleus. As transcripts are necessarily diverse in sequence, general recognition of the single stranded RNA messages must be sequence non-specific. Modifications at the 5' end of the message aid in transcript recognition by providing unique molecular handles for cellular proteins. In this proposal, we will focus on the recognition of one such molecular handle (the 5' m7G cap), the further modification of this handle (the O2-methylation of the first transcribed nucleotide), and the general sequence non- specific recognition of the transcript itself. These events will be studied in three model systems by combining the structural information of X-ray crystallographic analysis with the functional information obtained from biochemical assays. The concerted application of structural and functional analysis on structurally diverse but functionally similar proteins is a powerful method for elucidating structural features essential to protein function. Specific aim 1 is to elucidate the structural determinants of m7G cap binding and discrimination using the cap-specific RNA methyltransferase VP39 and the guanosine specific ribonuclease T1 as model systems. Specific aim 2 is to test the mechanism for single-stranded sequence non-specific RNA binding observed in a recent VP39/RNA co-crystal structure and then to determine the generality of this mechanism through the structural and biochemical characterization of a monoclonal anti-RNA antibody D44. Specific aim 3 examines the structural details and mechanism of O2 ribose RNA methylation by inducing catalysis in a VP39/RNA co- crystal. The results of these studies will prove invaluable in the analysis of current functional information and future structural information obtained from cellular RNA processing machinery.

DESCRIPTION (provided by applicant): The founding neural cell type in the developing retina is specified by the expression of a basic Helix-Loop Helix (bHLH) transcription factor called Atonal in the fly and Atonal homolog 5 (Ath5) in vertebrates (1-11). In the fly the founder cells later differentiate as R8 photoreceptors (12) and in vertebrates they become retinal ganglion cells (RGCs, 13). In both taxa this involves an initial rising and widespread expression of Atonal followed by its clearance from all cells except the future R8/RGC (2, 14-17). The accurate execution of this patterned selection process is crucial for normal retinal histogenesis (18). In the fly this process takes place in a moving wave called the morphogenetic furrow (12, 19-21). Here we focus on the role of the Ras/MAPK pathway in this event. MAPKs (Mitogen Activated/ Microtubule Associate Protein Kinase) are the final cytoplasmic element in the Ras signal transduction cascade (22-25). MAPKs are activated by dual phosphorylation in the cytoplasm and then phosphorylate both cytoplasmic and nuclear targets including transcription factors (26, 27). Nuclear translocation is thought to be regulated by this phosphorylation, and to rapidly follow it (in minutes, 28, 29, 30). However, in the morphogenetic furrow of the developing Drosophila eye phosphorylated MAPK antigen is held in the cytoplasm for hours (31). In the last funding period we developed a reagent to detect nuclear MAPK non-antigenically and have used this reagent to show that MAPK nuclear translocation is regulated by a second mechanism that is independent of phosphorylation. We also show that if this cytoplasmic hold is overcome, Atonal expression is disrupted and the founder cells differentiate precociously as neurons. Consequentially developmental patterning in the retina is disrupted. Preliminary data suggests that MAPK cytoplasmic hold is mediated by the sequestration of a critical nuclear transport factor: Dim7 (32, 33). We now propose four specific aims: 1) Tests for MAPK cytoplasmic hold at other times in development. 2) Define the amino-acid residues in MAPK which mediate hold. 3) Test hypotheses for the function of Dim7 in MAPK cytoplasmic hold. 4) Determine which developmental signals control Dim7 sequestration. Our long-term objective is a deep understanding of retinal histogenesis leading to possible intervention for the induction of regeneration in patients with retinal degeneration or damage.