Donald Ready - US grants

The long term goal of this project is to understand basic mechanisms of cell death. Many age-related degenerative conditions are related to cellular attrition. This decline is particularly acute in the nervous system, which has a severely restricted capacity for the addition of new cells; cell death is a significant element in many neurodegenerative diseases. A rational therapy to reduce or eliminate cell death will require an understanding of how cells die. Although accumulating evidence points to a "suicide program" which cells turn on if deprived of special signals, the broadest outlines of this program are still a matter of conjecture. The work proposed here aims to begin a cellular and mutational analysis of cell death in the developing Drosophila retina. The compound eye of the fly is a sensitive pattern amplifier of a simple neural module, the unit eye, or ommatidium. Each ommatidium is an exact repeat of a stereotyped unit crystal. Ommatidial development is well characterized at the cellular level, and cell death plays a key role in eliminating surplus cells from the eye lattice. A roughened lattice results if these extra cells are not eliminated, and allows a rapid and powerful screen for mutations that disable normal cell death. The baseline of cell death in normal eyes will be accurately mapped using a variety of methods, including quantitative light and electron microscopy, indicators for intracellular free calcium, and specially constructed genetic transformants. Rough-eyed mutants will be screened to select eyes with surplus cells and these will be examined to select candidates defective in normal cell death. Putative "cell death" mutants will be compared closely to the normal baseline. Genetic methods will be used to determine the site of action of the gene product. Additional alleles of the gene will be generated, particularly with a view to future molecular characterization of the selected gene(s). Many fundamental cellular mechanisms were established before arthropods and chordates diverged, and it is reasonable to expect that a process as basic as cell death will show strong parallels in the two groups. The ability to bring to bear a combination of cellular, genetic and molecular methods in the Drosophila eye has proved useful in exploring how cell fates are specified during development, and it promises to be informative regarding the machinery of the ultimate fate.

This group of investigators proposes to purchase an electron microscope and a confocal light microscope. The electron microscope will be used particularly for the study of frozen hydrated viruses and related specimens. The confocal microscope will be used in the study of cell substructure.

Neuroglian, a molecule identified in the fruit fly, is a member of the immunoglobulin superfamily which consists of a large number of proteins that share a common repeated structure/functional domain: the immunoglobulin-like domain. Members of this family play important roles in cell recognition and cell adhesion in the developing brain and nervous system. Although a large number of immunoglobulin-like molecules have been characterized, the mechanisms of action of these molecules is not fully understood. Molecular genetics, tissue culture, protein purification and X-ray crystallography will by applied to define specific functional domains in the neuroglian molecule and to describe the structure of these domains. A complete analysis of the structure and function of fruit fly neuroglian will advance our general understanding of the mechanism of action of immunoglobulin-like cell adhesion molecules. These studies relate not only to mechanisms underlying nervous system development, but also to more general mechanisms involved in cell adhesion and cell recognition in other tissues.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this research program is an integrated understanding of the cellular and molecular mechanisms that govern the development and maintenance of healthy photoreceptors. Here, we propose to investigate two cellular mechanisms essential to photoreceptors at all stages of life, directed membrane traffic and cytoskeletal regulation. These mechanisms are highly conserved in all animals and we will use methods available in Drosophila, an established model system, to extend our understanding of the structure and regulation of the multiprotein complexes that target the photosensory protein, rhodopsin, to the specialized photosensory membrane organelle. [unreadable] [unreadable] Experiments proposed here build on our demonstration of essential roles for Rab11 and Moesin, key regulators of membrane traffic and cytoskeletal organization, in photoreceptor development. We propose three specific aims to further an understanding of proteins interacting with Rab11 and Moesin and the consequences of their malfunction in developing and adult photoreceptors: 1) We will investigate the role of Rab11 and its associated proteins in rhodopsin transport. 2) We will extend our observation that Arrestin1 is essential for photoreceptor survival. 3) We will continue to investigate the role of Moesin and the specialized membrane-cytoskeletal assembly it organizes at the contact between the photoreceptor's signaling membrane and its cytoplasm. [unreadable] [unreadable] Insights into the coordination of membrane traffic and the cytoskeleton will be valuable in the rational design of therapies to treat retinal disease and injury. [unreadable] [unreadable]