Acquisition of Transmission Electron Microscopy Equipment for Invertebrate and Vertebrate Pigment Cell Studies, Undergraduate Laboratory Demonstrations, and Undergraduate Research

Kentucky Wesleyan College, Owensboro KY

Investigators

Abstract

A grant has been awarded to Drs. David F. Oetinger and Steven D. Wilt at Kentucky Wesleyan College. This grant will allow the investigators to purchase instrumentation to equip a transmission electron microscopy facility. Specifically, the equipment will include a transmission electron microscope for study of cellular components--in the 1,000 to 300,000 magnification range; an ultramicrotome with diamond knife for cutting tissue sections in the range of 60 to 80 nanometers in thickness; a second ultramicrotome for preparatory work and training purposes; a glass knife-maker, also for preparatory work and training; and appropriate darkroom equipment. Dr. Oetinger will study the structure and development of ommochrome pigment granules in the cells of freshwater sowbugs (isopods). Subsequently, he will seek a better understanding of how infection with larval worm parasites (acanthocephalans) interferes- structurally and biochemically with normal pigment granule formation. Dr. Wilt is interested in the retinal pigment epithelium (RPE) of vertebrate eyes-specifically the junctions between cells that form a "fence," restricting diffusion of lipids and proteins between adjacent cells. He will use cell cultures and chick embryos to determine the role of tight junction molecules in the development and maintenance of tight junctions. Pigmented epidermis from an age-series of aquatic isopods will be fixed, stained, embedded and sectioned. Ultrastructural examination will assess location, size, shape and number of pigment granules within cells. In certain insects, ommochrome pigments are known to be stored waste products from tryptophan metabolism: that hypothesis will be tested for freshwater isopods (crustaceans) using concurrent biochemical extraction techniques and ultrastructural examination of pigment granules. So too, study of the relationship between developing acanthocephalan larvae and altered pigmentation will be studied at the ultrastructural level. Dr. Wilt will use a Rous sarcoma retroviral expression system to overexpress subdomains of the tight junction molecule (ZO-2). Then, using traditional electron microscopy and immunogold labelling methods, he will determine the role of ZO-2 in the development and regulation of RPE tight junctions ultrastructurally. Cellular pigments and pigmented cells play of variety of biological roles in invertebrates and vertebrates: cryptic coloration, physiological color changes, ultraviolet light screening, and as important components of visual receptors. In the isopod-acanthocephalan system, Oetinger is seeking an understanding of how a parasite can mediate changes in a host's morphology--of selective advantage to the parasite. In Wilt's research, the integrity of the retinal pigment epithelium is critical for optimal vision. A better understanding of the ultrastructural-molecular relationships that maintain that integrity is important to our understanding of the evolution of the vertebrate eye and subsequent defects in structure and function.