KODAMA, Ryuji

Wings of the lepidopteran insects (butterflies and moths) develop from the wing imaginal disc, which is a hollow sac made of simple epithelium. Due to its simple construction, this tissue is a good material to study cellular interactions in the course of morphogenesis.

The outline shape of the adult wing is often different from that of the pupal wing. This difference is brought about by the programmed cell death of the marginal area of the pupal wing. The marginal dying area is called the degeneration region and the internal area, which develop into the adult wing, is called the differentiation region.
The cell deaths in the degeneration region proceeds very rapidly and completes in a half to one day period in Pieris rapae or several other species examined. It was shown that the dying cells in the degeneration re-gion have characteristics common with the apop-totic cell death in mammalian cells. The cells in the degeneration region are actively en-gulfed by the macrophages in the cavity beneath the wing epithelium. The macrophages seem to be concentrated beneath the degeneration region by the strong adhesion between basal surfaces of the dorsal and ventral epithelium in the differentiation region.

A collaborative work with the laboratory of Dr. K. Watanabe (Hiroshima University) concerns mostly on the development of trachea and tracheole pattern in the swallow tail butterflies. Trachea and tracheoles are both important in delivering air into the wing and their pattern coincide with that of the boundary of degeneration and differentiation zones at the distal end of the wing. According to the observations, the pattern formation of wing epithelium is often dependent on tracheal and tracheole patterns. Basic research on the development of tracheal pattern formation is being done by the scanning electron microscopy and the bright field light microscopy of the fixed or fresh specimens to describe the exact pathway and the time course of the formation of elaborate pattern of trachea and tracheoles and to establish the cytological and developmental relationship between the formation of tracheal pattern and epithelial cell pattern, such as scale cell pattern.

In collaboration with the Division of Molecular Neurobiology, the localization of a sodium channel protein is being investigated using immunohistological staining. DAB stained Vibratome section was further fixed and embedded in epoxy resin and then sectioned with ultramicrotome and observed under a transmission electron microscopy. Electron microscopic observation was utilized in order to identify the stained cells and surrounding cells by their ultrastructural characteristics.

Scanning electron microscopic imaging of a mutant mouse phenotype is also being done according to a request of another laboratory.


Control of the distribution of palmitoylated proteins in neuronal growth cones

Kohji Ueno

Signalling proteins such as G proteins and G protein-coupled receptors are modified with palmitate via thioester linkages. Protein palmitoylation is thought to be important in the regulation of signal transduction. We have previously found that protein palmitoylase is expressed in neural cells during mouse embryogenesis. In developing neurons, growth associated protein (GAP)-43 and Go, which are palmitoylated proteins, are mainly concentrated in the growth cones. Addition of an inhibitor of protein palmitoylase to the medium of cultured primary neuronal cells reduces the axonal growth of neurons. From these findings, we speculated that the localization of the palmitoylated proteins in growth cones is critical for the development of axons.

In this study, we are attempting to elucidate the mechanism that determines the localization of the palmitoylated proteins in growth cones. For thisanalysis, we have established a method to chemically modify a biotinylated peptide composed of residues 1-15 of the GAP-43 N-terminal with fatty acids via thioester linkages. Cys 3 and Cys 4 of GAP-43 are modified with palmitate in developing neurons. By using the method, we have prepared the peptides which are modified with myristate (C14:0), palmitate (C16:0), palmitoleate (C16:1), stearate (C18:0) and arachidate (C20:0) via thioester linkages. The method could modify the peptide with not only saturated but also unsaturated fatty acids.

With the acylated peptides, we have assayed the binding activity of ERM (Ezrin/Radixin/Moesin) proteins, which are thought to be general cross-linkers between plasma membranes and actin filaments, because ERM proteins are reported to be concentrated in growth cones and ERM proteins contain a domain which has a similar structure to a fatty acid thioesters binding protein. Recombinant full-length ERM proteins, which were expressed in bacteria, had the binding activity with the palmitoylated peptides, whereas the binding activity were less with the synthetic peptides modified with myristate, palmitoleate, stearate and arachidate. These result suggested that ERM proteins have a potentiality to recognize the fatty acid residue of the acylated peptides. Recombinant erythrocyte membrane protein band 4.1, which contains a similar structure to ERM proteins, had low binding activity with the palmitoylated peptide. From these results we suggested that a domain of ERM proteins is responsible for the interaction with the palmitoylated peptide.