The experiments described in this grant application are meant to further characterize those mechanisms which regulate photoreceptor outer segment (OS) renewal. The results of this work will provide us with additional insight into potential defects that could cause human retinal degenerative disease. A study of saponin-extracted Xenopus laevis retinas isolated at different times of the light:dark cycle is intended to demonstrate diurnal changes that occur in the distribution of ciliary and OS actin filaments in rods and cones. Myosin subfragment-1 decoration will demonstrate filament polarity in this tissue. Of particular interest is the question of whether actin filaments transiently elongate into new ciliary membrane evaginations at the base of the OS. It is these structures which become rod discs or cone lamellae. Immunoelectron microscopy with anti-actin antibodies will be performed in order to document total actin content of the evaginations. Immunoelectron microscopy will also be used to localize myosin and alpha-actinin to the actin filament rich domain in the distal cilium of developing rat photoreceptors. The presence of these two actin-associated proteins, which have recently been localized to adult photoreceptor cilia, would provide additional evidence that the components of a functional actin filament network are in place prior to the differentiation of an outer segment and that this network is necessary for disc assembly. The distribution of actin mRNA will be studied in rat photoreceptors using biotinylated riboprobes and in situ hybridization on tissue sections at both the light and electron microscope levels. This will demonstrate if actin mRNA is localized not only in the myoid, but also in the apical inner segment, in close proximity to the site of OS disc morphogenesis. The localized synthesis of actin molecules might facilitate the processes of actin polymerization and filament elongation which are believed to play a role in the regulation of disc assembly. The distribution of actin mRNA will also be studied in rat retinal pigment epithelium using in situ hybridization techniques. This will demonstrate if actin mRNA is concentrated in the apical pigment epithelium, in close proximity to the site of phagocytosis of shed OS material. Actin filament containing pseudopodia are believed to mediate phagocytosis and the localized synthesis of actin molecules could facilitate the actin polymerization necessary for this phenomenon.
| Agarwal, N; Jomary, C; Jones, S E et al. (1996) Immunocytochemical colocalization of clusterin in apoptotic photoreceptor cells in retinal degeneration slow rds mutant mouse retinas. Biochem Biophys Res Commun 225:84-91 |
| Chaitin, M H; Wortham, H S; Brun-Zinkernagel, A M (1994) Immunocytochemical localization of CD44 in the mouse retina. Exp Eye Res 58:359-65 |
