| Possible roles of cytoskeletal proteins in photoreceptor outer segments (OS) renewal were studied. Single, isolated whole rod photoreceptors were obtained from adult rabbit, rat, cat, Xenopus, and Rana retinas and labelled with the f-actin probe, fluorescent phalloidin, revealing an f-actin domain at the rod OS base, where the first step of OS assembly--i.e. plasma membrane evagination--occurs. The hypothesis that f-actin there functions in OS assembly was tested by culturing Xenopus eyecups with cytochalasin D (CD); dissociating the retinas; and labelling isolated photoreceptors with fluorescent phalloidin. F-actin domains in the isolated rods collapsed with this drug treatment. Injecting CD into rabbit eyes did not halt membrane assembly in rod and cone OSs, but new OS discs overgrew their normal diameters. These results showed that f-actin at the ROS base is sensitive to CD and somehow regulates ROS morphogenesis.; The possible role of microtubules (MTs) in ROS assembly was examined using the extracellular dye, Lucifer Yellow (LY), which directly labels newly synthesized ROS membranes in organ-cultured Xenopus eyecups; the axial dimension of the resulting fluorescent LY band is thus a measure of ROS assembly. Cultured eyecups were simultaneously incubated in LY plus either control (DMSO only) medium; anti-actin drug medium (CD or phalloidin); or anti-MT drug medium (colchicine, podophyllotoxin, vinblastine, nocodazole, or taxol). CD greatly reduced LY band displacement vs. the controls (correlating with its deregulatory effect on OS morphogenesis); phalloidin had no effect (correlating with its cell membrane impermeability); and all anti-MT drugs resulted in only a slight decrease. While the data suggest that MTs are not important for ROS assembly, further experiments are required to rule out a role in ROS assembly for drug-resistant MTs, perhaps working in concert with a kinesin-like translocator.; In a separate study, the intermediate filament protein, glial fibrillary acidic protein (GFAP), was localized in rabbit retina cryosections using a polyclonal antiserum and a monoclonal antibody, both of which were shown to be highly sensitive to aldehyde fixation. Fixation sensitivity of GFAP epitopes may thus explain the different patterns of immunoreactivity reported in mammalian retina. |
