Identification and immunolocalization of actin cytoskeletal components in light- and dark-adapted octopus retinas

Photoreceptors in the octopus retina are of the rhabdomeric type, with rhab domeres arising from the plasma membrane on opposite sides of the cylindric al outer segment. Each rhabdomere microvillus has an actin filament core, b ut other actin-binding proteins have not been identified. We used immunoblo tting techniques to identify actin-binding proteins in octopus retinal extr acts and immunofluorescence microscopy to localize the same proteins in fix ed tissue. Antibodies directed against cc-actinin and vinculin recognized s ingle protein bands on immunoblots of octopus retinal extract with molecula r weights comparable to the same proteins in other tissues. Anti-filamin id entified two closely spaced bands similar in molecular weight to filamin in other species. Antibodies to the larger of the Drosophila ninaC gene produ cts, p174, identified two bands lower in molecular weight than p174. Anti-v illin localized a band that was significantly less in molecular weight than villin found in other cells. Epifluorescence and confocal microscopy were used to map the location of the same actin-binding proteins in dark- and li ght-adapted octopus photoreceptors and other retinal cells. Antibodies to m ost of the actin-binding proteins showed heavy staining of the photorecepto r proximal/supportive cell region accompanied by rhabdom membrane and rhabd om tip staining, although subtle differences were detected with individual antibodies. In dark-adapted retinas anti-a-actinin stained the photorecepto r proximal/supportive cell region where an extensive junctional complex joi ns these two cell types, but in the light, immunoreactivity extended above the junctional complex into the rhabdom bases, Most antibodies densely stai ned the rhabdom tips but anti-villin exhibited a striated pattern of locali zation at the tips. We believe that the actin-binding proteins identified i n the octopus retina may play a significant role in the formation of new rh abdomere microvilli in the dark, We speculate that these proteins and actin remain associated with an avillar membrane that connects opposing sets of rhabdomeres in light-adapted retinas. Association of these cytoskeletal pro teins with the avillar membrane would constitute a pool of proteins that co uld be recruited for rapid microvillus formation from the previously avilla r region. (C) 1999 Academic Press.