De Velasco B, Martinez J M, Ochoa G H, Miller A M, Clark Y M, Matsumoto B, Robles L J
Biology Department, California State University, Dominguez Hills, 1000 East Victoria Street, Carson, CA, 90747, USA.
Exp Eye Res. 1999 Jun;68(6):725-37. doi: 10.1006/exer.1999.0654.
Photoreceptors in the octopus retina are of the rhabdomeric type, with rhabdomeres arising from the plasma membrane on opposite sides of the cylindrical outer segment. Each rhabdomere microvillus has an actin filament core, but other actin-binding proteins have not been identified. We used immunoblotting techniques to identify actin-binding proteins in octopus retinal extracts and immunofluorescence microscopy to localize the same proteins in fixed tissue. Antibodies directed against alpha-actinin and vinculin recognized single protein bands on immunoblots of octopus retinal extract with molecular weights comparable to the same proteins in other tissues. Anti-filamin identified two closely spaced bands similar in molecular weight to filamin in other species. Antibodies to the larger of the Drosophila ninaC gene products, p174, identified two bands lower in molecular weight than p174. Anti-villin 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 light-adapted octopus photoreceptors and other retinal cells. Antibodies to most of the actin-binding proteins showed heavy staining of the photoreceptor proximal/supportive cell region accompanied by rhabdom membrane and rhabdom tip staining, although subtle differences were detected with individual antibodies. In dark-adapted retinas anti-alpha-actinin stained the photoreceptor proximal/supportive cell region where an extensive junctional complex joins these two cell types, but in the light, immunoreactivity extended above the junctional complex into the rhabdom bases. Most antibodies densely stained the rhabdom tips but anti-villin exhibited a striated pattern of localization at the tips. We believe that the actin-binding proteins identified in the octopus retina may play a significant role in the formation of new rhabdomere 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 proteins with the avillar membrane would constitute a pool of proteins that could be recruited for rapid microvillus formation from the previously avillar region.
章鱼视网膜中的光感受器属于微绒毛型，微绒毛从圆柱形外段相对两侧的质膜产生。每个微绒毛都有一个肌动蛋白丝核心，但尚未鉴定出其他肌动蛋白结合蛋白。我们使用免疫印迹技术鉴定章鱼视网膜提取物中的肌动蛋白结合蛋白，并使用免疫荧光显微镜在固定组织中定位相同的蛋白。针对α-辅肌动蛋白和纽蛋白的抗体在章鱼视网膜提取物的免疫印迹上识别出单一条带，其分子量与其他组织中的相同蛋白相当。抗细丝蛋白鉴定出两条紧密间隔的条带，其分子量与其他物种的细丝蛋白相似。针对果蝇ninaC基因较大产物p174的抗体鉴定出两条分子量低于p174的条带。抗绒毛蛋白定位出一条分子量明显低于其他细胞中绒毛蛋白的条带。落射荧光显微镜和共聚焦显微镜用于绘制相同肌动蛋白结合蛋白在暗适应和光适应的章鱼光感受器及其他视网膜细胞中的位置。针对大多数肌动蛋白结合蛋白的抗体显示光感受器近端/支持细胞区域有强烈染色，伴有微绒毛膜和微绒毛尖端染色，尽管个别抗体检测到细微差异。在暗适应的视网膜中，抗α-辅肌动蛋白染色光感受器近端/支持细胞区域，此处有广泛的连接复合体连接这两种细胞类型，但在光照下，免疫反应性延伸到连接复合体上方进入微绒毛基部。大多数抗体使微绒毛尖端密集染色，但抗绒毛蛋白在尖端呈现条纹状定位模式。我们认为在章鱼视网膜中鉴定出的肌动蛋白结合蛋白可能在黑暗中形成新的微绒毛中起重要作用。我们推测这些蛋白和肌动蛋白在光适应的视网膜中仍与连接相对的微绒毛组的无绒毛膜相关联。这些细胞骨架蛋白与无绒毛膜的结合将构成一组蛋白库，可从先前的无绒毛区域募集用于快速微绒毛形成。
