Double immunogold localization of opsin and actin in the cilium of developing mouse photoreceptors

A 9 + 0 cilium represents the only connection between the light-sensitive rod outer segment (ROS) and the visual cell body. Differentiation of a ROS derives from a remodeling of the plasma membrane at the distal end of the cilium. Prior to this event, an actin-rich domain can be demonstrated within the distal cilium using immunocytochemical techniques. This actin is in the filamentous form and is also observed in mature photoreceptors where it has been implicated in ROS disc morphogenesis. In separate studies, the visual pigment protein, opsin, has also been localized to the distal ciliary membrane before disc synthesis begins. For the current report, we have used double label immunoelectron microscopy to investigate the presence of opsin and actin in the cilia of developing mouse photoreceptors during the period preceding ROS differentiation. Initially, we used post-embedding immunolabeling for the localization of both proteins on ultrathin sections of Lowicryl embedded tissues. However, increased sensitivity for the detection of membrane opsin was obtained when the retinas were immersion labeled prior to resin embedment. Although it remains unclear whether the appearance of ciliary opsin and actin are synchronized, the results of this study confirm that opsin and actin are each sequestered within their respective ciliary domains prior to the differentiation of an ROS.