Prominin-1 Localizes to the Open Rims of Outer Segment Lamellae in Xenopus laevis Rod and Cone Photoreceptors

The extraordinary compartmentalization of rod and cone photoreceptors has long drawn attention. Their unique light-sensitive organelles, termed outer segments (OS), respond to varied wavelengths of the visual spectrum.1 Outer segments are both biochemically and biosynthetically distinct organelles that connect to the cell body (the inner segment [IS]) by a narrow 9+0 nonmotile connecting cilium.2–4 For more than 100 years, the pathways of photoexcitation and the anatomic structures in rods and have been found to be parallel but distinct.5 Rods are more sensitive to dim light, typically in the blue-green region of the visual spectrum, whereas cones respond to long (red-orange), intermediate (blue-green), and short (blue-purple) regions of the visual spectrum6,7 as a consequence of their expression of various opsin genes.8 In addition, rods and cones have distinct morphologies. Rod photoreceptors have long cylindrical OS consisting of a stack of disks surrounded by, but separate from, the plasma membrane that encases them. Only at the base do the disks assume a lamellar shape with continuity between the lamellar surface and the plasma membrane.9 By contrast, in frogs and lower vertebrates in general, the cone outer segment (COS) are conical and the lamellae do not separate from the plasma membrane, so that the interlamellar space is permeable to extracellular molecules.10 In higher vertebrates, the COS lamellae are largely isolated within the enclosing plasma membrane and undergo episodic fusion with it to become accessible to extracellular molecular penetration for reasons that are undefined.11–14 This anatomic difference has consequences for the renewal of rod and cone OS membranes. Newly synthesized rhodopsin is deposited into the basal lamellae of growing rod outer segment (ROS) but then becomes locked into the disk as it separates from the plasma membrane and is moved outward by formation of new disks below.15,16 Newly inserted cone opsins, by contrast, distribute throughout the length and breadth of the COS as a consequence of the continuity of the lamellae and plasma membrane.10,17 ROS disk protein remains confined within a given disk until it arrives at the top of the stack and is then shed and engulfed into the adjacent pigment epithelial cells.18–20 Thus the morphogenesis of these highly related organelles poses distinct challenges to interpret how rods and cones accomplish the sculpting and renewal of two such distinct structures. Moreover, the entire process of morphogenesis occurs at the distal tip of the connecting cilium that connects the OS to the inner segment (IS), so that much of the cells' machinery is unavailable to accomplish this task. It has long been suspected that unidentified proteins account for this difference. Prominin-1, a pentaspan transmembrane protein,21–23 may be a candidate that participates in the morphogenesis or maintenance of OS structure in rod and cone photoreceptors. Maw et al.24 discovered that prominin-1 is localized to the evaginations of the plasma membrane that form the new basal lamellae at the base of murine ROS. Zacchigna et al.25 reported that knocking out prominin-1 in mice causes progressive degeneration of the photoreceptors, as evidenced by thinning of the outer nuclear layer. It is not clear whether cones also degenerate, but cone opsin missorting is observed in prominin−/− mice, and the morphology of both ROS and COS is abnormal before the cells die. A frameshift mutation (PROML11878, now designated c.1841delG) of prominin-1 is responsible for an autosomal recessive retinal degeneration in a consanguineous Indian family.24 An autosomal dominant mutation, R373C, of prominin-1 in humans caused retinal degeneration and abnormal structure of ROS in mice carrying this mutation.26 A third mutation, c.869delG, of human prominin-1 causes autosomal recessive retinitis pigmentosa in patients.27 However, despite these studies, little is known about the precise localization and the function of prominin-1 in photoreceptors. In Drosophila melanogaster, prominin was found to bind to spacemaker (spam), an intercellular glycoprotein.28 In the presence of spacemaker, the fly's rhabdomeres are separated. However, in insects not expressing spacemaker (for example, Apis mellifera, the honeybee), rhabdomeres adhere to each other, thereby participating in the two alternative arrangements of the compound eye's photoreceptors.28 Mammalian agrin and perlecan share similarity with the C terminus of spacemaker,28 but no homolog of spacemaker has yet been identified in vertebrates. These intriguing findings suggest that prominin may bind to extracellular proteins in the vicinity of the ROS basal disks or COS lamellae in vertebrates as well. Besides the expected interacting partners of prominin-1 in the extracellular space, photoreceptor cadherin 21 (PCDH21), a transmembrane protein, and filamentous actin, an intracellular protein, were found to bind to prominin-1 in mice.26 Why prominin-1 distributes to the ROS basal lamellae and how it is retained there is unknown, since it is reported to be excluded from the mature upper disks. Distribution of prominin-1 in the continuous membrane system of the COS has not been previously examined by high-resolution techniques. Prominin-1 has recently been observed at the base of COS in mice,25 but without much detail of its subcellular localization, probably because of the small diameters of murine COS. We recently described the cloning of three homologs of prominin in Xenopus laevis: xlProminin-1, xlProminin-2, and xlProminin-3.29 xlProminin-1 was identified as the ortholog of prominin-1 in this frog by phylogenetic analysis and was found to be expressed in the retina.29 In the present study, we generated antibodies against xlProminin-1 and studied its protein expression in X. laevis tissues with immunoblotting. We also used fluorescence and electron microscopic immunocytochemical methods to localize prominin-1 in X. laevis photoreceptors. We present evidence that xlProminin-1 localizes to the open rims of COS lamellae and open basal lamellae of ROS. We then generated transgenic X. laevis expressing the xlProminin-1-hrGFP II fusion protein in either rods or cones by using cell-specific promoters. Comparison of results obtained in transgenic X. laevis tadpoles and immunolocalization in normal tadpoles allowed us to evaluate the distribution of the transgene product independently.