Vertebrate photoreceptors are highly polar neurons with distinct morphology and subcellular organization. The connecting cilium of a photoreceptor cell is a modified primary cilium that forms a bridge between the inner and the outer segment (36
). It contains a microtubule-based axoneme, which initiates from the basal body in the inner segment and continues into the outer segment (26
). The outer segment is comprised of an ordered array of stacked membrane discs; approximately 10% of disks are replenished each day and in a mouse photoreceptor ~70 opsin molecules per second are transported to the outer segment (38
). In addition to the anterograde transport of opsin and other phototransduction proteins, bidirectional movement of arrestin and transducin has been demonstrated through the connecting cilium (39
). The connecting cilium therefore represents a critical junction in the cell biology, and consequently, the viability of the photoreceptors. The present study develops our understanding of the role of RPGR in primary cilia, particularly the photoreceptor cilium. We have shown that the distribution of RPGR-ORF15 includes the ciliary basal bodies, which function as a gateway to the cilium (38
). Moreover, we have identified binding partners of RPGR-ORF15, including microtubule motors and SMC proteins, which are involved in microtubule-based movement of chromosomes but whose ciliary function has not been realized. Thus, our findings suggest a significant role for RPGR in regulating transport along the photoreceptor cilium.
We have consistently observed multiple specific isoforms of RPGR-ORF15 that are generated, at least in part, by alternative splicing. Accumulating evidence indicates that distinct ORF15 isoforms may be localized to different subcellular compartments within the photoreceptors and perform specific functions (11
). A common theme is now emerging regarding RPGR’s role in intraphotoreceptor transport. The interactions of RPGR with PDE6D (18
), RPGRIP1 (17
) and NPHP5 (13
), the localization of one RPGR-ORF15 isoform to centrosomes of dividing cells and its association with nucleophosmin (22
) are consistent with a role in microtubule dynamics. The studies described in this report strongly suggest specific function of RPGR-ORF15 in regulating the ciliary transport at the level of basal bodies.
Basal bodies of primary cilia are the docking sites for proteins involved in assembly, maintenance, and function of the cilia. There is a selective transport of cargo from the basal body to the axoneme, which is partly carried out by the IFT polypeptides and polarity proteins (30
). IFT88 is required for assembly and maintenance of the photoreceptor outer segment and photoreceptor viability (41
). Based on our observations of RPGR-ORF15, IFT88 and kinesin-2 proteins (KIF3A and KAP3) as part of a multi-protein complex in the retinal axoneme, we hypothesize that RPGR-ORF15 is involved in the selection of cargo, which is carried by kinesin-2 along the cilium. Our hypothesis is consistent with a previous report that IFT88 associates with kinesin-2 in the retina (42
). Nevertheless, it should be noted that two of the potential cargo proteins, opsin and arrestin, were not detected as part of the RPGR-ORF15 complex(es) (data not shown).
The association of RPGR-ORF15 isoforms with both anterograde (kinesin-2) and retrograde (cytoplasmic dynein-dynactin complex) molecular motors is an interesting and significant finding. While the kinesin-2 complex has been shown to participate in compartmentalized ciliogenesis in Drosophila
sensory cilia and inter-segmental transport in mouse retina (43
), the function of dynein-dynactin complex in the retina is poorly understood. The dynactin subunits p50-dynamitin and p150Glued
are responsible for tethering cargo to the dynein motor (45
) and regulate transport of several microtubule-associated proteins (46
). The dynein-dependent localization of RPGR-ORF15 to basal bodies, as observed for the BBS4 protein (47
), provides further evidence in support of the functional relevance of RPGR-dynein association.
The interaction of SMC1 and SMC3 with RPGR-ORF15 and their localization to the photoreceptor axoneme suggest a broader role for SMC proteins in microtubule dynamics. SMC1 and SMC3 are large coiled-coil proteins associated with chromosomes, share structural similarity with the microtubule motor protein kinesin, and are involved in ATP-dependent chromosomal movement along spindle microtubules during cell division (34
). The mechanism by which neurons establish their polarity is similar to spindle organization during mitosis (48
). Our immunolocalization of SMC1 and SMC3 to primary cilia in the retina as well as in cultured mammalian cells demonstrates that these proteins are also associated with ciliary microtubules. SMC proteins, including 1 and 3, are listed as part of the sensory cilia in a recent genomic study (49
), which supports our findings.
Abnormal sperm tails and instability of sperm axonemes have been observed in patients with XLRP (33
). RPGR-ORF15 staining in the tip and the axoneme of mouse sperm flagella is consistent with these clinical findings. The flagellar tip is the site for axoneme turnover, a process similar to the turnover in photoreceptor outer segments (50
). Notably, abnormal nasal ciliary axonemes and hearing defects are also detected in some patients with RPGR
). Taken together, it appears that mutations in RPGR
lead to defects in microtubule-stability/maintenance but not cilia biogenesis. Consistent with this hypothesis, cilia formation is not compromised in the Rpgr−/−
) or in XLRP patients (53
In summary, we have demonstrated that RPGR-ORF15 isoform(s) are present in the axoneme and basal bodies of primary cilia and associated with proteins that are components of basal bodies and microtubule-based motor assemblies. These results suggest that RPGR-ORF15 functions in regulating transport along primary cilia, including the photoreceptor cilium. The photoreceptor degeneration (and sperm defects) observed in XLRP patients with RPGR mutations is therefore predicted to result from defects in transport assemblies in the photoreceptor cilia. A genetic test of this hypothesis must await an animal model in which the RPGR-ORF15 isoform is deleted or nonfunctional – unlike the present Rpgr
knockout mouse (24