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1From The Berman-Gund Laboratory for the Study of Retinal Degenerations and the 2Howe Laboratory, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; and the 3Medical Research Council (MRC) Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom.
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Abstract |
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METHODS. RPGR in mice and other mammalian species was examined by immunofluorescence. RPGR variants were distinguished by using isoform-specific antibodies. Different tissue processing procedures were evaluated. Immunoblot analysis of serial cross-sections of photoreceptors was performed as a complementary approach to subcellular localization.
RESULTS. RPGR was found in the connecting cilia of rods and cones with no evidence for species-dependent variation. RPGR ORF15 was the predominant variant in photoreceptor connecting cilia whereas constitutive RPGR (default) was the sole variant in the transitional zone of motile cilia in airway epithelia. Removal of soluble materials in the interphotoreceptor matrix facilitated detection of RPGR in the connecting cilia in photoreceptors.
CONCLUSIONS. RPGR localizes in photoreceptor connecting cilia and in a homologous structure, the transitional zone of motile cilia. These data are important for understanding the multitude of clinical manifestations associated with mutations in RPGR. Interphotoreceptor matrix surrounding the connecting cilia is a key variable for in situ detection of a protein in the connecting cilia.
The in vivo function of RPGR is not fully understood. The N-terminal sequence of RPGR is similar to that of RCC1, a nuclear protein that functions as a guanine nucleotide exchange factor (GEF) for the small guanosine triphosphatase (GTPase) Ran. A GEF activity of RPGR toward any small GTPases has not been demonstrated directly. Previous studies of an RPGR-mutant mouse model found evidence of protein mislocalization in photoreceptors,2 prompting the suggestion that RPGR may have a role in regulating protein trafficking. An RPGR-interacting protein (RPGRIP) has been identified.11 12 13 Mutations in RPGRIP also lead to a photoreceptor-specific disease,14 suggesting that these two proteins physiologically interact in vivo.
RPGR presents an unusual challenge to cell biological and biochemical studies. First, RPGR undergoes unusually complex splicing. The initial study of RPGR identified a transcript consisting of 19 exons.1 This transcript is expressed in a wide variety of tissues1 15 16 17 and is known as the constitutive or default variant. A second major transcript, referred to as ORF15, uses a portion of intron 15 as its terminal exon and has been shown to contain a mutation hot spot.18 Additional alternative splicing occurs in both the default and ORF15 transcripts.16 19 20 Second, RPGR is expressed at very low levels. In mice, RPGR default mRNA is barely detectable in the retina by Northern blot analysis, and the size of RPGR ORF15 mRNA remains unclear.17 Low-level expression of RPGR is also indicated by the difficulty in generating RPGR antibodies that recognize a predominant protein species on immunoblots of total retinal homogenates.
The complexity of RPGR expression and its low abundance have produced inconsistent results regarding its intracellular localization and have made functional studies difficult. We have reported that RPGR is concentrated in the photoreceptor connecting cilia2 13 20 and have proposed that RPGR localization in the cilia is probably mediated through binding to RPGRIP.13 By examining the cone-rich ground squirrel retinas, we found that RPGR is also enriched in cone connecting cilia, although cone localization has not been directly addressed by colocalization studies. Other groups have reported RPGR in the outer segments of photoreceptors12 and in Golgi membranes or nuclei in cultured cells.16 Recently, it has been reported that RPGR exhibits species-specific subcellular localization—that is, RPGR localizes in the connecting cilia of mouse but in outer segments of bovine and human photoreceptors.21 Resolution of these discrepancies is important for understanding RPGR function and disease mechanisms. We therefore performed a comprehensive study to evaluate these differences critically.
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Materials and Methods |
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Antibodies
A polyclonal ORF15 antibody was generated by immunizing a guinea pig with a glutathione S-transferase (GST) fusion protein encompassing residues 702-790 of the mouse RPGR ORF15.18 The immune serum was first passed through a GST-affinity column to remove GST-specific antibodies. Affinity purification of ORF15 antibody in the cleared serum was performed with the same immunizing antigen immobilized in an agarose bead column (Aminolink; Pierce, Rockford, IL). The mouse RPGR NT antibody was generated in rabbit by using a soluble GST-fusion protein targeting to the N-terminal 295 residues of the mouse rpgr sequence (GenBank accession, AAC4019; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). It was affinity purified using a His-tagged recombinant protein covering the same region. Bovine RPGR ORFC2 antibody was generated in rabbits with a fusion protein corresponding to the C-terminal 80 residues of bovine RPGR ORF1518 and was affinity purified.
Mouse blue and green cone opsin antibodies were raised in chicken using synthetic peptides corresponding to the C-terminal sequences CRKPMADESDVSGSQKT and FGKKVDDSSELSSTSKT, respectively. Total IgY was isolated from pooled egg yolks.22 Specific antibodies were then purified from the total IgY, using the respective immobilized immunizing peptides (Sulfolink columns; Pierce).
Other antibodies were raised in rabbit using recombinant proteins expressed in Escherichia coli as immunogens. Their lengths and locations along the RPGR polypeptide sequence are shown in the antibody map in Figure 1A . All antibodies were used after affinity purification. The chicken anti-RP1 antibody (the generous gift of Eric Pierce, University of Pennsylvania) was described.23 The rootletin antibody has been described previously.24
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Immunofluorescence staining of cryosections was performed as described.2 13 Briefly, sections were blocked in 5% normal goat serum in PBS for 15 minutes, followed by incubation in primary antibodies diluted in 5% normal goat serum. Incubation in primary antibodies was typically at room temperature for 2 to 3 hours. Primary antibodies were used at the following dilutions: NT, 1:2000; S1, 1:5000; S3, 1:5000; ORF, 1:200; C2, 1:200; RPGRIP, 1:5000; blue cone opsin, 1:5000, green cone opsin, 1:5000; and RP1, 1:2000. Sections were washed twice in PBS and incubated with fluorochrome-conjugated secondary antibodies (Molecular Probes, Eugene, OR). Sections were washed again and mounted in an aqueous mounting medium. For immunoblot analysis, tissues were homogenized in an SDS-protein sample buffer. Total retinal homogenates or axoneme-enriched fractions13 were used for detection of RPGR and RPGRIP on immunoblots.
Alternative Protocol for RPGR Immunostaining
Because cone photoreceptor morphology was poorly preserved in unfixed cryosections, we developed a new protocol for double-labeling cone opsins and RPGR. Retinas were gently dissected while eyes were submerged in PBS. To maintain the shape of the retina, the lens was left in place so that the retina retained the shape of a cup wrapped around the lens. The superior pole of the retina was marked with a burn spot made with an electric cauterizer. The retinal cup was left in Dulbecco’s PBS (Gibco-BRL; Mg2+, Ca2+-free, supplemented with 1 mM EDTA) for a total of 10 minutes with occasional swirling and then transferred to a cold fixative mixture containing methanol, acetone, and glacial acetic acid at a ratio of 1:1:0.05. The retinal cup was fixed in this mixture for 10 minutes with occasional swirling and then washed once in PBS and 0.1% Tween 20 and once in PBS and processed for immunofluorescence. Immunostaining of retinal cups was performed in small vials with occasional swirling but otherwise followed the protocols for staining cryosections. After completion of immunostaining, the retinal cup was fixed in 4% formaldehyde and PBS for 30 minutes, cryoprotected in 30% sucrose, and cryosectioned along the superior-to-inferior axis. The sections were mounted in an aqueous medium.
Serial Retinal Sectioning with Immunoblot Analysis
This technique has been described in detail.25 Briefly, rat eyes were enucleated and dissected under a dim red light. Retinas were flatmounted by placing them between two glass slides separated by 0.5-mm spacers and frozen on dry ice. For sectioning, the bottom slide with the vitreal side of the retina attached was mounted on the cryomicrotome specimen holder. Retinas were trimmed to remove any folded edges, and sequential 5-µm tangential retinal sections were cut. Each section was collected in 50 µL of SDS-PAGE sample buffer. Aliquots from each sample were subjected to SDS-PAGE (4%–12% gradient gels) followed by immunoblot analysis. Optimal transfer of RPGRIP required Towbin buffer with 15% methanol and 0.1% SDS. All other proteins were transferred in the same buffer but without SDS. Antibodies used for immunoblot analysis were: RPGR (NT); RPGRIP, a polyclonal antibody previously described13 ; cytochrome c oxidase subunit IV (monoclonal antibody 20E8-C12; Molecular Probes); and rhodopsin (rho 4D2).26 Band densities were quantified on a densitometer (Personal Densitometer SI; Molecular Dynamics, Sunnyvale, CA, with ImageQuant software).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In mice, constitutive RPGR migrates at 95 to 100 kDa2 13 20 and RPGR ORF15 migrates at approximately 200 kDa.20 As shown in Figure 1B , these antibodies recognized the correct-sized proteins as the major bands on immunoblots. Specificity of these antibodies was further confirmed by absence of these major bands in rpgr-/- retinal homogenates detected by the NT and ORF15 antibodies (Fig. 1B , WT KO) and by the S1 and S3 antibodies (WT).20 In addition, a band migrating at approximately 70 kDa appeared to represent a minor variant of RPGR. Detection by the C2 antibody showed that the bovine RPGRIP ORF15 was approximately 220 kDa, matching the murine variant. The C2 antibody also cross-reacted well with porcine RPGR. The RPGRIP antibody was generated against the conserved RPGR-binding domain of the murine sequence, and it cross-reacted with a major protein, enriched in photoreceptor axonemal preparations, from each of the other mammalian species. This observation, and the high degree of conservation among mammalian RPGRIP in the RPGR-binding domain, suggested that these proteins were bona fide RPGRIP. The rodent RPGRIP migrated at a higher molecular weight, which may be explained by a stretch of approximately 90 glutamic acid residues not found in bovine or human RPGIRP.
RPGR in Mouse Rods
We examined RPGR localization with the ORF15 antibody on frozen retinal sections and found staining largely confined to the junction between the inner and outer segments. Colocalization with RPGRIP showed a largely overlapping pattern (Fig. 2A) indicating that RPGR ORF15 was in the connecting cilia. Staining with RPGR NT (Fig. 2B) and S1 and S320 antibodies showed a similar pattern. The S3 antibody produced consistently weaker signals than ORF15 or S1, which suggested either the lesser quality of the S3 antibody or a smaller amount of constitutive RPGR in the connecting cilia. Subsequent analysis of RPGR in the motile cilia indicated the latter to be true (see Fig. 7A ).
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RPGR in Mouse Cones
To demonstrate directly that RPGR was enriched in connecting cilia of cone photoreceptors as well, we performed double labeling for RPGR and cone opsins. A major obstacle to this endeavor was that these two antibodies required very different fixation conditions. Whereas RPGR signal in the connecting cilia was obliterated by pre-embedding fixation, cone outer segments were poorly preserved in unfixed cryosections. Because RPGR staining was quenched by pre-embedding fixation in whole eyes but was retained if photoreceptors were first dissociated in an aqueous solution, we hypothesized that matrix proteins congealing around the connecting cilia may be preventing antibody access. Therefore, we developed a new protocol in which retinas were first dissected in a physiological buffer to remove some of the interphotoreceptor matrix material before proceeding to immunostaining (see the Discussion section). This protocol was satisfactory for the detection of both proteins. Careful examination of serial confocal photomicrographs clearly demonstrated that the proximal end of each cone outer segment (Fig. 3 , red) is linked to a minute structure strongly positive for RPGR (Fig. 3 , green) indicating RPGR localization in the connecting cilia of cones. Separate double-labeling experiments suggested that the connecting cilia of both green and blue cones are enriched for RPGR and that RPGR ORF15 as well as constitutive RPGR isoforms are expressed in cones.
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RPGR in the Transitional Zone of Motile Cilia
Finally, we sought to determine whether RPGR is found in analogous structures in other types of cilia, such as primary or motile cilia. We chose the airway epithelium to examine RPGR localization because the airway has well-developed motile cilia and because of reports in the literature linking mutations in RPGR to possible immotile cilia defects.9 10 We performed immunostaining for RPGR isoforms on frozen sections of mouse trachea (Fig. 7) . The RPGR ORF15 antibody showed no staining (not shown). With the RPGR NT, S1, and S3 antibodies, we observed staining appearing as a narrow band across the apical domain of all cells bearing cilia in WT mice (Fig. 7A) . Staining with S3 was much stronger than with either NT or S1. Goblet cells, interspersed between ciliated cells, did not show any staining. To determine the precise structure that stained positive for RPGR, we performed double-labeling with 
-acetylated tubulin which is enriched in the axonemal microtubules running along the entire length of the motile cilia. As shown in Figure 7B , RPGR staining overlapped the basal portion of the motile cilia. To confirm that RPGR was indeed in the basal cilia, rather than in the basal bodies, we performed double-labeling with rootletin, which was previously shown to be absent from basal bodies.24 Double-labeling with rootletin (Fig. 7C) showed a narrow gap between RPGR and rootletin, indicating that RPGR was located distal to basal bodies in motile cilia. We conclude that RPGR is localized specifically in the basal portion of motile cilia. This region of the cilia is known as the transitional zone and is structurally analogous to the connecting cilia of photoreceptors.28 29
Western blot analysis of mouse trachea homogenate with the S1 antibody found only the constitutive variant (Fig. 7D ; at 95–100 kDa). This result and the observation that the ORF15 antibody showed negative staining in the airway epithelium demonstrate that RPGR-constitutive was the sole variant detected in this tissue.
By immunoblot analysis, mouse testis expresses a distinct variant of RPGR which migrates at approximately 160 kDa.2 Immunofluorescence staining of sperm, however, did not find RPGR concentrated in the flagellar structure. It is not clear whether RPGR is associated with motile cilia in tissues other than the trachea, perhaps because RPGR is not expressed at high levels in those tissues. During mouse development, the constitutive RPGR variant appears to be abundantly expressed as indicated by immunoblot analysis and is associated with centrosome-related structures in all cells as revealed by double labeling with rootletin24 on sections of day-10 to day-12 embryos (data not shown). In the developing neural tube, RPGR staining is restricted to the apical domain of the neuroepithelium lining the lumen, indicating that RPGR may be associated with neuronal primary cilia.
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Discussion |
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How might we reconcile the disparate results from the literature? Because of its unique anatomy, the connecting cilium is surrounded by a large volume of interphotoreceptor matrix material. It appears that removal of at least some of this material is a key variable in achieving consistent labeling of a protein confined to the connecting cilium on thick (5–10-µm) sections. This proposal is supported by a number of observations. Both RPGR and RPGRIP are extremely sensitive to pre-embedding fixation if the subsequent immunolocalization is to be performed on thick sections. However, destruction of antigenic epitopes by fixatives is not the cause, because RPGR or RPGRIP could be detected satisfactorily on dissociated photoreceptors or by immunoelectron microscopy, even after prolonged fixation. Soluble interphotoreceptor matrix proteins would have been removed in dissociated photoreceptors, and, on ultrathin electron microscopic sections, the cilia would be sectioned by exposing the antigen to the antibody directly. Also supporting this view is the observation that RPGR in the transitional zone of motile cilia, surrounded by airway surface fluid that is not preserved by aqueous fixatives,30 is insensitive to fixation. We hypothesized that matrix material congealing around the cilia and blocking antibody penetration was the primary reason for failing to detect a protein in the connecting cilia.
To confirm our hypothesis, we dissected and rinsed the retinas in an aqueous medium to remove matrix proteins. The retinas were subsequently fixed for 30 minutes in 4% formaldehyde-PBS and immunostained for RPGR. This procedure was quite satisfactory for detection of RPGR (Fig. 3) . We conclude that removal of interphotoreceptor matrix materials facilitated immunodetection on thick sections of proteins confined to the connecting cilium. In practical terms, sections should be cut without prior fixation. A brief on-slide fixation with a low-concentration fixative serves both to preserve the tissues and to allow some diffusion of the matrix material. During storage of the sections, air oxidation and formation of a disulfide bond appear to play a major part in rendering the matrix material impermeable to antibodies, because treatment with a reducing agent is an effective means of antigen retrieval. Alternatively, the protocol we have developed for RPGR/cone opsin double labeling should be generally applicable for detecting a protein in the connecting cilium.
The conclusion of a ciliary localization for RPGR in photoreceptors was strengthened by the finding that RPGR is also localized in the transitional zone of motile cilia, a region that is structurally analogous to the photoreceptor connecting cilium.29 In photoreceptors, RPGRIP has been suggested to serve as an anchoring protein of RPGR.13 RPGRIP expression is, however, largely confined to photoreceptors.13 It appears, therefore, that another protein may be responsible for recruiting RPGR to the basal motile cilia. Search of the mammalian genome database identified an uncharacterized protein designated KIAA1005 (GenBank accession no. AB023222). This protein is homologous to RPGRIP and is widespread and we speculate that it may serve the role of an anchor for RPGR in ciliated cells outside the photoreceptors.
The finding of RPGR in the airway motile cilia supports the notion that certain RPGR mutant alleles may be responsible for the recurrent respiratory tract infections seen in some families.9 10 This effect appears to be allele specific, because recurrent respiratory tract infections are not known to be commonly associated with mutations in RPGR. To our knowledge there has been no report linking mutations in RPGR to situs inversus or male infertility, commonly found in immotile cilia syndrome. In mouse photoreceptors, loss of RPGR leads to a subtle defect and the deleterious effect must accumulate over a period of several months before photoreceptor cell death becomes apparent. It thus has been suggested that RPGR function is not central but facilitative to a cellular process and that RPGR may be described as a longevity gene.2 By analogy with RPGR function in the photoreceptor connecting cilium, loss of RPGR in motile cilia may produce a subtle defect easily masked by the regenerability of the airway epithelium. It is thus reasonable to suggest that mutant alleles linked to a motile cilium defect, if causality can be established, may be gain-of-function mutations.10
Comparison of the immunofluorescence data obtained with the S1 and S3 antibodies suggests that the ORF15 variant of RPGR is the predominant form in the photoreceptor connecting cilium. In the connecting cilium, the S1 antibody (recognizing both the constitutive and the ORF15 variants) gave a stronger signal than the S3 antibody (recognizing only the constitutive variant). On the contrary, S3 elicited a much stronger signal in the airway epithelium, where only the constitutive variant was expressed (Fig. 7) . We explain this difference by suggesting that the S1 epitope in the connecting cilium is more abundant than the S3 epitope, which supports the idea that the ORF15 variant is the predominant RPGR form in the connecting cilium.
The specific localization of proteins in the connecting cilia provides important clues about their in vivo functions. The photoreceptor connecting cilium is a thin bridge linking the cell body and the light-sensing outer segment. The outer segment undergoes constant renewal, a process in which new membranes are added at the base to form new discs, and older ones are shed at the tip.31 32 This process requires the directional transport of proteins to the outer segments from the biosynthetic inner segment. These proteins must pass through or along the cilia against steep concentration gradients. Thus, the connecting cilium must engage in active protein transport and restrict protein redistribution.33 A second role of the connecting cilium relates to disc morphogenesis. Nascent disks are formed by evagination of the plasma membranes at the distal cilia. Cellular machinery located in the cilia regulates an F-actin network located at the distal end of the cilia that is essential for the initiation of new disc membranes.34
Given our data on RPGR localization, future functional studies of RPGR and its interacting proteins should focus on whether they participate in these two major processes occurring at the connecting cilia. Finally, the differential expression of the ORF15 and constitutive isoforms by the sensory and motile cilia suggest a distinct role for the ORF15 variant in photoreceptor cells.
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Acknowledgements |
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Footnotes |
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Submitted for publication November 25, 2002; revised December 30, 2002; accepted January 7, 2003.
Disclosure: D.-H. Hong, None; B. Pawlyk, None; M. Sokolov, None; K.J. Strissel, None; J. Yang, None; B. Tulloch, None; A.F. Wright, None; V.Y. Arshavsky, None; T. Li, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Tiansen Li, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114; tli{at}meei.harvard.edu.
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95–100 kDa). (E) Schematic representation of RPGR (green) localization in a ciliated epithelial cell lining the trachea.
