Abstract
purpose. The retinitis pigmentosa guanosine triphosphatase (GTPase) regulator (RPGR) is essential for photoreceptor survival. There is as yet no consensus concerning the subcellular localization of RPGR. This study was undertaken as a comprehensive effort to resolve current controversies.
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.
RPGR is encoded by the X-linked RP3 locus and has an essential role in maintaining photoreceptor viability. Mutations in the human
RPGR gene cause retinitis pigmentosa (RP),
1 a form of hereditary photoreceptor degeneration that leads to blindness. Targeted disruption of the mouse
rpgr gene and naturally occurring mutations in dogs also lead to photoreceptor degeneration,
2 3 suggesting conserved RPGR function in mammalian species. Both in patients and in mice without RPGR, an early cone photoreceptor defect, in addition to the rod disease, has been noted, indicating that RPGR function is required in both rods and cones. Consistent with this notion, retinal histopathology of a carrier female revealed patchy cone photoreceptor loss independent of rod degeneration.
4 Considerable clinical heterogeneity has been reported. For example, patients in some families present primarily a cone-dominant disease.
5 6 7 8 Thus allelic difference may be at work to produce the highly varied clinical outcome. There have been occasional reports of families with
RPGR mutations in which affected individuals also have recurrent respiratory infections, indicative of an immotile cilium defect.
9 10 If the RPGR mutations are indeed causal of the respiratory problem in these families, it suggests a role for RPGR in the airway epithelium as well.
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 tissues
1 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 cilia
2 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 photoreceptors
12 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.
For typical in situ detection of RPGR and RPGRIP, eyes were embedded in optimal cutting temperature (OCT) compound without fixation and quick-frozen in liquid nitrogen. Cryosections at 5 to 10 μm were cut and collected on pretreated glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA). Sections were stored at −20°C and used within 2 to 3 days. Just before use, sections were fixed on slides for 2 to 5 minutes with 1% formaldehyde in phosphate-buffered saline (PBS) at pH 7.0. Use of higher formaldehyde concentrations was found to quench the signal. After sections had been stored for longer than 1 week, an additional treatment for 30 minutes in 0.1 M 2-mercaptomethanol, PBS, and 0.1% Triton X-100 was performed after fixation. Sections were then washed once in PBS and carried through to immunofluorescence staining. For detection of RPGR in motile cilia, freshly dissected tracheas were rinsed in PBS and fixed for 10 minutes in 4% formaldehyde and PBS. Tracheas were cryoprotected in 30% sucrose, embedded in OCT, and sectioned at 5- and 10-μm thickness.
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 fractions
13 were used for detection of RPGR and RPGRIP on immunoblots.
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.
The complexity of RPGR expression and low abundance have produced inconsistent results regarding RPGR intracellular localization and made the functional studies difficult. Different research groups have placed RPGR at various subcellular locations. The current controversy primarily concerns whether RPGR is localized in the connecting cilia versus the outer segments and whether RPGR localization varies among different mammalian species. These issues have important implications for understanding RPGR function in photoreceptor cells. Using complementary techniques of immunofluorescence and serial retinal sectioning, we demonstrated that major RPGR isoforms are localized in the photoreceptor connecting cilia. Each method has its own advantages and shortcomings. Immunofluorescence provides the high resolution necessary to localize RPGR in the connecting cilium. However, the connecting cilium where RPGR is highly concentrated occupies only a minute volume of the entire photoreceptor cell. If a fraction of RPGR exists diffusely in other parts of the cell, it could escape detection by immunofluorescence. It could not, however, escape immunoblot detection in serial sections, which accounts for material contained in the entire volume of a given cellular layer (section). In addition, the serial sectioning approach is not subject to epitope masking and gives a more quantitative measure of the data.
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.
Supported by National Institutes of Health Grants EY10309 and EY10336, the Foundation Fighting Blindness, the British Retinitis Pigmentosa Society (AFW), Fight For Sight (D-HH), and the Knights Templar Eye Foundation (D-HH, KJS).
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@meei.harvard.edu.
The authors thank Yun Zhao for helpful advice.
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