Human Molecular Genetics, 2002, Vol. 11, No. 24 3065-3074
© 2002 Oxford University Press
Localization in the human retina of the X-linked retinitis pigmentosa protein RP2, its homologue cofactor C and the RP2 interacting protein Arl3
1Division of Pathology, Institute of Ophthalmology, UCL, London EC1V 9EL, UK, 2Department of Biochemistry, New York University Medical Center, New York, NY 10016, USA, 3Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, UK, 4Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA and 5Division of Molecular Genetics, Institute of Opthalmology, UCL, London EC1V 9EL, UK
Received July 23, 2002; Accepted September 6, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the retinitis pigmentosa 2 (RP2) gene cause a severe form of X-linked retinal degeneration. RP2 is a ubiquitous 350 amino acid plasma membrane-associated protein, which shares homology with the tubulin-specific chaperone cofactor C. RP2 protein, like cofactor C, stimulates the GTPase activity of tubulin in combination with cofactor D. RP2 has also been shown to interact with ADP ribosylation factor-like 3 (Arl3) in a nucleotide and myristoylation-dependant manner. In this study we have examined the relationship between RP2, cofactor C and Arl3 in patient-derived cell lines and in the retina. Examination of lymphoblastoid cells from patients with an Arg120stop nonsense mutation in RP2 revealed that the expression levels of cofactor C and Arl3 were not affected by the absence of RP2. In human retina, RP2 was localized to the plasma membrane of cells throughout the retina. RP2 was present at the plasma membrane in both rod and cone photoreceptors, extending from the outer segment through the inner segment to the synaptic terminals. There was no enrichment of RP2 staining in any photoreceptor organelle. In contrast, cofactor C and Arl3 localized predominantly to the photoreceptor connecting cilium in rod and cone photoreceptors. Cofactor C was cytoplasmic in distribution, whereas Arl3 localized to other microtubule structures within all cells. Arl3 behaved as a microtubule-associated protein: it co-localized with microtubules in HeLa cells and this was enhanced following microtubule stabilization with taxol. Furthermore, Arl3 co-purified with microtubules from bovine brain. Following microtubule depolymerization with nocodazole, Arl3 relocalized to the nuclear membrane. These data suggest that RP2 functions in concert with Arl3 to link the cell membrane with the cytoskeleton in photoreceptors as part of the cell signaling or vesicular transport machinery.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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X-linked retinitis pigmentosa (XLRP) is a heterogeneous disease causing a severe form of retinal degeneration. Mutations in the retinitis pigmentosa 2 (RP2) gene have been shown to account for up to 15% of XLRP (1–3). The gene product, RP2, is a ubiquitously expressed 350 amino acid protein (1,4); however, the localization within tissues is unknown. RP2 has sites for N-terminal acyl modification by myristoylation and palmitoylation and is targeted to the plasma membrane in cultured cells (4,5). Furthermore, a pathogenic mutation
S6 (1,6) in RP2 prevents the plasma membrane targeting of RP2 (4,5), suggesting that the plasma membrane localization is essential for RP2 function in the retina.
The 
/ß tubulin heterodimer is assembled via interaction with several tubulin-specific chaperones termed cofactors A–E (8,9) in a reaction in which release of the heterodimer from a cofactor-containing supercomplex is accompanied by GTP hydrolysis by ß-tubulin. In addition, cofactors C and D together with cofactor E have been demonstrated to stimulate the GTPase activity of native tubulin (10). RP2 shares homology with the tubulin-specific chaperone cofactor C over 151 amino acids (1) and the existence of pathogenic mutations in RP2 at conserved residues (1–3,7) is consistent with a functional homology between the two proteins. A recent study has demonstrated that, in the presence of cofactor D, RP2 also stimulates the GTPase activity of tubulin, but cannot substitute for cofactor C in the tubulin heterodimerization reaction (11). Evidence for a shared structural element between the two homologues comes from the observation that a pathogenic mutation R118H in RP2 (1–3,7) at a residue conserved in cofactor C abolishes the tubulin–GTPase stimulating (tubulin–GAP) activity in both RP2 and cofactor C (11). These data suggest that this residue acts as an ‘arginine finger’ to trigger the tubulin–GAP activity and that the R118H mutation in RP2 may cause retinitis pigmentosa due to this loss of tubulin–GAP activity.
The mammalian Arl [ADP ribosylation factor (Arf)-like] proteins constitute a family of Ras-related small GTP-binding proteins with at least eight members (12–15). They share 40–60% amino acid sequence identity with Arf proteins but do not possess the biochemical activities that characterize Arf proteins (16–18). The in vivo functions of members of the Arl protein family are relatively unexplored, but it has recently been shown that Arl2 is essential in tubulin biogenesis (19). In vitro, Arl2 modulates the tubulin-GAP activity of cofactors C and D (20) and mutations in its putative yeast homologues affect microtubule stability (21–23). In contrast, GTP–Arl3 binds specifically to RP2 but does not affect the tubulin–GAP activity of RP2 and cofactor D (11). Interestingly, Arl3 binding is enhanced if RP2 is not myristoylated (11).
Here we demonstrate that RP2 is localized to the plasma membrane in cells throughout the retina. In contrast, we find that cofactor C resides predominantly in the cytoplasm and also the photoreceptor connecting cilium, whilst Arl3 is targeted to the connecting cilium and other microtubule structures within all cells. We also show that Arl3 associates with microtubules, and suggest that RP2 functions to link the cell membrane with the cytoskeleton in photoreceptors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Arl3 and cofactor C expression is unaffected inlymphoblastoid cells from RP2 patients
Given the functional overlap between RP2 and cofactor C and the interaction of RP2 with Arl3 (11), we considered whether the ablation of RP2 in a cell line might affect the expression of the other two proteins. We therefore examined the expression of Arl3 and cofactor C in lymphoblastoid cells from males with the Arg120stop mutation in RP2 (24). Total cell lysates from lymphoblastoid cells were analysed by western blotting and relative expression levels were compared (Fig. 1). As previously reported (24), there was no RP2 detectable in cells from patients with the Arg120stop mutation (Fig. 1A), using RP2 antiserum S974 (4). To determine whether the expression levels of cofactor C were affected by the absence of RP2 in these cells, the lysates were blotted using an anti-cofactor C antiserum. A single band of the predicted size for cofactor C (
40 kDa) was observed in both patient and control cells (Fig. 1A). There was no significant difference in expression levels of cofactor C in the patient cells when compared to cells from control males, at any protein loading or exposure time tested (data not shown). A single band of the predicted size for Arl3 (21 kDa) was found in all of the cells and at similar intensities when using the anti-Arl3 antiserum (Fig. 1A). This indicates that the expression levels of Arl3 were also unaffected by the absence of RP2. Therefore, in lymphoblastoid cells with no detectable RP2, cofactor C expression does not appear to be upregulated to compensate for the lack of RP2. The level of Arl3 is similarly unaffected by the absence of RP2.
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Characterization of an anti-peptide serum to RP2
A rabbit polyclonal antiserum to RP2 was developed by immunization with a synthetic peptide to the C-terminus of human RP2 (residues 337–350) and was named hRP2-337–350. This serum recognized RP2 in western blots of tissues and cell lysates (Fig. 1B) and was specific as determined by competition of the RP2 reactivity following pre-incubation with the peptide and a lack of reactivity in the pre-immune serum. In contrast to S974, which only reacts well with human RP2 (4), hRP2-337–350 recognized RP2 from other mammalian species, including mouse (Fig. 1B).
Immunohistochemistry of RP2 in human retina
The expression pattern of RP2 was investigated using adult human retinal sections from paraffin-embedded, formalin-fixed tissue. Specific immunolabelling with hRP2-337–350 (Fig. 2, RP2) was detected in rod and cone photoreceptors extending from the tips of the outer segments (OS), through the inner segments (IS) and outer nuclear layer (ONL) and into the synapses in the outer plexiform layer (OPL). Immunolabelling was also observed in the bipolar, horizontal and amacrine cells in the inner nuclear layer (INL), extending to the inner plexiform layer (IPL) and finally though the ganglion cell layer (GCL) and into the nerve fibre layer (NFL). RP2 immunolabelling was absent from nuclei and was localized predominantly to the plasma membrane of rod and cone photoreceptors extending from the OS and IS (Fig. 2, Zoom IS+OS) and throughout the ONL (Fig. 2, Zoom ONL). RP2 immunoreactivity could also be detected in the retinal pigment epithelium (RPE), as a diffuse staining (data not shown), but could not be localized to any particular region of the cell due to their heavily pigmented nature. When the pre-immune serum was used for immunolabelling only a faint, background level of staining was detected (Fig. 2, Pre-Imm). This was also observed when the primary antibody was omitted or when immunolabelling was performed in the presence of the competing peptide against which hRP2-337–350 was raised.
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RP2 is present on the plasma membrane ofrod and cone photoreceptors
In order to define the precise localization of RP2 within the photoreceptors, double immunofluorescence labelling was performed with the hRP2-337–350 antiserum and a panel of well-characterized retinal markers on Vibratome sections of paraformaldehyde-fixed human retina. The lectin wheat germ agglutinin (WGA) binds proteins with oligosaccharide chains in the extracellular matrix and was used as a marker for the lateral plasma membrane of the photoreceptor IS and OS (Fig. 3A, WGA). RP2 immunolabelling was localized to the plasma membrane in the IS and OS (Fig. 3A, RP2), as demonstrated by double labelling with WGA (Fig. 3A, Merge). RP2 staining did not extend into the extracellular space but was more concentrated on the intracellular face of the membrane, as would be expected from the previous localization of RP2 in cultured cells to the cytosolic face of the plasma membrane (4,5).
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Immunofluorescent labelling with the monoclonal antibody 1D4 detected rhodopsin in the rod photoreceptor OS (Fig. 3B, 1D4). RP2 staining was evident at the plasma membrane of the IS and extended to the very tips of the OS (Fig. 3B, RP2). The signals for RP2 and 1D4 overlapped at the plasma membrane (Fig. 3B, Merge), demonstrating the presence of RP2 in the rod photoreceptor outer segment plasma membrane. The RP2 staining pattern did not, however, co-localize with rhodopsin within the OS, suggesting that RP2 is not a component of rod OS disks.
The monoclonal antibody 7G6 was used as a marker for cone photoreceptors (Fig. 3C, 7G6). RP2 localized to the plasma membrane of cells in the ONL (Fig. 3C, RP2). When double-labelled with 7G6 (Fig. 3C, Merge), RP2 was observed to stain the plasma membrane of cone cells in the ONL, extending from the cell body and along the cone inner fibre to the cone pedicles. RP2 staining was absent from the nuclei and only very faint staining could be observed in the cytoplasm of rod and cone photoreceptors.
To complement the staining obtained using hRP2-337–350, the sheep anti-RP2 antiserum S974 was used in double-labelling experiments (Fig. 3D, RP2+RP2). In order to circumvent problems of non-specific cross-reactivity with commercially available anti-sheep sera (24), affinity-purified antiserum S974 was directly labelled with Alexa 488. In the ONL (Fig. 3D, RP2+RP2) double labelling of RP2 by the S974 and hRP2-337–350 antisera was observed at the plasma membrane. The RP2 staining was clearly absent from the nuclei but weak cytoplasmic staining could be seen in some cells. Co-localization of the two RP2 antisera was also observed in the IS and OS of the photoreceptors (Fig. 3D, RP2+RP2) and in places this was punctuate in appearance. A third anti-RP2 antiserum (11) was also used to confirm the plasma membrane localization of the protein (data not shown). The specificity of hRP2-337–350 in immunofluorescent staining was confirmed in control experiments as follows. When immunolabelling was performed in the presence of excess competing peptide, the staining was greatly diminished (Fig. 3, RP2+Peptide). There was also no staining observed when the primary antibody was omitted or when pre-immune serum was used. No cross-reaction was detected between the primary and secondary antisera used in the double-labelling experiments. Therefore, the distribution of RP2 in the retina determined by confocal immunofluorescence microscopy confirmed the immunolabelling observed in the paraffin embedded sections (Fig. 2) and was consistent with three different specific antibodies.
Distribution of cofactor C in the human retina
RP2 shares sequence homology with human cofactor C (1) and both proteins possess tubulin–GAP activity (11). Immunofluorescent labelling of paraformaldehyde-fixed human retina using anti-cofactor C antiserum demonstrated that cofactor C was distributed throughout the retina (Fig. 4, Cofactor C). The signal was most intense in the rod and cone photoreceptors, extending from the IS, through the ONL and into the OPL. The strongest cofactor C signal was localized to the photoreceptor connecting cilium as demonstrated by co-localization with ß-tubulin (Fig. 4, ß Tubulin) at the tips of the inner segments. Moderate staining was also detected in the OS, INL and IPL. Apart from the region of the connecting cilium, there was only limited co-localization between tubulin and cofactor C. Cofactor C appeared to have a diffuse cytoplasmic staining pattern in most cells. The cofactor C staining was specific as it could be prevented by preincubation competition with a molar excess of recombinant cofactor C.
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Arl3 localizes to the connecting cilium and microtubule structures in the human retina
Because human RP2 has been shown to interact with GTP-bound Arl3 (11), immunofluorescent labelling of Arl3 was performed with anti-Arl3 antiserum on Vibratome sections of paraformaldehyde-fixed human retina. The Arl3 antibody labelled cells throughout the retina (Fig. 5A, Arl3), but the most intense staining was seen in the connecting cilium, the myoid region of the IS and in cone photoreceptors. Moderate staining was observed in the OS and in the INL and IPL. Arl3 co-localized with ß-tubulin (Fig. 5A, Merge), particularly in the outer retinal layers, suggesting that Arl3 is localized to microtubule structures in the retina. Further evidence for the co-localization of Arl3 with the connecting cilium was provided by double labelling retinal sections with anti-Arl3 and an antibody to acetylated
-tubulin (Fig. 5B).
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To further investigate the intense Arl3 staining in cone photoreceptors, the cone-specific 7G6 antiserum was used as a marker (Fig. 5C). Arl3 intensely labelled 7G6 positive cells, staining from the cone inner segment, along the inner fibre and into the cone pedicle confirming localization of Arl3 in cone photoreceptors. No staining was detected in any layer of the retina when immunolabelling was performed using Arl3 pre-immune sera.
Arl3 is a microtubule-associated protein
As Arl3 appeared to be localized to microtubule structures within the retina, the association of Arl3 with microtubules was investigated further by immunocytochemistry and by biochemical association with microtubules in vitro. Immunofluorescence analyses of Arl3 and -tubulin in HeLa cells using affinity-purified anti-Arl3 antiserum detected an Arl3-specific signal that co-localized with microtubules, in interphase cells (Fig. 6Aa and b). The authenticity of this signal was confirmed in several ways. A strong coincident microtubule stain was observed in the mitotic spindle of dividing cells (Fig. 6Ac and d). This strong signal may correspond either to a higher level of Arl3 binding to spindle microtubules or to the greater density of microtubules in spindles. Stabilization of microtubules with taxol led to an increase in Arl3 staining of bundled microtubules similar to the -tubulin staining pattern (Fig. 6Ag and h). Moreover, following microtubule depolymerization with nocodazole, the Arl3-specific signal was dramatically re-localized to the nuclear envelope in a staining pattern that did not correspond to the diffuse -tubulin localization (Fig. 6Ae and f). To test if Arl3 was in a stable association with microtubules and tubulin we investigated the co-purification of Arl3 with microtubules and tubulin from bovine brain. Arl3 co-purified with microtubules that had been purified by cycles of polymerization and depolymerization (Fig. 6B), suggesting that Arl3 is a microtubule-associated protein (MAP). Furthermore, a fraction of Arl3 also co-purified with tubulin dimers that had been further purified by phosphocellulose chromatography, suggesting a significant interaction between Arl3 and tubulin. However, Arl3 could be biochemically resolved from tubulin by passage over a MonoQ anion exchange column. Collectively, these data show that Arl3 associates with tubulin and microtubules in vitro and in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have demonstrated that RP2 is localized to the plasma membrane of all cells in the adult human retina. This finding is not unexpected as, although the primary pathology for retinitis pigmentosa is thought to lie in the rod photoreceptors, RP2 is ubiquitously expressed (1,4). In addition, in cultured cells, RP2 is targeted to the plasma membrane by a dual acylation motif for myristoylation and palmitoylation (4,25). This dual acylation of RP2 appears to mediate the targeting to the plasma membrane in all the cells of the retina. The significance of the plasma membrane targeting of RP2 had already been suggested by studies that showed the disease-causing
S6 mutation in RP2 prevented dual acylation of the protein and its targeting to the membrane (4,5). The localization of RP2 on the plasma membrane of photoreceptors and other cells in the retina would, therefore, be expected. Three independent, specific antibodies to RP2 under different fixation conditions confirmed this staining pattern in the plasma membrane of photoreceptors and other cells in the retina. No sorting of RP2 to macro-domains of the membrane within the neuronal cells (e.g. axons or dendrites) of the retina was observed. In contrast to cultured cells, where RP2 is predominantly but not exclusively on the plasma membrane (4), only weak RP2 cytoplasmic staining was observed and the nucleus was negative in all the cells in the retina. This discrepancy is likely to reflect differences in sensitivity of immunolabelling between cells and tissue. RP2 is a low-abundance protein in tissues, while cells in culture express much higher levels of the protein (4,25). Therefore, it is probable that in retina it is only possible to clearly detect the strongest signal, which is on the plasma membrane. Thus, while RP2 may also be present in the cytoplasm and other intracellular organelles at low levels, it is not possible to resolve the localization of these components.
There is a functional overlap in vitro between RP2 and its homologue cofactor C (11): both proteins stimulate the GTPase activity of native tubulin in the presence of cofactor D, and both proteins can complement for their putative yeast homologue CIN2. The two proteins, however, are only similar over part of their sequence and may not share full functional equivalence. Cofactor C functions in the heterodimerization of newly formed tubulin subunits, whereas RP2 does not. Similarly, RP2 may have functions that cannot be substituted by cofactor C. This would appear to be the case as cofactor C is present in both rod and cone photoreceptors and does not appear to be able to compensate for the loss of RP2 function in patients. Furthermore, when RP2 is absent from lymphoblastoid cells cofactor C is not upregulated to compensate for the loss of RP2 function. Cofactor C is localized in the cytoplasm and the connecting cilium of photoreceptors, in contrast to RP2 which localizes to the plasma membrane.
Arl3–GTP interacts with RP2 in cell lysates and with in vitro translated products (11). The localization of Arl3 and RP2 in the retina, however, is quite distinct. Arl3 behaves as a MAP, decorating microtubule structures and was not observed on the plasma membrane either in the retina or in cell culture. The binding of Arl3 to RP2 is enhanced when RP2 is unmyristoylated (11). Therefore, Arl3 may not interact with acylated RP2 on the plasma membrane in vivo but in another cellular locale, possibly on or close to the microtubule network.
Arl3 is a member of the ADP-ribosylation factor (Arf)-like family. Arf proteins are molecular switches that mediate multiple steps in membrane traffic, amongst other cellular functions. Arf proteins are characterized by N-myristoylation at glycine 2 and this N-terminal acyl moiety is only accessible to the membrane in the GTP bound state of the protein (26). It has not been possible to myristoylate Arl3 in vitro but the structure of Arl3–GDP (27) suggests that, similar to Arf proteins, Arl3 will undergo a conformational transition upon GTP binding that will expose an N-terminal helix. If Arl3 is myristoylated in vivo, it is possible that RP2 would bind GTP–Arl3 on a membrane. No guanine nucleotide exchange factor (GEF) for Arl3 has been identified and the proportion of Arl3 that exists in the GTP-bound state is undetermined. Nevertheless, we did not observe Arl3 on the plasma membrane under any conditions, suggesting that the plasma membrane is not the site of the interaction between RP2 and Arl3. Arl3 relocalized to the nuclear envelope of HeLa cells following microtubule depolymerization and a small proportion of Arl3 was seen on the nuclear envelope in some cells under normal growth conditions. It was not possible to determine whether Arl3 was localized to the nuclear envelope in photoreceptors at the resolution of light microscopy. The significance of this nuclear envelope localization of Arl3 is unclear at present, but it will be important to determine if this is a site for the interaction of RP2 and Arl3. The identification of an Arl3 GEF and the characterization of the N-myristoylation state of Arl3 will help clarify the role of these modifications on the interaction with RP2.
Arl3 localized to microtubule structures within the retina, particularly within cone photoreceptors. The connecting cilium of rod and cone photoreceptors was strongly labelled and this would suggest a role for Arl3 in the maintenance and function of the photoreceptor cytoskeleton. These data, combined with the ability of RP2 to act, with cofactor D, as a GTPase-activating protein (GAP) for tubulin, indicate that RP2 is likely to be involved in modulating the function of the cytoskeleton. Photoreceptors have a specialized microtubule architecture with several unique properties, most prominent of which is the connecting cilium. The connecting cilium is a key player in the vectorial transport of proteins to the OS and mutations in several connecting cilium-associated proteins have been shown to cause retinal degeneration (28–30). The localization of Arl3 to the connecting cilium suggests that this is another potential site for an interaction with RP2, which could be important for photoreceptor maintenance. In addition to binding RP2, Arl3 interacts with PDE
(31). RPGR, which is a major cause of RP (3), also interacts with PDE (32). RPGR is targeted to the connecting cilium by RPGRIP (29), suggesting that the connecting cilium is a possible site for a convergence of pathways involving RP2 and RPGR, perhaps involving vectorial vesicular transport.
Alternatively, RP2 may interact with microtubules near the plasma membrane of the OS in the incisures that are characteristic of some photoreceptors (33). Rod photoreceptors have a specific set of glutamic-acid-rich proteins (GARPs) that are localized to these incisures and organize a dynamic protein signalling complex on the plasma membrane near the disc rim (34). As RP2 is associated with lipid rafts (Chapple et al., submitted), which are also rich in signalling components, it is tempting to speculate that RP2 could link the cytoskeleton to these signalling complexes and a functional deficit is only observed in rods because the GARPs are rod-specific. The localization of RP2 to the plasma membrane and its interacting protein to microtubules further strengthens the hypothesis that RP2 functions to link the membrane to the cytoskeleton either through membrane traffic or cell signalling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibody generation
Rabbit polyclonal antiserum, hRP2-337–350, was raised against peptide VDSFYNFADIQMGI, which corresponds to amino acid residues 337–350 at the C-terminus of human RP2, conjugated to keyhole limpet haemocyanin (KLH; Genosys Biotechnologies Ltd, Cambridge, UK). To confirm the specificity of immunolabelling, western blots on tissue and cell extracts were also probed with no primary antibody, pre-immune serum and hRP2-337–350 preabsorbed with excess peptide as previously described (35). Production and characterization of affinity-purified sheep polyclonal RP2 antiserum S974, rabbit anti-Arl3 and rabbit anti-cofactor C have been described previously (4,11).
SDS–PAGE and western blotting
Human lymphoblastoid cell lines from RP2 patients with premature truncation mutations and male controls were maintained in suspension culture in RPMI 1640 Glutamax-I (Life Technologies, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, Poole, UK). For western blot analysis the cells were Dounce homogenized on ice in 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 12.5 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, containing a protease inhibitor cocktail and added to sample buffer (100 mM Tris–HCl, pH 6.8, 2% glycerol, 1% SDS, 1% 2-ß mercaptoethanol, 0.01% bromophenol blue, final concentration). The concentration of protein in the homogenates was determined using the BCA protein assay (Pierce, Tattenhall, UK), to ensure even loading on the gels. Protein samples were resolved on 12% SDS–polyacrylamide gels and electroblotted onto nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Non-specific binding sites on the nitrocellulose were blocked by incubation with 5% marvel, 1 x phosphate-buffered saline (PBS), 0.1% Tween-20, overnight at 4°C. The blots were hybridized with affinity-purified sheep anti-RP2 polyclonal antiserum S974 (1 : 1000), rabbit anti-Arl3 crude polyclonal antiserum (1 : 1000), rabbit anti-cofactor C crude polyclonal antiserum (1 : 2000) or mouse anti-ß tubulin monoclonal antiserum (1 : 2000; Clone TUB2.1, Sigma) for 1 h. Subsequently, the membranes were incubated with peroxidase conjugated anti-sheep (Sigma), anti-rabbit (Pierce) or anti-mouse (Sigma) antibodies for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham Pharmacia, Little Chalfont, UK).
Immunohistochemistry
Adult human retinae were fixed with 10% neutral-buffered formalin within 2 min of enucleation. After at least 24 h of fixation, the samples were dehydrated with increasing concentrations of industrial methylated spirits (IMS), equilibrated in xylene and embedded in paraffin wax. Cross-sections, 8 µm in thickness were floated out onto 20% methanol, expanded on water preheated to 40°C and mounted onto electrostatically charged glass slides (VWR International Ltd, Poole, UK). After dewaxing successively in xylene, 100 and 95% IMS, the samples were stained using the streptavidin-biotin method, as described previously (36) using hRP2-337–350 (1 : 100). To verify the specificity of the immunostaining, sections were also stained with no primary antiserum, pre-immune serum, and hRP2-337–350 preabsorbed with the peptide epitope (20 µg/µl). The immunolabelled retinal sections were visualized with an Olympus BX50 light microscope using bright field optics.
Immunofluorescence labelling and confocal scanning microscopy
Adult human retinae were fixed with 4% paraformaldehyde in PBS pH 7.3, within 2 min of enucleation for at least 2 h. After thorough rinsing in PBS, the retina tissue was embedded in 5% low melting point agarose and cut into 80 µm sections using a Vibratome. The sections were blocked with 5% normal donkey serum (Jackson Immunoresearch, Luton, UK) and 2% BSA overnight at 4°C prior to antibody incubations. The hRP2-337–350 antiserum (1 : 100), anti-Arl3 antiserum (1 : 200) and anti-cofactor C antiserum (1 : 300) were used in double-labelling experiments with other antisera. Rhodopsin-specific antiserum, 1D4 (1 : 200; National Cell Culture Center, USA), was used as a marker for rod photoreceptor outer segments. Rod and cone photoreceptor inner and outer segment extracellular matrix oligosaccharides were detected using WGA (1 : 200; Vector Laboratories, Burlingame, USA). Cone photoreceptors were detected with the cone-specific antiserum, 7G6 (1 : 100; kindly provided by Dr P. MacLeish, Morehouse School of Medicine Neuroscience Institute, USA). ß-Tubulin was detected using clone TUB2.1 antiserum (1 : 500; Sigma) and acetylated -tubulin was labelled using clone 6-11B-1 antiserum (1 : 400; Sigma). Sheep anti-RP2 S974 directly conjugated to Alexa 488 (Molecular Probes, Cambridge, UK) was used as a second antiserum for RP2 staining. Primary antibodies were detected with Cy3-conjugated donkey anti-rabbit (1 : 100), Cy2 conjugated donkey anti-mouse (1 : 100) or Cy2-conjugated streptavidin (1 : 100; all Jackson Immunoresearch). All antibody incubations were performed in blocking buffer overnight at 4°C, with extensive washes in PBS in between. Nuclei were labelled with DAPI in the final PBS wash and sections were mounted in mounting media containing 15 mM sodium azide (Dako, Ely, UK). To verify the specificity of the immunostaining, sections were also stained with no primary antiserum, pre-immune serum, and hRP2-337–350 pre-absorbed with the peptide epitope (20 µg/µl). In addition, rabbit primary antibodies were incubated with mouse secondaries and vice versa to ensure there was no cross-reactivity between antisera in the double-labelling procedures. Labelled retinal sections were visualized with a Zeiss LSM 510 laser scanning confocal microscope.
Immunocytochemistry
HeLa cells were maintained in modified Eagle's medium supplemented with 10% fetal calf serum. For immunocytochemistry, cells were grown on coverslips and fixed in 4% paraformaldehyde in PBS followed by detergent permeabilization in 0.2% Triton X-100. Drug-treated cells were incubated with 10 µM of either taxol or nocodazole for 2 h prior to fixation. Affinity-purified anti-Arl3 antibody was used at a dilution of 1 : 20 and a monoclonal anti- tubulin antibody (Sigma clone B-5-1-2) was used at a dilution of 1 : 2000. Cells were subsequently incubated with FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse antibodies (Jackson ImmunoResearch) at dilutions of 1 : 50 for the detection of Arl3 and tubulin respectively. Fluorescence images were captured using a Zeiss Axiophot microscope.
Microtubule purification
A microtubule pellet was purified from bovine brain using established protocols (37). Tubulin was further purified from microtubule-associated proteins by passage through a phosphocellulose column (38) and application to an anion exchange column (MonoQ, Amersham Pharmacia), which was developed with a linear gradient of NaCl. To demonstrate the presence of Arl3 in the tubulin samples, 4 µg of each fraction and 50 µg of HeLa cell lysate were loaded on an SDS–polyacrylamide gel and subjected to western blot analysis. Immunolabelling was detected using affinity-purified anti-Arl3 (1 : 20) and a peroxidase-conjugated anti-rabbit secondary antibody (Amersham Pharmacia). Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham Pharmacia).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr Peter MacLeish for the 7G6 marker. We would like to thank to Dr J. van der Spuy for assistance in immunohistochemical methods, and would also like to thank Mr R. Alexander and Miss R. Hart. This work was supported by grants from the Wellcome Trust and Fight for Sight (to M.E.C., K.R.W. and A.J.H.) and a grant (to N.J.C.) from the National Institutes of Health. C.G. is a Fight for Sight Prize Student.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +44 2076086944; Fax: +44 2076086862; Email: michael.cheetham{at}ucl.ac.uk
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REFERENCES |
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