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Address correspondence to Tiansen Li, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114. Tel.: (617) 573-3904. Fax: (617) 573-3216. E-mail: tli{at}meei.harvard.edu
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Abstract |
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Key Words: photoreceptor; cilium; striated fiber; basal body; centriole
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Introduction |
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A number of functions have been ascribed to the rootlets. Their cytoskeletal nature and the cross-linking between the rootlets and other cytoskeletons indicate an important role as an anchor and support structure for the cilia. Rootlets are associated with both motile and immotile cilia, the latter including the connecting cilia of vertebrate photoreceptor cells (Tokuyasu and Yamada, 1959; Cohen, 1960, 1965). In mammalian photoreceptors, the rootlets are very prominent and span much of the cells' lengths (Spira and Milman, 1979). The connecting cilia may be particularly dependent on rootlets for support, being a thin bridge linking the cell body and a comparatively giant organelle, the light-sensing outer segment (Besharse and Horst, 1990). Interaction between photoreceptor rootlets with membrane-bound vesicles, ER, Golgi, and mitochondria has been reported (Spira and Milman, 1979; Wolfrum, 1992), indicating a role in the proper positioning or anchoring of cellular organelles. A role for rootlets in intracellular protein transport has also been suggested (Fariss et al., 1997).
Though these proposals seem plausible, a definitive determination of the in vivo rootlet function requires knowledge of its structural constituent and the ability to manipulate it genetically. The question of what constitutes the ciliary rootlets in higher eukaryotes has remained unresolved despite numerous investigations. For example, centrin has been found in the flagellar rootlets of green algae (Salisbury, 1995), but is not present in the ciliary rootlets of vertebrate photoreceptors (Wolfrum, 1995). Antigenic epitopes in rootlet-associated proteins have been documented using mAbs raised against partially purified rootlet preparations. An mAb (Clone CC310) recognizes a 175-kD protein associated with the rootlets in the chick oviduct (Klotz et al., 1986). This antibody recognizes ciliary rootlets in a variety of species, including mammals. Using a similar approach, a 195-kD protein was found in the rootlets of human oviduct epithelium (Hagiwara et al., 2000). Although these antibodies have been useful as markers for the rootlets, the identities of proteins that they recognize remain unknown. In the present work, we characterized a novel coiled-coil protein present in the ciliary rootlet. Our data suggest that this protein, which we have named rootletin, is a structural component of the rootlet. We propose that the ciliary rootlet is composed of homopolymeric protofilaments of rootletin.
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Results |
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Rootletin is a component of the rootlet in all ciliated cells
To confirm that rootletin is indeed a component of the rootlet, two antibodies directed against different regions of mouse rootletin were generated (Fig. 1 A). Immunoblots using either Root10 or Root6 antibody revealed a polypeptide migrating at 220 kD. Among multiple tissues examined, the retina exhibited the highest level of expression (Fig. 2 A). Smaller amounts of rootletin were detected in the brain, trachea, and kidney. Rootletin in the retina was primarily derived from photoreceptor cells because its level was greatly diminished in rd mouse retinas in which the photoreceptors had degenerated (unpublished data). Rootletin was found in the insoluble fraction of cell lysate. It was resistant to detergent extraction, but readily solubilized in high salt solutions, indicating ionic interaction is important in rootletin polymer formation. Rootletin was fully soluble in the presence of chaotropic agents or under denaturing conditions (Fig. 2 B).
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By immunoelectron microscopy (Fig. 3, AD), rootletin was found in the rootlet only and did not extend into the basal bodies. On cross sections (Fig. 3 E), rootlets were seen as bundles of individual thin filaments (protofilaments) with a diameter of 910 nm. The shape and dimension of the bundles were highly variable. Rootlets measured on cross sections were as wide as 300 nm or as narrow as 50 nm. The number of protofilaments in a bundle also varied widely. Interestingly, both longitudinal and cross-sectional views showed that rootlets were closely flanked by membranous saccules (Fig. 3, C and E). The saccules did not completely encircle the rootlet bundles; in some areas rootlets were exposed directly to the surrounding cytoplasm. The saccules were frequently seen continuous with rough ER, and their outer surfaces were also studded with ribosomes.
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Formation of rootlet from rootletin monomers
To examine how rootlets were formed from rootletin monomers, we analyzed a series of deletion constructs by protein interaction assays and by transient expression in COS cells. Deletion constructs of rootletin are schematically shown in Fig. 6 A. Four of them (R1, R2, R3, and R4) were tested for protein interactions in yeast two-hybrid assays. In these assays, each fragment was inserted into both a bait and a prey vector, and the bait and prey plasmids were cotransformed into yeast. Protein interactions were indicated by the rescue of nutritional markers in the cotransformants. The results are summarized in Fig. 6 B. The globular domain (R1) did not interact with any part of rootletin. In contrast, fragments derived from the rod domain (R2, R3, and R4) exhibited homotypic binding. In large-scale two-hybrid library screens, baits R3 and R4 also identified rootletin as an interacting partner, demonstrating the specificity of the interactions. These data indicate that the rod domain of rootletin mediates the formation of homodimers. Because each fragment binds only to itself, the rootletin dimer should be parallel and in axial register.
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Because the rod domain of rootletin exhibits homotypic binding, it is likely that truncated or otherwise mutated versions of rootletin will still interact with the full-length rootletin. As such, they may interfere with rootlet formation. To test this hypothesis, we cotransfected each of the rod domain fragments (R2, R3, and R4) in combination with the full-length rootletin. As shown in Fig. 6 D, each induced aggregates containing both the truncated and full-length rootletin, and inhibited formation of rootlets. These data suggest that certain mutant forms of rootletin readily incorporate with the normal rootletin and may act as strong dominant-negatives.
Although recombinant rootletin forms homopolymeric fibers resembling rootlets, rootlets in vivo may in fact be formed as a copolymer with an unidentified but homologous protein. To search for proteins that might copolymerize with rootletin, we performed immunoprecipitation of retinal homogenate and examined if another protein coprecipitated stoichiometrically with rootletin. On Coomasie dye staining, one visible polypeptide (excluding the immunoglobulin heavy and light chains) migrated at 220 kD and was confirmed to be rootletin by immunoblotting (Fig. 6 E). The only mammalian homologue of rootletin is C-Nap1, which migrates at >250 kD. A protein of this size was not present in the immunoprecipitate. Together, the available data suggest that rootlets in vivo are composed of homopolymers of rootletin.
The globular head domain of rootletin interacts with kinesin light chain 3
The head domain of rootletin does not contribute to the formation of the rootletin filaments, indicating that this domain may protrude from the fibrous polymer and potentially interact with other proteins. To search for interacting partners, we performed yeast two-hybrid screening of a mouse retinal cDNA library using the head domain (R1) as a bait. 13 independent clones were identified from a screen of over 106 cotransformants that encoded the full-length cDNA for kinesin light chain 3 (KLC3),* a protein initially found in spermatids (Junco et al., 2001). To confirm the physical interaction between these two proteins, KLC3 was expressed either singly or in combination with the full-length rootletin in COS cells. When expressed alone, KLC3 fusion protein appeared as amorphous aggregates in COS cells (Fig. 7 A). Coexpression with rootletin lead to distribution of KLC3 along the rootlet fibers (Fig. 7 B). These data confirm the physical interaction between KLC3 and rootletin. To investigate if KLC3 was present in photoreceptors, we conducted immunostaining and immunoblotting with KLC3 antibodies. KLC3 was detected as a single band on immunoblots from retinal homogenate and was abundant in the inner segments of photoreceptors (Fig. 7 C). Therefore, KLC3 is localized in the same subcellular compartment as rootletin in the photoreceptors. These data suggest that the interaction between KLC3 and rootletin could potentially be physiologically relevant.
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Discussion |
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Rootletin is predicted to form extended coiled-coil structures. Coiled-coil interactions produce stable polymers, as exemplified by intermediate filaments, collagen fibers, and the tail domain of myosin heavy chains, among others. Thus, coiled-coil interaction in the extended -helical rod domain of rootletin is a key feature in rootlet formation. We propose a model to account for the assembly of rootlets from rootletin monomers (Fig. 9). The first step is the formation of rootletin homodimers that are parallel and in axial register. Formation of higher order elongated polymers does not require head-to-head or head-to-tail interactions because the globular head domain is dispensable. Instead, lateral binding within the rod domain of rootletin dimers, which primarily involves ionic interactions, is important. The resulting rootletin protofilament has a diameter of
10 nm, similar to intermediate filaments. By analogy, the number of rootletin monomers in a cross section of the protofilament may be close to 32. The periodicity of cross striation likely corresponds to the axial stagger; the repeat segments of lower packing density incorporate more stain and manifest as darker (electron dense) bands. Finally, the rootlet itself is assembled from bundled rootletin protofilaments. These bundles are highly variable with respect to the shape and the number of protofilaments within, giving rise to variable appearance of rootlets. By morphological criteria, individual rootlet protofilament would be indistinguishable from intermediate filaments. Previous studies have suggested that in cells in which the rootlets are less developed, there tends to be a concomitant increase in the fibrous component of the apical cortex. These fibrous components may in fact be nonbundled rootlet protofilaments.
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The globular head domain is found to physically interact with KLC3. Although originally characterized as a protein enriched in spermatids [Junco et al., 2001], our analysis shows that KLC3 is also abundant in photoreceptors. This interaction may have a role in transporting rootletin to the proximal basal body, where rootlet formation initially occurs. Alternatively, the rootlet may be associated with the kinesin motor complex and is involved in the trafficking of other proteins. In this regard, it is interesting to note that the rootlet has been suggested to play a role in the transport of a photoreceptor-specific protein (Fariss et al., 1997). A number of studies have also identified ATPase activity overlapping with the cross striation of the rootlet (Matsusaka, 1967), consistent with an association of rootletin with motor proteins. The physiological significance in the rootletinKLC3 interaction remains to be established.
Our finding that rootletin normally localizes to the proximal ends of centrioles in nonciliated cells raises the possibility of a centriolar function for rootletin. In this putative role, a possible connection to the function of C-Nap1 is intriguing. Rootletin substantially overlaps with C-Nap1 in many molecular and cell biological aspects. The two proteins are similar in primary sequence and in overall domain organization. Both proteins are found at the centrioles and disassemble from the mitotic spindle at anaphase during mitosis. C-Nap1 is proposed to play a role in cell cycle-regulated centrosome cohesion, i.e., the linkage between the two centrioles. Yet, C-Nap1 is restricted to the proximal ends of individual centrioles and is therefore not the linker itself (Mayor et al., 2000). In contrast, striated rootlet fibers are seen linking the two basal bodies (Fig. 3 A). In nonciliated cells, rootletin polymers appear to overlap with the two centrioles (Fig. 5 A). Thus, rootletin appears able to fulfill the role of a centriolar linker. In this putative scenario, the linker is composed of rootletin polymers that may interact with C-Nap1 at the points of centriolar attachment.
Rootlets are associated with all types of cilia, be they motile, sensory, or primary. Our data suggest that both recessive null and dominant negative mutations of rootletin may disrupt the formation of rootlets. Clinical entities such as the immotile cilium syndrome, primary ciliary dyskinesia, and retinitis pigmentosa are candidates for having a genetic defect in rootletin. How such a deficit will in turn affect the structure and function of the cilia and the host cells will provide much insight into the physiological role of the rootlet. The rootlet is well known, yet still mysterious. Identification of rootletin as its structural component marks a major step forward toward understanding this organelle.
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Materials and methods |
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Antibodies, immunofluorescence, immunoprecipitation, and immunoblotting
cDNA fragments coding for amino acid residues 101923 (Root10) and 18192006 (Root6) of mouse rootletin were inserted into the expression vector pET28b (Novagen). Recombinant proteins were expressed as His-tagged fusion proteins in the Escherichia coli host BL21(DE3)pLysS. The recombinant proteins were purified through a Ni2+-charged nitriloacetic acid agarose column and were used to immunize rabbits and chickens. Rootletin-specific antibodies were affinity-purified from antisera or egg yolk extracts. Different rootletin antibodies gave similar results on immunoblots and on immunofluorescence. Unless otherwise noted, data in this report were obtained using both rabbit anti-Root6 and anti-Root10 antibodies. MAb CC310 (Klotz et al., 1986) and anti-KLC3 pAb (Junco et al., 2001) were described previously. Monoclonal antiacetylated -tubulin, monoclonal anti
-tubulin, monoclonal anti-Golgi58, polyclonal antiactin, monoclonal antivimentin, and monoclonal anticytokeratin 7 antibodies were obtained from Sigma-Aldrich. Monoclonal anti-KDEL (ER marker) antibody was obtained from Calbiochem. Alexa 488 and Alexa 594conjugated secondary antibodies were obtained from Molecular Probes, Inc.
Immunofluorescence and immunoblotting were performed essentially as described previously (Hong et al., 2001). Tissues were collected from normal mice (C57BL/6) after euthanasia by CO2 inhalation. To prepare dissociated photoreceptors, retinas were dissected out and collected in a microcentrifuge tube containing 0.5 ml PBS. The tube was shaken vigorously for a few seconds and allowed to settle. The buffer containing broken photoreceptor cells was transferred onto glass slides (Superfrost Plus; Fisher Scientific International). Materials adhering to the glass slides contained many outer segments attached to the connecting cilia, rootlets, and sometimes remnants of inner segment membranes. They were fixed in 4% formaldehyde/PBS for 10 min before immunostaining. Cultured cells were fixed in a mixture of methanolacetone at -20°C for 10 min before immunostaining. Cell nuclei were counterstained with Hoechst dye 33342. Slides were mounted in an aqueous mounting medium, viewed, and photographed on a fluorescent microscope (model IX70; Olympus) equipped with a digital camera (Carl Zeiss MicroImaging, Inc.) or on a confocal laser scanning microscope (model TCS SP2; Leica), using the AxioVision 2.0 software and the Leica Confocal Software, respectively.
To assess the solubility of rootletin, retinas were homogenized in a buffer containing 10 mM Tris, pH 7.5, 10 mM EGTA, 2 mM MgCl2, 1 mM DTT and protease inhibitors. After a low speed centrifugation to remove nuclei, the supernatant was centrifuged at 170,000 g for 40 min. Pellets were then extracted at RT for 10 min in various buffers as shown in Fig. 2 B. The resulting supernatants and pellets were analyzed by immunoblotting.
For immunoprecipitation, mouse retinas were homogenized in a buffer containing 10 mM Tris, pH 7.4, 300 mM NaCl, 1 mM DTT and protease inhibitors. The homogenate was centrifuged at 18,000 g for 5 min. The supernatant was precleared by incubating with protein G agarose followed by centrifugation at 2,000 g for 2 min. The supernatant was incubated with either rabbit antiRoot10 antibody or nonimmune rabbit IgG, and then with protein G-agarose for 1 h each at RT. After washing, the bound materials were eluted by incubating with SDS protein sample buffer at 70°C for 20 min. Samples were separated by SDS-PAGE and analyzed either by staining with Coomasie dye or by immunoblotting using the chicken antiRoot6 antibody.
Transient expression of recombinant rootletin
COS-7 cells were maintained in DME supplemented with 5% FBS at 37°C in 5% CO2. Transfection was performed using the GeneshuttleTM-40 reagent (Qbiogene, Inc.) according to the manufacturer's instructions. To express rootletin or its fragments, the cDNAs were inserted into the mammalian expression vector pcDNA3.1(-) (Invitrogen) or pEGFP-C2 (CLONTECH Laboratories, Inc.). Cells were processed 30 h after transfection. Expression constructs used in the expression studies were as follows (the numbers denote amino acid residues): R1, 3533; R2, 5011000; R3, 9991500; R4, 14952009; R123, 11684; R234, 5012009; FL, 12009.
Electron microscopy and immunoelectron microscopy
For transmission electron microscopy, enucleated eyes were fixed for 10 min in 1% formaldehyde, and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5). After removal of the anterior segments and lens, the eye cups were left in the same fixative at 4°C overnight. Eye cups were washed with buffer, post-fixed in osmium tetroxide, dehydrated through a graded alcohol series, and embedded in Epon. Sections were stained in uranyl acetate and lead citrate before viewing on an electron microscope (model 100CX; JEOL USA, Inc.). To examine ultrastructural localization of rootletin, mouse eyes were processed for immunoelectron microscopy as described previously (Hagstrom et al., 2001) and stained using the rabbit antirootletin Root6 antibodies. To examine the ultrastructure of recombinant rootletin polymers, the pEGFP-R234 expression construct was transiently expressed in COS-7 cells. In this construct, the head domain of rootletin dispensable for polymer formation was replaced by GFP for ease of tracking cells expressing the recombinant protein. Cells were treated with 20 µM nocodazole for 1.5 h before harvesting. Cells were scraped off the culture vessel, pelleted, and fixed for 1 h in a solution containing 0.5% saponin, 2% tannic acid, 1% formaldehyde, and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (Maupin and Pollard, 1983). Cells were post-fixed in 1% osmium tetroxide, dehydrated through a graded alcohol series, and embedded in Epon. Sections were stained in uranyl acetate and lead citrate before viewing.
Yeast two-hybrid screening
Cloning vectors, yeast host cells, and reagents were purchased from CLONTECH Laboratories, Inc. A GAL4-based two-hybrid system (System 3) was used. A retinal cDNA library was constructed using poly(A)+ RNA from C57BL/6 mouse retinas and inserted into the pACT2 plasmid vector. The bait plasmid was constructed by inserting a cDNA encoding the bait protein into the pGBKT7 plasmid vector. Library screening was performed according to the manufacturer's instructions. To test interactions directly, these fragments were also inserted into the prey vector pGADT7 and cotransformed with the bait plasmids into yeast host cells. Positive colonies were identified based on their ability to express nutritional markers HIS3 and ADE2 as well as a lacZ reporter. Each cotransformation experiment was plated out on both SD-4 (-Leu, -Trp, -Ade, and -His) and SD-2 (-Leu and -Trp) plates. The latter served as a control for successful cotransformation. A baitprey combination was deemed interacting if the following criteria were met: (1) colonies were recovered from SD-4 plates; (2) colonies were completely white (indicating functioning adenine biosynthetic pathway); and (3) colonies turned blue upon exposure to X-Gal substrate (CLONTECH Laboratories, Inc.). Negative pairings yielded no colonies on SD-4 plates.
GenBank accession number
Mouse rootletin (GenBank/EMBL/DDBJ accession no. AF527975).
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Footnotes |
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Acknowledgments |
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This work was supported by a National Institutes of Health grant (EY10309). T. Li is a Research to Prevent Blindness Sybil B. Harrington Scholar.
Submitted: 26 July 2002
Revised: 27 September 2002
Accepted: 27 September 2002
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References |
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Besharse, J.C., and C.J. Horst. 1990. The photoreceptor connecting cilium: a model for the transition zone. In Ciliary and Flagellar Membranes. R.A. Bloodgood, editor. Plenum, New York. 389417.
Cohen, A.I. 1965. New details of the ultrastructure of the outer segments and ciliary connectives of the rods of human and macaque retinas. Anat. Rec. 152:6380.[Medline]
Engelmann, T.W. 1880. Zur anatomie und physiologie der flimmerzellen. Arch gel Physiol. 23:505535.
Fawcett, D.W., and K.R. Porter. 1954. A study of the fine structure of ciliated epithelia. J. Morphol. 94:221282.
Fry, A.M., T. Mayor, P. Meraldi, Y.D. Stierhof, K. Tanaka, and E.A. Nigg. 1998. C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2. J. Cell Biol. 141:15631574.
Hagstrom, S.A., M. Adamian, M. Scimeca, B.S. Pawlyk, G. Yue, and T. Li. 2001. A role for the Tubby-like protein 1 in rhodopsin transport. Invest. Ophthalmol. Vis. Sci. 42:19551962.
Hong, D.H., G. Yue, M. Adamian, and T. Li. 2001. A retinitis pigmentosa GTPase regulator (RPGR)-interacting protein is stably associated with the photoreceptor ciliary axoneme and anchors RPGR to the connecting cilium. J. Biol. Chem. 276:1209112099.
Junco, A., B. Bhullar, H.A. Tarnasky, and F.A. van der Hoorn. 2001. Kinesin light-chain KLC3 expression in testis is restricted to spermatids. Biol. Reprod. 64:13201330.
Lechtreck, K.F., and M. Melkonian. 1998. SF-assemblin, striated fibers, and segmented coiled coil proteins. Cell Motil. Cytoskeleton. 41:289296.[CrossRef][Medline]
Lupas, A. 1996. Coiled coils: new structures and new functions. Trends Biochem. Sci. 21:375382.[CrossRef][Medline]
Matsusaka, T. 1967. ATPase activity in the ciliary rootlet of human retinal rods. J. Cell Biol. 33:203208.
Maupin, P., and T.D. Pollard. 1983. Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation. J. Cell Biol. 96:5162.[Abstract]
Mayor, T., S. York-Dieter, K. Tanaka, A.M. Fry, and E.A. Nigg. 2000. The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion. J. Cell Biol. 151:837846.
Nussbaum, M. 1887. Ueber die theilbarkeit der lebendegen materie. ii. Beitrage zur naturgeschichte des Genus Hydra. Arch mikr Anat. 29:265366.
Salisbury, J.L., A. Baron, B. Surek, and M. Melkonian. 1984. Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle. J. Cell Biol. 99:962970.[Abstract]
Sjostrand, F.S. 1953. The ultrastructure of the inner segments of the retinal rods of the guinea pig eye as revealed by electron microscopy. J. Cell. Comp. Physiol. 42:4570.
Tokuyasu, K., and E. Yamada. 1959. The fine structure of the retina studied with the electron microscope. J. Biophys. Biochem. Cytol. 6:225230.
Wolfrum, U. 1995. Centrin in the photoreceptor cells of mammalian retinae. Cell Motil. Cytoskeleton. 32:5564.[Medline]
Worley, L.G., E. Fischbein, and J.E. Shapiro. 1953. The structure of ciliated epithelial cells as revealed by the electron microscope and in phase contrast. J. Morphol. 92:545577.