Evidence for Kinesin-related Proteins Associated with ... - Science Direct

Exp. Eye Res. (1997) 64, 895–903

Evidence for Kinesin-related Proteins Associated with the Axoneme of Retinal Photoreceptors V I R G I L M U R E S A N*, E L E N A B E N D A L A-T U F A N I S CO, B R I A N A. H O L L A N D E R    J O S E P H. C. B E S H A R S E† Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, U.S.A. (Received Cleveland 17 September 1996 and received in revised form 4 November 1996) Situated at the junction between inner and outer segment, the connecting cilium of retinal photoreceptors supports regulated transport of molecules that function distally, while restricting diffusion of membrane proteins from one plasmalemmal domain to the other. Both functions are thought to be performed by a group of proteins stably or transiently associated with the axoneme. We have identified two types of unique polypeptides which associated with the axoneme in a nucleotide-dependent manner : they bind to the axonemes in the presence of adenosine monophosphate (AMP)-PNP, and are solubilized in the presence of adenosine triphosphate (ATP). The first group contained glyconjugates, previously shown to be part of the axoneme–plasmalemma cross-linkers at the connecting cilium. The second group cross-reacted with antibodies to two different conserved peptide sequences (called LAGSE and HIPYR) of kinesin-related proteins, and included polypeptides of C 85–97 kDa. Immunofluorescence microscopy of whole-mounted axonemes with the two anti-kinesin antibodies showed labeling throughout the axoneme, including the connecting cilium-basal body region. These results suggest that the identified proteins may serve as motor molecules for transport of material to the outer segment via the connecting cilium. # 1997 Academic Press Limited Key words : kinesin-related proteins ; retinal photoreceptors ; connecting cilium axoneme ; axonemeplasmalemma cross-linkers ; membrane traffic ; microtubule-based transport.

1. Introduction Vertebrate retinal photoreceptors are highly-polarized neuroepithelial cells with four longitudinallydisplayed, functionally-distinct, but interconnected compartments (reviewed in Besharse and Horst, 1990). Light absorption and the phototransduction cascade occur in the outer segment, which is connected to the rest of the cell (i.e., inner segment, cell body, and synaptic terminal) via a modified ciliary structure, the connecting cilium. Structurally, the connecting cilium has the main features of the transition zone of motile cilia and flagella, to which it topologically corresponds (Ro$ hlich, 1975 ; Besharse and Horst, 1990). Its main component is a cytoskeletal structure, the axoneme, consisting of a characteristic core of 90 interconnected microtubule doublets, which maintain a strong association with the overlaying plasmalemma. This association is mediated by large, multimolecular protein complexes, held together by unusually strong interactions (Horst, Forester and Besharse, 1987 ; Muresan and Besharse, 1994). It is probably the elaborate transmembrane assemblage that is cross-linked to the axoneme which confers diffusion barrier properties to the connecting cilium. * Current address : Department of Cell Biology, Harvard Medical School, Boston, MA 02115, U.S.A. † For correspondence : The University of Kansas Medical Center, Department of Anatomy and Cell Biology, 3901 Rainbow Boulevard, Kansas City, KS 66160-7400, U.S.A.

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While effectively restricting diffusion of membrane proteins from one plasmalemmal domain to another (Spencer, Detwiler and Bunt-Milam, 1988), the connecting cilium is thought to support transport from the inner segment of molecules destined to function distally. Molecular motors may play a role in such transport. The photoreceptor microtubular network has a unique organization, with all microtubules emanating from a basal body located in the distal region of the inner segment (Troutt and Burnside, 1988 ; Troutt et al., 1990 ; Muresan, Joshi and Besharse, 1993). Thus, all axonemal microtubules are oriented with their plus-ends toward the outer segment, suggesting that motors involved in distal transport along the axoneme should have properties of kinesins. At the same time, kinesins may not be involved in transport events from the cell body towards the connecting cilium, which would require minus-end directed motors, if transport should occur via microtubules. The recent identification of unique kinesin-related proteins associated with Chlamydomonas flagellar axonemes (Bernstein and Rosenbaum, 1994 ; Bernstein et al., 1994 ; Fox, Sawin and Sale, 1994 ; Johnson, Haas and Rosenbaum, 1994 ; Walther, Vashishtha and Hall, 1994), and preliminary identification of kinesin-like immunoreactivity in outer segments of retinal photoreceptors (Corless and Worniallo, 1992 ; Eckmiller, 1993), prompted us to investigate whether similar kinesin-related proteins # 1997 Academic Press Limited

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are associated with the connecting cilium axoneme of photoreceptors. We have used polyclonal antibodies to conserved polypeptides from the kinesin motor domain (Sawin, Mitchison and Wordeman, 1992) to identify cross-reacting proteins in an enriched photoreceptor axoneme preparation, and have shown that a subset of these bind to the axoneme in a nucleotide dependent manner specific for kinesins. These proteins may serve as molecular carriers of material transported along the connecting cilium axoneme to the outer segment. In addition, we have shown that other axonemeassociated proteins, including components of the axoneme–plasmalemma cross-linkers, are dissociated from the axoneme in a nucleotide-dependent fashion. This suggests that ATP may be involved in the regulation of axoneme–plasma membrane interactions at the connecting cilium. Preliminary reports of some of these data have been presented previously (Muresan, 1993 ; Thurm et al., 1995). After completion of this study, Beech et al. (1996) reported the localization of kinesin-related proteins to the inner segment and connecting cilium of fish photoreceptors.

2. Materials and Methods Buffers and Antibodies The following buffers were used throughout this study : buffer A (10 m Pipes, pH 7±0, 5 m MgCl , # 0±1 m phenylmethylsulfonyl fluoride), for RIS–ROS preparation ; buffer B (10 m Pipes, pH 7±0, 5 m MgCl , 1 m dithiothreitol, 0±1 m phenylmethyl# sulfonyl flouride, 2 % Triton X-100), for RIS-ROS extraction ; transfer buffer [192 m glycine, 25 m Tris, pH 8±3, 20 % methanol, 0±05 % sodium dodecyl sulfate (SDS)] ; Tris buffered saline (TBS : 25 m Tris, pH 7±4, 137 m NaCl, 3 m KCl, 1 m MgCl ). # Affinity-purified anti-LAGSE and anti-HIPYR antibodies, raised in rabbits against two decapeptides (LNLVDLAGSE and HIPYRESKLT, respectively) corresponding to conserved sequences from the kinesin motor domain (Sawin, Mitchison and Wordeman, 1992) were provided by Dr Kenneth E. Sawin, University of California, San Francisco. These same peptides (LNLVDLAGSE and HIPYRESKLT) were synthesized in milligram quantities by the University of Kansas Medical Center Biotechnology facility for use as a specificity control in antibody binding experiments. A rabbit polyclonal antibody to a conserved peptide of γ-tubulin (purified IgG fraction) was obtained from Dr Harish C. Joshi, Emory University School of Medicine, Atlanta. A monoclonal antibody recognizing all β-tubulin gene products (Joshi and Cleveland, 1989) was from Amersham Corp. (Arlington Heights, IL, U.S.A.). The monoclonal antibody K26 (hybridoma supernatant), recognizing an axoneme-associated epitope in bovine photoreceptors and ciliated epithelial cells, was previously described (Horst, Johnson and Besharse, 1990).

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Polyclonal antibodies were detected with alkaline phosphatase-conjugated anti-rabbit IgG (1 : 3000 dilution ; for Western blotting), and affinity purified goat anti-rabbit IgG--rhodamine (Boehringer Mannheim Biochemicals, Indianapolis, IN, U.S.A.) (for immunofluorescence). The monoclonal antibodies were detected with anti-mouse-Ig-digoxigenin [affinity purified F(ab«) fragment], followed by anti-digoxigenin# rhodamine (Boehringer Mannheim). Galanthus nivalis agglutinin (GNA), used as a digoxigenin conjugate, was detected in lectin blots with anti-digoxigenin antibody coupled to alkaline phosphatase (Boehringer Mannheim). Isolation of Photoreceptor Axonemes Dark-adapted, frozen bovine retinas (Excel Corporation, Rockville, MO, U.S.A.) were used to prepare a cytoskeletal fraction enriched in photoreceptor axonemes (Muresan and Besharse, 1994). Briefly, RIS–ROS were purified by sucrose density centrifugation from 50 retinas, thawed and suspended in buffer A. RIS-ROS were then extracted for 1 hr by mixing 1 : 1 (v}v) with buffer B, and fractionated by a second sucrose density centrifugation step. The axoneme fraction was obtained as a Triton X-100 insoluble residue at the interface of the 50 and 60 % sucrose layers. Solubilization of Axoneme-associated Proteins with Adenosine Triphosphate (ATP) Axonemal samples were pelleted by centrifugation (1 hr, 13 000 g), resuspended and incubated for 1 hr on ice in buffer B alone or buffer B supplemented with either 1 m adenosine monophosphate (AMP)–PNP or 10 m Mg#+}ATP. In some experiments, incubations were done in 1 m AMP–PNP plus 1 m AlCl and 4 m NaF (to generate 1 m AlF – ). Where $ $& noted, 0±5  NaCl was included in the incubation buffers. Solubilized and nonsolubilized material was recovered after centrifugation and analysed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic Separation and Immunoblot Axonemal samples were incubated in sample buffer either 4 min at 95°C or 40 min at 60°C, and analysed in minigels by SDS-PAGE according to Laemmli (1970). Proteins were transferred for 1 hr at 80 mA onto Immobilon4-P transfer membranes (Millipore Co., Bedford, MA, U.S.A), using the TE 70 SemiPhore4 Semi-Dry Blotter (Hoefer Scientific Instruments, San Francisco, CA, U.S.A.) or by the method of Towbin, Staehelin and Gordon (1979). Strips were cut from the dried membrane blot and processed for blotting with antibodies and lectins. GNA blots were produced with probes and reagents from the Glycan Differentiation Kit (Boehringer Mannheim) according to manufacturer’s instructions. For immunoblotting, mem-

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brane strips were blocked 2 hr at 23°C in TBS, 1 % bovine serum albumin (BSA), then incubated overnight at 4°C with the anti-kinesin antibodies. Membranes were washed three times with TBS, 1 % BSA, 0±05 % Tween 20, incubated with the secondary antibody, and processed for detection of alkaline phosphatase reaction product by conventional methodology. After completion of initial experiments, Western blot analysis with anti-LAGSE and antiHYPIR antibodies was repeated using a chemiluminescent detection system (Amersham Life Sciences) according to the manufacturers instructions.

Johnson and Besharse, 1990). Many of these form multimeric conglomerates of unusual stability, maintained together by a combination of ionic and hydrophobic interactions (Muresan and Besharse, 1994). In an attempt to investigate these interactions, we have used salts, covering a wide range of chaotropic strength of the anion (Muresan and Besharse, 1994), as well as physiological agents such as ATP which may regulate in vivo protein–protein interactions (reported here). Axonemal samples pelleted by centrifugation were resuspended and incubated at 4°C in sucrose-free extraction buffer B, supplemented with 10 m Mg#+}ATP. As shown in Fig. 1, numerous proteins were partially or totally rendered soluble by this treatment. Among these, a protein doublet of about 97 kDa appeared particularly sensitive to ATP. The non-hydrolyzable ATP analog AMP–PNP also induced solubilization of some axonemal proteins, although to a considerably lesser extent than ATP [Fig. 1(A)]. However, most of these proteins were also solubilized simply by re-suspension of the axonemal pellets in buffer B alone. Since we have shown that sucrose stabilizes axonemal preparations (Muresan and Besharse, 1994), the above result may be attributed to axoneme destabilization during incubations done in the absence of sucrose. Interestingly, the phosphate analogue AlF – largely prevented dissociation of $& proteins from the axonemes [Fig. 1(B)]. This is in line with the proposed effect of this compound of stabilizing microtubules and inducing strong binding of motor molecules, such as kinesin, to microtubules

Immunolabeling of Intact Axonemes Purified, intact axonemes were whole-mounted by diluting axonemal samples 1 : 5 in buffer B, and allowing them to dry onto glass slides. Specimens were washed three times with TBS plus 1 % BSA, and incubated overnight at 4°C with appropriate dilutions of the primary antibody. After three washes with TBS plus 0±1 % Triton X-100, secondary antibodies were applied at dilutions suggested by the manufacturers. In controls, primary antibodies were omitted. 3. Results Nucleotide Sensitivity of Axoneme-associated Polypeptides It has previously been shown that the photoreceptor axoneme consists of a large number of proteins tightly associated with the axonemal microtubule backbone (Horst, 1987 ; Forestner and Besharse, 1987 ; Horst,

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F. 1. Solubilization of axoneme-associated proteins with ATP. (A) Axonemal pellets (P) and soluble fractions (S) were obtained from samples incubated in buffer B alone (BUF, lanes 1, 2) or buffer B plus either 1 m AMP–PNP (lanes 3, 4) or 10 m Mg#+}ATP (lanes 5, 6), and analysed by SDS-PAGE. Note that ATP specifically renders soluble several proteins (arrows), including a doublet at about 97 kDa. (B) Solubilization of proteins from the axoneme is largely prevented in samples incubated in buffer B plus 1 m AMP–PNP and 1 m AlF – (lanes 1, 2). The position of molecular size markers (in kDa) is indicated at $& right.

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F. 2. Axoneme-linked glycoconjugates are solubilized by ATP. SDS-PAGE analysis (A) and lectin blot (B) of axonemal pellets (P) and solubilizates (S) from samples extracted with either 1 m AMP–PNP}AlF – (lanes 1, 2) or 10 m Mg#+}ATP plus 0±5  $& NaCl (lanes 3, 4). The transblot was probed for mannose-containing proteins with GNA. ATP}NaCl solubilizes several axonemal glycoconjugates [(B), lane 4]. Note that the proteins indicated by arrowheads [(B), lane 1] appear to dissociate into components of higher electrophoretic mobility (arrows in [(B), lane 4] upon solubilization with ATP. Molecular size standards (in kDa) are positioned at left.

(von Massow, Mandelkow and Mandelkow, 1989 ; Chabre, 1990 ; Song et al., 1991 ; MalekzadehHemmat, Gendry and Launay, 1993). It has previously been shown that several photoreceptor membrane glyconjugates remain attached to the axoneme upon Triton X-100 extraction (Horst, Forestner and Besharse, 1987). These glycoproteins have been attributed to the transmembrane assemblage that is cross-linked to the connecting cilium axoneme, and maintain a strong association with other constituent molecules of the cross-linkers. We have previously shown that some of these multimolecular complexes are not dissociated upon SDS denaturation and migrate as high-molecular-mass complexes in SDS-PAGE (Muresan and Besharse, 1994). Most of the glycoproteins detectable with the mannose-specific lectin from Galanthus nivalis (GNA) in the axonemal preparation were rendered soluble after incubation of axonemes in a buffer containing 10 m Mg#+}ATP and 0±5  NaCl (Fig. 2). None of these glycoconjugates were solubilized by AMP-PNP and AlF – (Fig. 2). Additionally, ATP and salt $& appeared to induce not only the solubilization of the axoneme-linked glycoconjugates, but also the dissociation of some of the SDS-resistant high-molecularmass complexes previously described (Muresan and Besharse, 1994) (Fig. 2). For example, the GNAstained protein bands indicated by arrowheads in lane 1 of Fig. 2(B) are not seen either in the pellet, or in the soluble fraction after extraction of axonemes with NaCl and Mg#+}ATP. At the same time, several new bands were detected in the solubilizate at positions

corresponding to lower molecular mass [arrows in Fig. 2(B), lane 4]. Presence of Kinesin-related Proteins in the Photoreceptor Axoneme Fraction The nucleotide-sensitive association of photoreceptor proteins with the connecting cilium axoneme, as described in Fig. 1, bears characteristics of the mechanochemical enzyme kinesin (Brady, 1985 ; Lasek and Brady, 1985 ; Vale, Reese and Sheetz, 1985) : binding to the axoneme in the presence of nonhydrolyzable analogues of ATP (e.g., AMP–PNP or AlF – ), and ATP-induced release from axonemes. $& Therefore, we have explored the possibility that kinesin-related proteins were present in the photoreceptor axonemal fractions. We have probed transblots of axonemal proteins with two affinity-purified rabbit polyclonal antibodies (anti-LAGSE and antiHIPYR), each recognizing a different, but conserved sequence in the motor domain of kinesin (Sawin, Mitchison and Wordeman, 1992). These antibodies have been used to identify kinesin-related proteins in various systems (Fox, Sawin and Sale, 1994 ; KingSmith, Bost-Usinger and Burnside, 1995 ; Beech et al., 1996). Several proteins in the molecular mass range of 50–115 kDa cross-reacted with both antibodies (Fig. 3). Of these, at least three (including a doublet of molecular mass C 97 kDa and a single band of C 85 kDa were partially solubilized from the axoneme by incubation with Mg#+}ATP, but not with a combination of AMP–PNP and AlF – (Fig. 3). Additional $&

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F. 3. The photoreceptor axonemal fraction contains nucleotide-sensitive proteins immunologically-related to kinesin heavy chain. Pelleted axonemes were extracted with AMP–PNP}AlF – (lanes 1–4) or Mg#+}ATP (lanes 5–8), $& separated into a pellet (P) (lanes 1, 2, 5, 6) and a soluble fraction (S) (lanes 3, 4, 7, 8), and analysed by Western blotting with anti-HIPYR (H) or anti-LAGSE (L) antibody. Note that at least three proteins in the soluble fraction react with both anti-kinesin antibodies (arrows, lanes 7, 8). This includes a doublet at C 97 kDa and a singlet at C 85 kDa. Additional bands such as that indicated by the arrow at C 70 kDa bind only one of the antibodies. Positions of molecular size standards (in kDa) are indicated at left.

experiments using chemiluminescence detection (data not shown) has revealed a similar pattern of bands in eluants of ATP treated axonemes but not in eluants with AMP–PNP plus AlF – or buffer alone. In the $& latter experiments we also found that 100 µg ml−" of LAGSE or HYPIR peptide blocked binding of their corresponding specific antibody but not that of the other antibody. We believe that these proteins are bona fide axonemal kinesin-related proteins and not contaminants of cytoplasmic photoreceptor kinesins. First, the axonemal fraction was obtained by detergent extraction of a RIS–ROS photoreceptor preparation which contains little cell body cytoplasm, being essentially equivalent to the flagellar preparation obtained from algae (see, for example, Fox, Sawin and Sale, 1994). In addition, we did not use AMP–PNP or AlF – during the actual preparation of axonemes, to $& avoid binding of any cytosolic motor protein to the axonemal microtubules. Second, the protein doublet of C 97 kDa associated with the photoreceptor axonemes migrated in SDS–PAGE at the same position as the two axoneme-specific 96}97 kDa kinesin-related proteins identified recently in eukaryotic flagella (Fox,

We have used the pan-kinesin antibodies to localize immunoreactive species in whole-mounted axonemes, prepared in the absence of exogenously added nucleotides. Labeling with anti-LAGSE and anti-HIPYR antibody was similar, although the latter showed a higher staining intensity. All fluorescent staining was localized at the axonemes, and usually labeled the entire axonemal structure, including the connecting cilium and the basal body region (Fig. 4). For a better distinction of the different domains of the photoreceptor axoneme, we have labeled, in parallel experiments, the basal body with anti-γtubulin antibody (Muresan, Joshi and Besharse, 1993) and the connecting cilium region with K26 antibody, recognizing with high specificity a protein associated with the axoneme-plasmalemma cross-linkers (Horst, Johnson and Besharse, 1990) (Fig. 4). The entire axoneme was labeled with an anti-β-tubulin antibody. Although positive staining with the anti-kinesin antibodies was detected throughout the axoneme, we have often seen intensifications in the connecting cilium–basal body region. The significance of kinesinrelated proteins at the basal body remains obscure. However, this result is in line with previous reports indicating the presence of kinesin at basal bodies of Chlamydomonas flagella (Vashishtha, Walther and Hall, 1996) and of primary cilia in various cultured cells (Neighbors, Williams and McIntosh, 1988). Often, the staining appeared discontinuous along the entire axoneme, including the connecting cilium region (Fig. 4). Since the anti-kinesin antibodies used in this study detected several proteins by Western blotting in the axonemal preparation, the immunofluorescence images show the global distribution of all these proteins along the axoneme. At present, we do not have more specific antibodies to discern among the different axoneme-associated kinesin-like proteins. 4. Discussion Our previous work on bovine photoreceptor axonemes (Horst, Forestner and Besharse, 1987 ; Horst, Johnson and Besharse, 1990 ; Muresan and Besharse, 1994) has emphasized the extraordinary stability of microtubule-membrane cross-linkers that associate with cell-surface glycoconjugates in the connecting cilium. This work has provided evidence that the cross-linkers are associated with a transmembrane complex that links cell-surface glycoconjugates of the cilium to the underlying axoneme. It is thought that this complex may be very important in maintenance of distinct domains essential for photoreceptor function.

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F. 4. Immunolocalization of kinesin-related proteins on photoreceptor axonemes. The gallery contains pairs of fluorescence (A, C, E, G, I, K, M, O) and phase contrast (B, D, F, H, J, L, N, P) images of whole-mounted axonemes probed with the antiHIPYR antibody (A–H), K26 antibody (I, J), anti-γ-tubulin antibody (K, L), and anti-β-tubulin antibody (M, N). The staining with anti-γ-tubulin and K26 antibody is localized to the basal body and connecting cilium region of the axoneme, respectively. Note that the anti-kinesin antibody shows a discontinuous distribution along the axoneme, including the region of basal body (BB) and connecting cilium (CC). No staining is seen in the absence of primary antibody (O, P). Bar ¯ 2 µm.

In addition, structural (Ro$ hlich, 1975 ; Besharse and Horst, 1990) and molecular (Horst, Johnson and Besharse, 1990) similarity between the connecting cilium and the transition zone of motile cilia suggests shared functions for these two domains. This study provides evidence that ATP destabilizes microtubule– membrane cross-linkers, permitting release of axoneme associated polypeptides. Solubilization of crosslinker components during ATP treatment may prove useful in further efforts to identify and purify molecular

constituents of the ciliary cross-linkers and suggests that their association with the axoneme may be regulated. A major finding of this study is the identification of kinesin-related proteins associated with the axoneme of retinal photoreceptors. At least three proteins, including a doublet of estimated molecular mass of C 97 kDa and a singlet of C 85 kDa were found to react with two different antibodies directed at conserved, but non-overlapping regions in the kinesin


motor domain. In addition, they remained bound to the axoneme in the presence of AMP–PNP and AlF – , $& but were partially solubilized by ATP. Preliminary results also suggest that some ATP solubilized proteins reassociate with the axoneme in the presence of AMPPNP. When ATP is removed and supernatants are incubated with whole axonemes in the presence of AMP–PNP and AlFl – , at least four protein bands $& including one at C 97 kDa are removed from the supernatant (unpublished data). These are expected features of members of the kinesin superfamily and provide compelling evidence for kinesin related proteins in photoreceptor axonemes. Recent work, emphasizing the large size and diversity of the family of kinesin-related heavy chains (Brady, 1995), has led to the identification of many members of the family, including heavy chains of a molecular size similar to those in our study. The C 97 kDa protein doublet of photoreceptor axonemes may correspond to the 97 kDa kinesin-related proteins of Chlamydomonas flagella (Fox, Sawin and Sale, 1994). As suggested for the algal proteins (Fox, Sawin and Sale, 1994), the two photoreceptor axonemal kinesins may form a dimeric complex. Furthermore, the C 85 kDa band appears to correspond to an 85 kDa polypeptide detected in fish photoreceptors with an antibody to KIF3A (Beech et al., 1996) and to the kinesin-related protein encoded by the Chlamydomonas FLA10 locus (Walther, Vashishtha and Hall, 1994 ; Kozminiski, Beech and Rosenbaum, 1995 ; Vashishtha, Walther and Hall, 1996). These proteins belong to a novel family of heterotrimeric kinesins first described in sea urchin eggs (KRP85}95) (Cole, et al., 1993) and mouse brain (KIF3A}B) (Yamazaki et al., 1995), with apparent role in membrane traffic in axons, axonemes, and spindles (Scholey, 1996). The precise location on the axoneme of the identified kinesin-related proteins is not known. However, their association with the photoreceptor axoneme suggests that they may serve as motors for material transported to the outer segment via the connecting cilium. If so, these kinesins should be distributed along the entire axoneme, consistent with our immunofluorescence results. The connecting cilium of vertebrate photoreceptors may serve as a transport route for membrane lipids, as well as cytosolic and membrane proteins that function in the outer segment (Besharse and Horst, 1990 ; Wetzel, Bendala-Tufanisco and Besharse, 1993, Besharse and Wetzel, 1995). The structural organization of the connecting cilium, with the axoneme and its associated structures occupying most of its intracellular space, appears not to favor a robust transport activity based upon simple diffusion of proteins from the inner segment to the outer segment (Muresan and Besharse, 1994). However, pathways for an active and sustained transport of material may either totally by-pass the connecting cilium (Besharse and Wetzel, 1995), or use the axoneme itself as a route

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for intracellular traffic (Besharse and Horst, 1990 ; Muresan, 1993). The identification of kinesin-related proteins in association with the photoreceptor axoneme provides a possible mechanism for directional transport along the axoneme. Such transport could in principle occur either between the doublet microtubules and the connecting cilium membrane, or through the interior cavity of the axoneme. The latter alternative is less probable, due to the presence of the basal body and associated material at the proximal end of the axoneme, which would block this particular route. However, a bidirectional intraflagellar transport of granule-like particles apparently moving between the axonemal microtubules and flagellar membrane along the length of flagella has been recently described in Chlamydomonas (Kozminski et al., 1993). In addition, electron microscopy data support such a model (Johnson and Rosenbaum, 1993 ; Kozminski et al., 1993, Kozminski, Beech and Rosenbaum, 1995). In order for a similar transport to occur in photoreceptor cells along the connecting cilium, there should be a means to remove, at least periodically, the restrictions imposed to the passage of the transported material by the presence of the massive, microtubule– membrane cross-linkers. The fact that ATP dissociates several proteins from the axoneme suggests that nucleotides may regulate the association of the axoneme with the overlaying connecting cilium plasmalemma. Although it is tempting to causally relate the ATP elution of kinesin-related proteins and other axonemal components, the nature of the effect of ATP may be entirely different for the two. ATP could thus serve not only as source of energy for the motordriven transport to the outer segment, but also as an agent capable of opening, in a highly regulated fashion, the connecting cilium gate. Its action could be either direct, or mediated through specific axonemeassociated kinases. Recently, the nucleotide-dependent binding to the axoneme of a ciliary protein from Tetrahymena was shown to be regulated via a kinase and a phosphatase, both associated in a large complex with the protein (Wang, Suprenant and Dentler, 1993 ; Wang, Hilmes and Dentler, 1994). Such a mechanism could act at the photoreceptor connecting cilium as well. Once the cargo has passed to the outer segment, the connection between the axoneme and plasmalemma could be reformed via a reversed process. The kinesin-related proteins identified in the axonemal preparation from retinal photoreceptors represent only a small fraction of the proteins solubilized from the axonemes by Mg#+}ATP. It is not uncommon for such a treatment to dissociate a large number of proteins from detergent-extracted cytoskeletal preparations (Heintzelman, Hasson and Mooseker, 1994). However, it is surprising that many of the polypeptides which cross-reacted with the pan kinesin antibodies remained insoluble upon nucleotide addition. Similar observations were made in Chlamydomonas axoneme

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preparations (Bernstein and Rosenbaum, 1994) and cytoskeletal preparations from fish photoreceptors (Beech et al., 1996). The fact that many of these proteins were recognized by both anti-LAGSE and anti-HIPYR antibodies suggests that they may indeed contain kinesin-related domains, but have lost the motor activity and serve other functions. It is known that several kinesin-related proteins, including conventional kinesin heavy chain, contain an additional microtubule binding site, situated outside the motor domain and insensitive to ATP, which allows kinesins to form cross-bridges between microtubules (Andrews et al., 1993 ; Noda et al., 1995). Kinesins with no motor activity could thus serve as stable cross-linkers between axonemal microtubules. In addition, nonmotile kinesin related proteins may be involved in stable microtubule–membrane interactions. It is known that conventional kinesin heavy chain binds to vesicular organelles very tightly, in an almost irreversible manner (Schnapp, Reese, and Bechtold, 1992 ; Morin, Johnson and Fine, 1993 ; Muresan et al., 1996). In addition, most kinesins have an extended structure, being able to span a distance of about 80 nm (Brady, 1991). If, for some reason, kinesin lost the capacity to detach from microtubules, it would become an ideal cross-linker of membranes to microtubules. Based on these considerations, one might speculate that some of the proteins in the axoneme preparation which bind the anti-kinesin antibodies, but are insensitive to nucleotides, may be part of the axoneme–plasmalemma cross-linker in the connecting cilium. In conclusion, we have shown that, in photoreceptor cells, ATP destabilizes proteins with characteristics of kinesins that are associated with the connecting cilium axoneme. Some of these may act as motors in the transport of material to the outer segment along the axoneme, while others may have a structural role at the connecting cilium.

Acknowledgements We would like to thank Drs Winfield Sale and Beth Burnside for helpful discussions and sharing results prior to publication, Dr Winfield Sale for providing samples of axoneme linked KRPs from Chlamydomonas, and Drs Kenneth E. Sawin and Harish C. Joshi for the use of antikinesin and anti-tubulin antibodies. This work was supported by NIH research grant EY03222 (JCB).

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