Evidence for Kinesin-related Proteins Associated with the Axoneme of Retinal Photoreceptors

Exp. Eye Res. (1997) 64, 895–903

Evidence for Kinesin-related Proteins Associated with the

Axoneme of Retinal Photoreceptors

VIRGIL MURESAN*, ELENA BENDALA-TUFANISCO, BRIAN A. HOLLANDER

JOSEPH. C. BESHARSE†

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 retinalphotoreceptors supports regulated transport of molecules that function distally, while restricting diffusionof membrane proteins from one plasmalemmal domain to the other. Both functions are thought to beperformed by a group of proteins stably or transiently associated with the axoneme. We have identifiedtwo 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 solubilizedin the presence of adenosine triphosphate (ATP). The first group contained glyconjugates, previouslyshown to be part of the axoneme–plasmalemma cross-linkers at the connecting cilium. The second groupcross-reacted with antibodies to two different conserved peptide sequences (called LAGSE and HIPYR) ofkinesin-related proteins, and included polypeptides ofC85–97 kDa. Immunofluorescence microscopy ofwhole-mounted axonemes with the two anti-kinesin antibodies showed labeling throughout theaxoneme, including the connecting cilium-basal body region. These results suggest that the identifiedproteins may serve as motor molecules for transport of material to the outer segment via the connectingcilium. # 1997 Academic Press Limited

Key words : kinesin-related proteins ; retinal photoreceptors ; connecting cilium axoneme; axoneme-plasmalemma cross-linkers ; membrane traffic; microtubule-based transport.

1. Introduction

Vertebrate retinal photoreceptors are highly-polarized

neuroepithelial cells with four longitudinally-

displayed, functionally-distinct, but interconnected

compartments (reviewed in Besharse and Horst,

1990). Light absorption and the phototransduction

cascade occur in the outer segment, which is con-

nected 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 over-

laying 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 MedicalSchool, 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.

While effectively restricting diffusion of membrane

proteins from one plasmalemmal domain to another

(Spencer, Detwiler and Bunt-Milam, 1988), the con-

necting 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 identi-

fication 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

0014–4835}97}06089509 $25.00}0}ey960261 # 1997 Academic Press Limited

896 V. MURESAN ET AL.

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 axoneme-

associated 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 inter-

actions at the connecting cilium. Preliminary reports

of some of these data have been presented previously

(Muresan, 1993; Thurm et al., 1995). After com-

pletion 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 anti-

bodies, raised in rabbits against two decapeptides

(LNLVDLAGSE and HIPYRESKLT, respectively) cor-

responding to conserved sequences from the kinesin

motor domain (Sawin, Mitchison and Wordeman,

1992) were provided by Dr Kenneth E. Sawin, Uni-

versity 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 experi-

ments. 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 anti-

body K26 (hybridoma supernatant), recognizing an

axoneme-associated epitope in bovine photoreceptors

and ciliated epithelial cells, was previously described

(Horst, Johnson and Besharse, 1990).

Polyclonal antibodies were detected with alkaline

phosphatase-conjugated anti-rabbit IgG (1:3000 di-

lution; for Western blotting), and affinity purified goat

anti-rabbit IgG--rhodamine (Boehringer Mannheim

Biochemicals, Indianapolis, IN, U.S.A.) (for immuno-

fluorescence). The monoclonal antibodies were

detected with anti-mouse-Ig-digoxigenin [affinity puri-

fied 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 Cor-

poration, 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 centri-

fugation 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 ax-

oneme 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, 13000 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, incu-

bations 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 SDS-

polyacrylamide 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 SemiPhore4Semi-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 manu-

facturer’s instructions. For immunoblotting, mem-

RETINAL PHOTORECEPTOR-ASSOCIATED KINESINS 897

brane strips were blocked 2 hr at 23°C in TBS, 1%

bovine serum albumin (BSA), then incubated over-

night at 4°C with the anti-kinesin antibodies. Mem-

branes 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 meth-

odology. After completion of initial experiments,

Western blot analysis with anti-LAGSE and anti-

HYPIR antibodies was repeated using a chemi-

luminescent detection system (Amersham Life

Sciences) according to the manufacturers instructions.

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,

97

66

45

1 2 3 4

P S

(A)BUF

P S

AMP–PNP

P S

ATP

P S

(B)AIF3–5

65 1 2

97

66

45

F. 1. Solubilization of axoneme-associated proteins with ATP. (A) Axonemal pellets (P) and soluble fractions (S) wereobtained from samples incubated in buffer B alone (BUF, lanes 1, 2) or buffer B plus either 1 m AMP–PNP (lanes 3, 4) or10 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 incubatedin 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.

Johnson and Besharse, 1990). Many of these form

multimeric conglomerates of unusual stability, main-

tained 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


97

66

45

1 2 3 4

P S

(A)AIF3–5

P S

ATP + NaCl

P S

(B)AIF3–5

P S

ATP + NaCl

1 2 3 4GNA

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 axonemalglycoconjugates [(B), lane 4]. Note that the proteins indicated by arrowheads [(B), lane 1] appear to dissociate into componentsof higher electrophoretic mobility (arrows in [(B), lane 4] upon solubilization with ATP. Molecular size standards (in kDa) arepositioned at left.

(von Massow, Mandelkow and Mandelkow, 1989;

Chabre, 1990; Song et al., 1991; Malekzadeh-

Hemmat, Gendry and Launay, 1993).

It has previously been shown that several photo-

receptor 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 assem-

blage 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 multi-

molecular 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 dis-

sociation of some of the SDS-resistant high-molecular-

mass complexes previously described (Muresan and

Besharse, 1994) (Fig. 2). For example, the GNA-

stained 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 photorecep-

tor 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 photo-

receptor axonemal fractions. We have probed trans-

blots of axonemal proteins with two affinity-purified

rabbit polyclonal antibodies (anti-LAGSE and anti-

HIPYR), 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; King-

Smith, 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 massC97 kDa and a single band of

C85 kDa were partially solubilized from the axoneme

by incubation with Mg#+}ATP, but not with a combi-

nation of AMP–PNP and AlF$–&

(Fig. 3). Additional


97

66

P S

AMP–PNP

P S

ATP

H1

L2

H3

L4

H5

L6

H7

L8

F. 3. The photoreceptor axonemal fraction containsnucleotide-sensitive proteins immunologically-related tokinesin heavy chain. Pelleted axonemes were extracted withAMP–PNP}AlF

$–&

(lanes 1–4) or Mg#+}ATP (lanes 5–8),separated into a pellet (P) (lanes 1, 2, 5, 6) and a solublefraction (S) (lanes 3, 4, 7, 8), and analysed by Westernblotting with anti-HIPYR (H) or anti-LAGSE (L) antibody.Note that at least three proteins in the soluble fraction reactwith both anti-kinesin antibodies (arrows, lanes 7, 8). Thisincludes a doublet atC97 kDa and a singlet atC85 kDa.Additional bands such as that indicated by the arrow atC70 kDa bind only one of the antibodies. Positions ofmolecular 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 con-

taminants 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

C97 kDa associated with the photoreceptor axo-

nemes 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,

Sawin and Sale, 1994). Samples of the latter protein,

provided by Dr Winfield Sale, comigrated on the same

gel with our doublet (data not shown).

Immunodetection of Kinesin-related Proteins in Whole-

Mounted Photoreceptor Axonemes

We have used the pan-kinesin antibodies to localize

immunoreactive species in whole-mounted axonemes,

prepared in the absence of exogenously added nucleo-

tides. 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-γ-

tubulin 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 kinesin-

related 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 immuno-

fluorescence 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 axo-

nemes (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.


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 anti-HIPYR antibody (A–H), K26 antibody (I, J), anti-γ-tubulin antibody (K, L), and anti-β-tubulin antibody (M, N). The stainingwith 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 ax-

oneme associated polypeptides. Solubilization of cross-

linker 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

C97 kDa and a singlet ofC85 kDa were found to

react with two different antibodies directed at con-

served, 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 AMP-

PNP. 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 atC97 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 pro-

teins 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. TheC97 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,

theC85 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 photo-

receptors 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

for intracellular traffic (Besharse and Horst, 1990;

Muresan, 1993). The identification of kinesin-related

proteins in association with the photoreceptor ax-

oneme provides a possible mechanism for directional

transport along the axoneme. Such transport could in

principle occur either between the doublet micro-

tubules 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 ad-

dition, 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 motor-

driven 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 axoneme-

associated 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 axo-

nemal preparation from retinal photoreceptors rep-

resent 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 prepara-

tions (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


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 con-

ventional 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 inter-

actions. 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 photo-

receptor cells, ATP destabilizes proteins with charac-

teristics 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 BethBurnside for helpful discussions and sharing results prior topublication, Dr Winfield Sale for providing samples ofaxoneme linked KRPs from Chlamydomonas, and DrsKenneth E. Sawin and Harish C. Joshi for the use of anti-kinesin and anti-tubulin antibodies. This work wassupported by NIH research grant EY03222 (JCB).

References

Andrews, S. B., Gallant, P. E., Leapman, R. D., Schnapp,B. J. and Reese, T. S. (1993). Single kinesin moleculescrossbridge microtubules in vitro. Proc. Natl. Acad. Sci.U.S.A. 90, 6503–7.

Beech, P. L., Pagh-Roehl, K., Noda, Y., Hirokawa, N.,Burnside, B. and Rosenbaum, J. L. (1996). Localizationof kinesin superfamily proteins to the connecting ciliumof fish photoreceptors. J. Cell Sci. 109, 889–97.

Bernstein, M. and Rosenbaum, J. L. (1994). Kinesin-likeproteins in the flagella of Chlamydomonas. Trends CellBiol. 4, 236–40.

Bernstein, M., Beech, P. L., Katz, S. G., and Rosenbaum, J. L.(1994). A new kinesin-like protein (Klp1) localized to asingle microtubule of the Chlamydomonas flagellum. J.Cell Biol. 125, 1313–26.

Besharse, J. C. and Horst, C. J. (1990). The photoreceptorconnecting cilium. A model for the transition zone. In:(Ed. Bloodgood, R. A.) Ciliary and Flagellar Membranes.Pp. 389–417. Plenum Press : New York, London.

Besharse, J. C. and Wetzel, M. G. (1995). Immuno-cytochemical localization of opsin in rod photoreceptorsduring periods of rapid disc assembly. J. Neurochem. 24,371–88.

Brady, S. T. (1985). A novel brain ATPase with propertiesexpected for the fast axonal transport motor. Nature317, 73–5.

Brady, S. T. (1995). A kinesin medley: biochemical andfunctional heterogeneity. Trends Cell Biol. 5, 159–64.

Brady, S. T. (1991). Molecular motors in the nervoussystem. Neuron 7, 521–33.

Chabre, M. (1990). Aluminofluoride and beryllofluoridecomplexes : new phosphate analogs in enzymology.Trends Biochem Sci. 15, 6–10.

Cole, D. G., Chinn, S. W., Wedaman, K. P., Hall, T., Vuong,T. and Scholey, J. M. (1993). Novel heterotrimerickinesin-related protein purified from sea urchin eggs.Nature. 366, 268–70.

Corless, M. and Worniallo, E. (1992). Myosin I and kinesinmotors may contribute to the axial displacement ofdisks in rod and cone outer segments [Abstract]. Invest.Ophthalmol. Vis. Sci. 33 (Suppl.), 739.

Eckmiller, M. S. (1993). A cytoskeletal system at incisures ofrod outer segments and its implications for renewal andretinal degeneration [Abstract]. Invest. Ophthalmol. Vis.Sci. 34 (Suppl.), 1359.

Fox, L. A., Sawin, K. E. and Sale, W. S. (1994). Kinesin-related proteins in the eukaryotic flagella. J. Cell Sci.107, 1545–50.

Heintzelman, M. B., Hasson, T. and Mooseker, M. S. (1994).Multiple unconventional myosin domains of the in-testinal brush border cytoskeleton. J. Cell Sci. 107,3535–43.

Horst, C. J., Forestner, D. M. and Besharse, J. C. (1987).Cytoskeletal-membrane interactions : a stable interac-tion between cell surface glycoconjugates and doubletmicrotubules of the photoreceptor connecting cilium. J.Cell Biol. 105, 2973–87.

Horst, C. J., Johnson, L. V. and Besharse, J. C. (1990).Transmembrane assemblage of the photoreceptor con-necting cilium and motile cilium transition zone containa common immunologic epitope. Cell Motil. Cytoskeleton17, 329–44.

Johnson, K. A. and Rosenbaum, J. L. (1993). Flagellarregeneration in Chlamydomonas : a model system forstudying organelle assembly. Trends Cell Biol. 3,156–161.

Johnson, K. A., Haas, M. A. and Rosenbaum, J. L. (1994).Localization of a kinesin-related protein to the centralpair apparatus of the Chlamydomonas reinhardtiiflagellum. J. Cell Sci. 107, 1551–6.

Joshi, H. C. and Cleveland, D. W. (1989). Differentialutilization of β-tubulin isotypes in differentiatingneurites. J. Cell Biol. 109, 663–73.

King-Smith, C., Bost-Usinger, L. and Burnside, B. (1995).Expression of kinesin heavy chain isoforms in retinalpigment epithelial cells. Cell Motil. Cytoksel 31, 66–81.

Kozminski, K. G., Johnson, K. A., Forscher, P. andRosenbaum, J. L. (1993). A motility in the eukaryotic


flagellum unrelated to flagellar beating. Proc. Natl. Acad.Sci. U.S.A. 90, 5519–23.

Kozminski, K. G., Beech, P. L. and Rosenbaum, J. L. (1995).The Chlamydomonas kinesin-like protein FLA10 isinvolved in motility associated with the flagellarmembrane. J. Cell Biol. 131, 1517–27.

Laemmli, U. K. (1970). Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature 227, 680–5.

Lasek, R. J. and Brady, S. T. (1985). Attachment of trans-ported vesicles to microtubules in axoplasm is facilitatedby AMP–PNP. Nature 316, 645–7.

Malekzadeh-Hemmat, K., Gendry, P. and Launay, J.-F.(1993). Rat pancreas kinesin : Identification and po-tential binding to microtubules. Cell. Mol. Biol. 39,279–85.

Morin, P. J., Johnson, R. J. and Fine, R. E. (1993). Kinesin israpidly transported in the optic nerve as a membraneassociated protein. Biochim. Biophys. Acta. 1146,275–81.

Muresan, V. (1993). Molecular dissection of the photo-receptor connecting cilium cytoskeleton: Biochemicalcharacterization of basal body and axoneme-plasmalemma cross-linkers in relation to structure andfunction. Ph.D. Dissertation. University of Kansas,Kansas City, KS, U.S.A.

Muresan, V., Joshi, H. C. and Besharse, J. C. (1993). γ-Tubulin in differentiated cell types : localization in thevicinity of basal bodies in retinal photoreceptors andciliated epithelial. J. Cell Sci. 104, 1229–37.

Muresan, V. and Besharse, J. C. (1994). Complex inter-molecular interactions maintain a stable linkage be-tween the photoreceptor connecting cilium axonemeand plasma membrane. Cell Motil. Cytoskeleton 28,213–30.

Muresan, V., Godek, C. P., Reese, T. S. and Schnapp, B. J.1996. Plus-end motors override minus-end motorsduring transport of squid axon vesicles on microtubules.J. Cell Biol. 135, 383–97.

Neighbors, B. W., Williams, R. C., Jr. and McIntosh, J. R.(1988). Localization of kinesin in cultured cells. J. CellBiol. 106, 1193–204.

Noda, Y., Sato-Yoshitake, R., Kondo, S., Nangaku, M. andHirokawa, N. (1995). KIF is a new microtubule-basedanterograde motor that transports membranousorganelles distinct from those carried by kinesin heavychain or KIF3A}B. J. Cell Biol. 129 : 157–67.

Ro$ hlich, P. (1975). The sensory cilium of retinal rods isanalogous to the transitional zone of motile cilia. CellTiss Res. 161, 421–30.

Sawin, K. E., Mitchison, T. J. and Wordeman, L. G. (1992).Evidence for kinesin-related proteins in the mitoticapparatus using peptide antibodies. J. Cell Sci. 101,303–13.

Schnapp, B. J., Reese, T. S. and Bechtold, R. (1992). Kinesinis bound with high affinity to squid axon organelles thatmove to the plus-end of microtubules. J. Cell Biol. 119,389–399.

Scholey, J. M. (1996). Kinesin-II, a membrane traffic motorin axons, axonemes, and spindels. J. Cell Biol. 133, 1–4.

Song, Y.-H., Heins, S., Mandelkow, E. and Mandelkow, E.-A.(1991). Aluminum fluoride, microtubule stability, andkinesin rigor. J. Cell Sci. 14, 147–50.

Spencer, M., Detwiler, P. B., and Bunt-Milam, A. H. (1988).Distribution of membrane proteins in mechanicallydissociated retinal rods. Invest. Ophthalmol. Vis. Sci. 29,1012–20.

Thurm, C. W., Muresan, V., Bendala-Tufanisco, E. andBesharse, J. C. (1995). Identification of kinesin-relatedproteins associated with the axoneme of the photo-receptor connecting cilium [Abstract]. Invest.Ophthalmol. Vis. Sci. 36 (Suppl), S267.

Towbin, H., Staehelin, T. and Gordon, J. (1979). Electro-phoretic transfer of proteins from polyacrylamide gels tonitrocellulose sheets : Procedure and some applications.Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4.

Troutt, L. L. and Burnside, B. (1988). Microtubule polarityand distribution in teleost photoreceptors. J. Neurosci. 8,2371–80.

Troutt, L. L., Wang, E., Pagh-Roehl, K. and Burnside, B.(1990). Microtubule nucleation and organization inteleost photoreceptors : microtubule recovery after elim-ination by cold. J. Neurocytol. 19, 213–23.

Vale, R. D., Reese, T. S. and Sheetz, M. P. (1985). Identi-fication of a novel force-generating protein, kinesin,involved in microtubule-based motility. Cell 42, 39–50.

Vashishtha M., Walther, Z. and Hall, J. L. (1996). Thekinesin-homologous protein encoded by the Chlamydo-monas FLA10 gene is associated with basal bodies andcentrioles. J. Cell Sci. 109, 541–9.

von Massow, A., Mandelkow, E.-M. and Mandelkow, E.(1989). Interaction between kinesin, microtubules, andmicrotubule-associated protein 2. Cell Motil. Cyto-skeleton 14, 562–71.

Walther, Z., Vashishtha, M. and Hall, J. L. (1994). TheChlamydomonas FLA10 gene encodes a novel kinesin-homologous protein. J. Cell Biol. 126, 175–88.

Wang, W., Suprenant, K. and Dentler, W. L. (1993).Reversible association of a 97-kDa protein complexfound at the tips of ciliary microtubules with in vitroassembled microtubules. J. Biol. Chem. 268,24796–807.

Wang, W., Himes, R. H. and Dentler, W. L. (1994). Thebinding of a ciliary microtubule plus-end bindingprotein complex to microtubules is regulated by ciliaryprotein kinase and phosphatase activities. J. Biol. Chem.269, 21460–6.

Wetzel, M. G., Bendala-Tufanisco, E. and Besharse, J. C.(1993). Tunicamycin does not inhibit transport ofphosphatidylinositol to Xenopus rod outer segments. J.Neurocytol. 22, 397–412.

Yamazaki, H., Nakata, T., Okada, Y. and Hirokawa, N.(1995). KIF3A}B: a heterodimeric kinesin superfamilyprotein that works as a microtubule plus end-directedmotor for membrane organelle transport. J. Cell Biol.130, 1387–99.

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Evidence for Kinesin-related Proteins Associated with the Axoneme of Retinal Photoreceptors