Kinesin II and regulated intraflagellar transport of Chlamydomonas aurora protein kinase

Junmin Pan and William J. Snell*

University of Texas Southwestern Medical School, 5323 Harry Hines Blvd, Dallas, TX 75235-9039, USA

* Author for correspondence (e-mail: william.snell{at}utsouthwestern.edu)

Accepted 21 February 2003


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 Materials and Methods
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The assembly and functioning of cilia and flagella depend on a complex system of traffic between the organelles and the cell body. Two types of transport into these organelles have been identified. The best characterized is constitutive: in a process termed intraflagellar transport (IFT), flagellar structural components are continuously carried into cilia and flagella on transport complexes termed IFT particles via the microtubule motor protein kinesin II. Previous studies have shown that the flagella of the unicellular green alga Chlamydomonas exhibit a second type of protein import that is regulated. During fertilization, the Chlamydomonas aurora protein kinase CALK undergoes regulated translocation from the cell body into the flagella. The motor that powers this second, regulated type of movement is unknown. Here, we have examined the cellular properties of the CALK in Chlamydomonas and used a kinesin II mutant to test the idea that the motor protein is essential for regulated translocation of proteins into flagella. We found that the CALK that is transported into flagella of wild-type gametes becomes part of a membrane-associated complex, that kinesin II is essential for the normal localization of this Chlamydomonas aurora protein kinase in unactivated gametes and that the cAMP-induced translocation of the protein kinase into flagella is disrupted in the fla10 mutants. Our results indicate that, in addition to its role in the constitutive transport of IFT particles and their cargo, kinesin II is essential for regulated translocation of proteins into flagella.

Key words: Chlamydomonas, Kinesin II, Regulated translocation, Aurora protein kinase, Flagella


    Introduction
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 Materials and Methods
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Flagella and cilia are complex, microtubule-filled cellular extensions that serve as both organelles of motility and sensory transducers. Because cilia and flagella do not contain protein synthetic machinery, all ciliary/flagellar proteins are synthesized in the cell body and undergo sorting (Bloodgood, 2000Go; Ersfeld and Gull, 2001Go; Hill et al., 1999Go; Snapp and Landfear, 1999Go) and transport processes that ensure their proper delivery to the organelles. Recently, a novel intraflagellar transport (IFT) system, first discovered in the green alga Chlamydomonas (Kozminski et al., 1993Go), has been shown to be responsible for constitutive transport of proteins into and out of cilia and flagella (Rosenbaum and Witman, 2002Go). The central elements of this constitutive transport system are: an anterograde motor protein, kinesin II; a retrograde motor protein, cytoplasmic dynein 1b; and transport entities called IFT particles, which are composed of 17 proteins (reviewed in Cole, 1999Go; Goldstein, 2001Go; Rosenbaum and Witman, 2002Go; Signor et al., 2000Go). The current model for flagellar assembly and maintenance proposes that kinesin II works continuously to ferry IFT particles and their cargo flagellar components to the tip of the flagellum. Once at the tip, the particles release their cargo and the cytoplasmic dynein 1b carries the particles and the kinesin II back to the cell body to complete the cycle. Genetic disruption of IFT components leads to the failure of ciliary/flagellar assembly in all organisms studied (Brazelton et al., 2001Go; Cole et al., 1998Go; Haycraft et al., 2001Go; Kozminski et al., 1995Go; Pazour et al., 1999Go; Pazour et al., 2000Go; Piperno and Mead, 1997Go; Qin et al., 2001Go). For example, mice with a lesion in IFT particle protein IFT88 fail to assemble cilia in their kidney tubules, leading to polycystic kidney disease, and Chlamydomonas strains with non-functional kinesin II are aflagellate (Matsuura et al., 2002Go; Pazour et al., 2000Go; Walther et al., 1994Go).

In addition to their role in cell motility, the flagella of Chlamydomonas are also central signaling organelles during fertilization. When vegetatively growing mt+ and mt cells are transferred into nitrogen-free medium they undergo gametogenesis to become mt+ and mt gametes. The gametes are in a fusion-incompetent (unactivated) state until they are mixed with gametes of the opposite mating type. Unactivated gametes are surrounded by a cell wall, their mating structures (cell fusion organelles) are not activated, and they have not yet undergone other cellular changes that render the cells capable of cell fusion (Pan and Snell, 2000bGo). When unactivated mt+ and mt gametes are mixed together, interactions between their mt+ and mt flagellar adhesion molecules, called agglutinins, cause the cells to adhere to each other via their flagella. In addition to bringing the cells into close contact, interactions between the flagellar agglutinins also initiate a signal transduction pathway leading to activation of a gamete-specific flagellar adenylyl cyclase. The resulting increases in cAMP bring about multiple changes in the cells as part of a process called gamete activation, which prepares the gametes for cell fusion. Incubation of gametes of either mating type alone with the cAMP analog dibutyryl cAMP (db-cAMP) also induces gamete activation (Pan and Snell, 2000bGo; Pasquale and Goodenough, 1987Go; Pijst et al., 1984Go).

Recently, we made the surprising discovery that a cell body cytoplasmic protein, the Chlamydomonas aurora kinase CALK, is rapidly translocated into the flagella during gamete activation (Pan and Snell, 2000aGo; Pan and Snell, 2000bGo). In vegetative cells, Chlamydomonas CALK, which is expressed as 78 kDa and 80 kDa isoforms, is present exclusively in the cell bodies. Even after gametogenesis, during which time mt+ and mt vegetative cells differentiate into mt+ and mt gametes, neither the 78 kDa nor the 80 kDa form of CALK were present in the flagella of the (unactivated) gametes. Only after gamete activation, induced either by mixing mt+ and mt gametes together or by adding dibutyryl cAMP to gametes of a single mating type, did we find that the 78 kDa isoform translocated into flagella. The 78 kDa isoform of CALK was detectable in flagella within 5 minutes; after 30 minutes of gamete activation, substantial amounts were present. The 80 kDa form continued to be detectable only in the cell body. To our knowledge, this is one of the first examples of a cytoplasmic protein whose translocation into a cilium or flagellum has been shown to be regulated.

Because kinesin II is known to be essential for constitutive transport of flagellar components required for flagellar structure and motility, we used fla10 mutant cells, which have a lesion in the motor domain of kinesin II, to test the idea that this plus-end-directed microtubule motor protein plays a role in the regulated movement of proteins during signal transduction. By use of cell fractionation and immunoblotting, we show that, like wild-type vegetative cells, fla10 vegetative cells contain CALK only in their cell bodies. On the other hand, and unlike wild-type cells, the 80 kDa isoform of CALK is present in the flagella of unactivated gametes in an unregulated fashion and is associated with the axonemal fraction of the organelles. Moreover, cAMP-induced transport of the 78 kDa form of CALK into the flagella is disrupted in fla10 gametes, indicating that kinesin II is essential for its regulated transport.


    Materials and Methods
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 Materials and Methods
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Cells, cell culture and gamete activation
Chlamydomonas reinhardtii strains 21gr (mt+) (CC-1690), 6145C (mt) (CC-1691) and fla10-1 (CC-1919) (mt) from the Chlamydomonas Genetics Center (Duke University) were cultured vegetatively on a 13:11 hour light/dark cycle. Gametogenesis was induced by transferring vegetative cells into nitrogen-free medium. Cell growth, induction of gametogenesis, cell wall loss and determination of zygote formation were carried out as previously described (Buchanan and Snell, 1988Go; Pan and Snell, 2000aGo; Pan and Snell, 2002Go; Snell, 1976Go). Gametes remain unactivated until they are mixed with gametes of the opposite mating type or incubated with dibutyryl cAMP. For gamete activation, mt+ and mt gametes in nitrogen-free medium were mixed together for 30 minutes or gametes of a single mating type were incubated in 15 mM db-cAMP and 0.15 mM papaverine for 30 minutes.

Isolation of flagella
Flagella were isolated essentially as described in Zhang et al. (Zhang et al., 1991Go). Briefly, 3-4 l of cell culture was concentrated to 30 ml by centrifugation at 3500 g for 5 minutes at 4°C, ice-cold 25% sucrose in 10 mM Tris, pH 7.2, was added to yield a final concentration of 7% sucrose, the cells were subjected to pH shock and the flagella samples were purified by underlaying/centrifugation twice with 25% sucrose in 10 mM Tris, pH 7.2. Flagella were harvested by centrifugation at 9000 g for 8 minutes. The sedimented flagella were resuspended in HMDEK buffer (20 mM HEPES, pH 7.2, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 25 mM KCl) containing a 1/100 dilution of the Sigma protease inhibitor cocktail for plant cells (Sigma catalogue number P9599) and flash frozen in liquid nitrogen. To obtain cell bodies, the cell bodies that were in the lower phase after the first underlaying/centrifugation were harvested by centrifugation at 3500 g for 5 minutes. The cell body pellet was resuspended in HMDEK buffer containing protease inhibitors and flash frozen in liquid nitrogen.

For cell fractionation, whole cells or cell bodies in HMDEK buffer were sonicated on ice (three times for 10 seconds each separated by 1 minute rests, output control 90%, Vibra Cell Sonicator; Sonics & Material, Danbury, CT, USA). The disrupted cell sample was centrifuged at 600 g for 3 minutes to remove unbroken cells, and the supernatant was then centrifuged at 170,000 g for 10 minutes at 4°C (TLA 100.3 rotor; TLX Ultracentrifuge, Beckman) to obtain the freely soluble cell fraction. The sedimented material was resuspended in HMDEK buffer plus 1% NP-40, kept on ice for 15 minutes and centrifuged as above to yield the NP-40-soluble and NP-40-insoluble cell fractions. Freely soluble and NP-40-soluble and -insoluble fractions were obtained from flagella by use of the same procedures.

Sucrose gradient analysis
Samples were loaded onto 2 ml 5-20% linear sucrose gradients prepared according to the method described in Beckman products application bulletin (DS-640A) for the tabletop ultracentrifuge (TL-100, Beckman). Samples (0.1 ml) were loaded on top of the gradient followed by centrifugation in rotor TLS-55 at 200,000 g for 4 hours at 4°C. After centrifugation, fractions were collected from above and analysed as described in the Results. Bovine serum albumin (BSA) and aldolase run in parallel tubes were used as standards. Protein concentration was determined by use of a Bio-Rad protein assay kit with BSA (Albumin Standard from Pierce) as a standard.

SDS-PAGE and immunoblotting
SDS-PAGE on 9% polyacrylamide gels and immunoblotting were carried out as previously described (Pan and Snell, 2000aGo). Typically 15-30 µg of protein was loaded in each lane. The rabbit polyclonal anti-Fla10N antiserum (Cole, 1999Go) was provided by D. Diener and J. Rosenbaum (Yale University, New Haven, CT), and the anti-LC8 antibody (Pazour et al., 1998Go) was provided by G. Pazour and G. Witman (University of Massachusetts Medical School, Worcester, MA). NIH Image (v. 1.62) was used to estimate the amount of 78 kDa CALK on scans of immunoblots of flagella isolated from activated wild-type and fla10 gametes. The results for flagellar 78 kDa CALK in each sample were normalized to the amounts of 78 kDa cell body CALK.


    Results
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 Materials and Methods
 Results
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 References
 
Properties of cell body and translocated flagellar 78 kDa and 80 kDa Chlamydomonas CALK
We used cell fractionation and immunoblotting with an anti-CALK antibody to establish the distribution and properties of CALK in wild-type gametes before gamete activation. Fig. 1A shows the 78 kDa and 80 kDa isoforms of CALK in cell bodies of unactivated gametes and documents the near absence of CALK in the flagella of these cells (Pan and Snell,2000aGo). To examine the properties of the CALK isoforms, we used cell disruption and detergent extraction in combination with centrifugation to obtain the freely soluble, NP-40-soluble (i.e. sedimentable after mechanical disruption but rendered soluble after addition of NP-40) and NP-40-insoluble fractions from unactivated gametes. As shown in Fig. 1B, each of the isoforms behaved similarly and both were present in either the freely soluble fraction (i.e. in the 170,000 g supernatant of mechanically disrupted cells) or the detergent-insoluble fraction of unactivated gametes. Little, if any, of either isoform was in the detergent-soluble fraction. Similar results were obtained with vegetative cells (data not shown). These results indicated that ~70% of CALK was freely soluble and ~30% was associated with detergent-insoluble cellular components.



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Fig. 1. Distribution of 78 kDa and 80 kDa isoforms of CALK in wild-type, unactivated gametes. (A) Both isoforms of CALK are primarily localized in the cell bodies of unactivated wild-type gametes. Flagella (30 µg) and cell bodies (15 µg) isolated from unactivated wild-type gametes were analysed by SDS-PAGE and immunoblotting with anti-CALK antibody. (B) Absence of membrane-associated CALK in unactivated gametes. Unactivated wild-type gametes were separated into freely soluble, NP-40-soluble and NP-40-insoluble fractions and analysed by SDS-PAGE and immunoblotting with anti-CALK antibody. (C) Sucrose gradient analysis of CALK of unactivated gametes. NP-40 extracts of unactivated wild-type gametes were separated on 5-20% sucrose gradients and equal portions of each fraction were analysed by anti-CALK immunoblotting. (We used NP-40 to disrupt the gametes in this experiment for consistency with the experiments in Fig. 2.) All 18 fractions from the gradient were analysed by SDS-PAGE and immunoblotting but only the top 9, which contained all of the CALK detected on the gradient, are shown here.

 



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Fig. 2. Translocated 78 kDa flagellar CALK required detergent for solubilization and is present in a more rapidly sedimenting complex in wild-type gametes. (A) 78 kDa CALK translocates into flagella during gamete activation. Cell bodies and flagella isolated from wild-type unactivated mt gametes and wild-type mt gametes activated by incubation in dibutyryl cAMP were analysed by anti-CALK immunoblotting. (B) Unlike cell body CALK, most translocated 78 kDa flagellar CALK in activated gametes requires detergent to be soluble. The freely soluble, NP-40-soluble (i.e. sedimented at 170,000 g after mechanical disruption and subsequently rendered soluble by addition of NP-40) and NP-40-insoluble fractions of flagella and cell bodies from activated wild-type mt gametes were analysed by anti-CALK immunoblotting. (C) Translocated 78 kDa flagellar CALK is part of a higher molecular mass complex. NP-40 extracts, which contained both freely soluble proteins and proteins that required NP-40 to be soluble, were prepared from flagella and cell bodies from activated mt+ and mt gametes, and fractionated on 5-20% sucrose gradients; the fractions were analysed by anti-CALK immunoblotting.

 
We also used velocity sedimentation on sucrose density gradients to study the properties of CALK in unactivated gametes. High-speed supernatants from gametes disrupted by detergent were subjected to centrifugation on 5-20% sucrose gradients and gradient fractions were analysed by immunoblotting. As shown in Fig. 1C, the isoforms of CALK behaved similarly and both were present near the top of the gradient, with peak amounts being present in fraction 3, with an S value of ~4-5.

Fig. 2A shows that, during gamete activation, the 78 kDa isoform of CALK was translocated into the flagella (Pan and Snell, 2000aGo). In this experiment, unactivated and activated gametes were deflagellated, and cell bodies and flagella were analysed by immunoblotting. Fractionation of activated gamete cell bodies indicated that gamete activation did not alter the properties of cell body CALK. Both isoforms of CALK in cell bodies were distributed between the freely soluble fraction and the detergent-insoluble fraction, and neither isoform was present in the detergent-soluble fraction (Fig. 2B).

Quite different results were obtained, however, when we examined the properties of the translocated 78 kDa flagellar CALK. As shown in Fig. 2B, only a small portion of the 78 kDa CALK that was translocated into the flagella during gamete activation was freely soluble. In addition, little was associated with the NP-40-insoluble, or axonemal, fraction of flagella. Instead, most of this form of CALK required detergent to become soluble. These properties were consistent with the idea that the translocated 78 kDa flagellar CALK became associated with the flagellar membrane.

We also studied the properties of 78 kDa flagellar CALK by use of velocity sedimentation to determine whether the CALK that translocated into the flagella had formed new associations with cellular components. Detergent extracts of whole flagella from activated gametes were subjected to fractionation on sucrose gradients and CALK location in the gradient was determined by immunoblotting as above (Fig. 2C). Whereas cell body CALK sedimented near the top of the gradient in the form of a single peak, 78 kDa flagellar CALK sedimented as two broad peaks with significant overlap. One component peaked in fraction 3 and the other in fraction 6 (Fig. 2C). Based on the sedimentation of protein standards on identical gradients, the larger CALK complex sedimented at ~6-7 S.

Kinesin II is essential for proper localization of 80 kDa CALK in unactivated gametes
Having established the localization and properties of CALK in wild-type cells, we next studied the protein in fla10 mutant cells, which contain the fla10-1 allele of kinesin II. The mutation in the fla10-1 allele produces a kinesin II with a lysine instead of an asparagine at residue 329, which is in the motor domain of the protein (Walther et al., 1994Go). Although the cellular effects of the mutation are especially noticeable when fla10 cells are incubated at elevated temperature, being associated with a complete cessation of IFT and resorption of flagella, the mutation has significant effects on cells cultured under standard culture conditions. At room temperature, vegetative fla10 cells produce substantially less Fla10 protein than the wild type (Cole et al., 1998Go; Kozminski et al., 1995Go; Walther et al., 1994Go), contain fewer IFT particles in their flagella and exhibit reduced IFT (Cole et al., 1998Go; Iomini et al., 2001Go; Kozminski et al., 1995Go).

Because the above studies were carried out on vegetative cells, we determined whether gametes of fla10 cells also showed reduced amounts of Fla10 protein compared with wild-type cells, and we also tested for differences between wild-type and fla10 gametes in their fertilization phenotype. As shown in Fig. 3A, and consistent with previous studies on vegetative fla10 cells, the flagella from fla10 gametes cultured at 23°C contained substantially less Fla10 protein than wild-type gametes. The lower panel of Fig. 3A is a loading control showing that the samples contained equivalent amounts of LC8, a dynein light chain. We evaluated the fertilization phenotype of fla10 gametes by mixing them with mt+ gametes and assessing flagellar adhesion and zygote formation. Examination of the mixed samples by phase-contrast microscopy indicated that the extent of flagellar adhesion in the fla10 samples was similar to that of the wild-type samples, consistent with previous results (Pan and Snell, 2002Go). However, the quality of the adhesive interactions was different. The fla10 gametes formed looser, more tenuous associations with each other than wild-type gametes (not shown). Analysis of the rate of zygote formation of fla10 gametes at 23°C (Fig. 3B) indicated that the mutant cells exhibited a lower rate of gamete fusion than their wild-type counterparts. These results were consistent with the idea that fla10 gametes exhibit alterations in flagellar signal transduction at 23°C.



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Fig. 3. Fla10 protein levels and the rate of zygote formation in wild-type and fla10 gametes. (A) Fla10 protein in the flagella of wild-type and fla10 unactivated gametes was determined by SDS-PAGE and immunoblotting with anti-Fla10N antibody (top). The lower panel shows the same blot probed with an anti-LC8 antibody as a loading control. (B) Wild-type and fla10 mt gametes were mixed with mt+ gametes and, at the indicated times, the extent of zygote formation was assessed. The figure shows zygote formation as percentage of the maximum zygote formation for each sample (70% for the wild type and 42% for fla10). {triangleup}, wild-type; {blacktriangleup}, fla10.

 

Examination of total cellular CALK in fla10 cells showed that the total amounts of protein and the relative amounts of the 78 kDa and 80 kDa isoforms were similar to the wild type in fla10 vegetative cells and fla10 gametes (Fig. 4A). Likewise, just as both isoforms were absent from the flagella of wild-type vegetative cells and gametes, both were also missing from the flagella of vegetative fla10 cells (Fig. 4B). However, the flagella of unactivated fla10 gametes contained substantial amounts of CALK, but only of the 80 kDa isoform; as with wild-type gametes, the 78 kDa isoform was not present in the flagella of unactivated fla10 gametes. These results indicated that wild-type levels of kinesin II are essential for excluding the 80 kDa form of CALK from the flagella of unactivated gametes. Moreover, the results showed that gametes contain mechanisms for interactions between CALK and kinesin II that are not present vegetative cells.



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Fig. 4. The 80 kDa isoform of CALK is mislocalized in fla10 gametes and is associated with the NP-40-insoluble, axonemal fraction of flagella. Wild-type and fla10 vegetative cells, and unactivated gametes (A) were analyzed by anti-CALK immunoblotting, as were flagella isolated from wild-type and fla10 vegetative cells and unactivated gametes (B). (C) Freely soluble, NP-40-soluble and NP-40-insoluble fractions of flagella and cell bodies isolated from unactivated fla10 gametes were analysed by anti-CALK immunoblotting.

 

We also examined the properties of the 80 kDa CALK that appeared in the flagella of fla10 gametes. To our surprise, this inappropriately localized 80 kDa CALK in unactivated gametes exhibited properties quite distinct from the 78 kDa CALK that translocated into the flagella of activated wild-type gametes. Whereas most of the translocated 78 kDa CALK in activated wild-type gametes was detergent soluble (Fig. 2B), none of the 80 kDa flagellar CALK in the unactivated fla10 gametes was soluble in the absence or presence of detergent, but was associated with the axonemal (detergent-insoluble) fraction (Fig. 4C).

Kinesin II and regulated translocation of 78 kDa CALK into flagella
Having established the dependence on kinesin II for exclusion of the 80 kDa CALK from gamete flagella, we next investigated the role of kinesin II in translocation of the 78 kDa CALK during gamete activation. fla10 gametes were activated by incubation in db-cAMP, the cells were deflagellated and the cell body and flagellar fractions were analysed by SDS-PAGE and immunoblotting to assess CALK distribution. As expected, in control wild-type gametes, the 78 kDa CALK translocated into the flagella upon activation (Fig. 5). However, in the activated fla10 gametes, translocation of the 78 kDa CALK was dramatically reduced (Fig. 5). Densitometric analysis (see Materials and Methods) indicated that the amount of 78 kDa CALK translocated into flagella during activation of fla10 gametes was ~10% that of wild-type gametes. Interestingly, the levels of kinesin II present in the flagella of fla10 cells was ~10% of that found in the flagella of wild-type cells (Fig. 3A).



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Fig. 5. Regulated translocation of 78 kDa CALK into flagella is disrupted in fla10 gametes. Wild-type and fla10 mt gametes were activated by incubation in dibutyryl cAMP and flagella and cell bodies were isolated and analysed by anti-CALK immunoblotting.

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Translocated 78 kDa flagellar CALK becomes part of a membrane-associated complex
Recently, we identified the protein CALK, in Chlamydomonas and discovered that the 78 kDa isoform of the protein moved into the flagella during gamete activation (Pan and Snell, 2000aGo). To learn more about the properties of cell body and translocated flagellar CALK, we carried out cell fractionation experiments. In unactivated gametes, we found that the 78 kDa and 80 kDa isoforms of CALK were present in the freely soluble fraction and the detergent-insoluble fraction of the unactivated cells. This observation that CALK is a soluble cytoplasmic protein in the cell body of vegetative cells and unactivated gametes, and is excluded from the flagella is strong evidence for a mechanism that regulates entry of proteins into the flagellum (Rosenbaum and Witman, 2002Go).

After gamete activation, only a small proportion of the 78 kDa CALK that had translocated into flagella was associated with the freely soluble fraction or the detergent-insoluble (axonemal) fraction of flagella. Most of the translocated 78 kDa flagellar CALK was associated with the detergent-soluble membrane fraction of these organelles. Furthermore, sucrose gradient analysis showed that, in contrast to the soluble cell body CALK, a sizeable proportion of the 78 kDa flagellar CALK was part of a higher molecular weight complex (Fig. 2) that was not observed in cell body samples.

These results suggested that translocated 78 kDa flagellar CALK formed new associations during the process of moving into the flagella. The observation that 78 kDa CALK required detergent to become soluble was consistent with the idea that it was associated with the flagellar membrane. The fact that it was part of a more rapidly sedimenting entity on sucrose gradients suggested that it had formed a complex with a flagellar membrane protein or that it had undergone a conformational change. It will be interesting to determine whether formation of this altered form of CALK plays a role in the presence of 78 kDa CALK in the flagella.

Kinesin II is required for exclusion of the 80 kDa form of CALK from the flagella of gametes
Our studies to test for a relationship between Chlamydomonas CALK and kinesin II indicated that kinesin II influences the cellular location of CALK. Unlike in wild-type cells, the 80 kDa isoform was present in the flagella of unactivated and activated fla10 gametes. Moreover, the mislocalization did not occur in vegetative fla10 cells, and the 80 kDa flagellar CALK of fla10 gametes was associated with the detergent-insoluble, axonemal fraction. Although these results do not easily lend themselves to a simple explanation, they document a genetic interaction between the two proteins and a role for kinesin II in the localization of CALK in gametes. The gamete specificity of the altered localization also suggests that gametes contain CALK-interacting molecules not found in vegetative cells, which would allow gametes to exploit CALK for as-yet-unidentified functions. One possible explanation, suggested above for 78 kDa CALK, is that (even though the steady-state flagellar levels are very low) the 80 kDa CALK normally cycles through the flagella of gametes and that kinesin II is essential for the exit of 80 kDa CALK from the flagella.

Regulated translocation of 78 kDa CALK into flagella during gamete activation requires kinesin II
Our studies to test the idea that kinesin II is the motor for regulated translocation of the 78 kDa isoform of CALK showed that gamete-activation-induced movement of 78 kDa CALK into flagella was significantly disrupted in fla10 cells (Fig. 5). Compared with wild-type cells, movement of IFT particles is substantially reduced in fla10 cells maintained under standard culture conditions (room temperature) and movement ceases at elevated temperatures. Cells might have a requirement for increased kinesin II at elevated temperature or the mutation in fla10 causes kinesin II to be somewhat unstable at room temperature and even more unstable at elevated temperature (Cole et al., 1998Go; Kozminski et al., 1995Go). It was the subsequent disassembly of the flagella in fla10 cells transferred to elevated temperature that originally led to the idea that kinesin II is the motor for anterograde IFT (Kozminski et al., 1995Go; Lux and Dutcher, 1991Go). Apparently, even though kinesin II is considerably diminished in fla10 cells at room temperature compared with wild-type cells (Cole et al., 1998Go; Kozminski et al., 1995Go; Walther et al., 1994Go), the reduced amount of kinesin II activity is still above a threshold needed to maintain flagella of full length. We also showed recently that activation of the flagellar adenylyl cyclase during flagellar adhesion between mt+ and mt gametes occurs in fla10 gametes at room temperature but is disrupted at elevated temperatures (Pan and Snell, 2002Go). Different circumstances hold with regulated translocation of 78 kDa CALK. Our results show that the low amount of kinesin II in fla10 cells at room temperature is associated with a disruption of induced movement of 78 kDa CALK into the flagella. Thus, the thresholds for constitutive movement of IFT particles and regulated translocation of 78 kDa CALK are different.

Our experiments do not address the question of the role of IFT particles in CALK translocation. It is possible, for example, that 78 kDa CALK does not interact with IFT particles but binds directly to kinesin II. Alternatively, 78 kDa CALK could move into flagella as cargo on IFT particles, and the reduced levels of IFT particles present in fla10 gametes (Cole et al., 1998Go; Iomini et al., 2001Go; Kozminski et al., 1995Go) might be insufficient to move the CALK.

We should note that the requirement shown here for kinesin II in regulated translocation of 78 kDa CALK contrasts with recent studies on agglutinin, a flagellar adhesion molecule that also undergoes cAMP-induced movement from the cell body to the flagella during gamete activation. We and others have shown that substantial amounts of the agglutinins, which are responsible for flagellar adhesion between mt+ and mt gametes, are stored in an inactive form at the cell body. During gamete activation, the pre-existing, inactive agglutinins are recruited from the cell body to the flagella, where they become active (Goodenough, 1989Go; Hunnicutt et al., 1990Go; Saito et al., 1985Go). We found that, independent of temperature, gamete-activation-induced recruitment of flagellar agglutinins was intact in fla10 gametes (Pan and Snell, 2002Go).

It will be interesting to determine whether the ability of fla10 gametes to recruit active agglutinins, but not 78 kDa CALK, reflects a difference in the amount of kinesin II required for these two activities or a difference in the motor molecules used for movement. Studies from Scholey's group have shown that, in addition to the heterotrimeric CeKinesin II, Caenorhabditis elegans cilia contain a dimeric kinesin, CeOsm-3, which is essential for assembly of the distal portions of the cilia in C. elegans (Perkins et al., 1986Go; Signor et al., 1999bGo). Perhaps Chlamydomonas uses Fla10 kinesin II for flagellar assembly and maintenance, initiation of signal transduction, and CALK translocation in gametes, and uses an as-yet-unidentified Chlamydomonas CeOsm-3-related protein for agglutinin recruitment.

CALK, kinesins and signal transduction
To our knowledge, the work reported here is the first evidence for a role of kinesin II in protein translocation induced during sensory transduction (Solter and Gibor, 1977Go) in cilia or flagella. Chemosensation in C. elegans has been shown to require a functional IFT system (Cole et al., 1998Go; Haycraft et al., 2001Go; Qin et al., 2001Go; Signor et al., 1999aGo; Signor et al., 1999bGo), but the requirement is indirect: the C. elegans IFT mutants fail to undergo sensory transduction because of their inability to assemble structurally intact cilia, not because of the failure of their assembled cilia to function (Collet et al., 1998Go; Haycraft et al., 2001Go; Perkins et al., 1986Go). Some of the chemosensory IFT mutants in C. elegans do assemble cilia of sorts, but the cilia are misshapen and their centers are filled with amorphous material (Perkins et al., 1986Go; Qin et al., 2001Go).

Although gamete-activation-induced CALK translocation is the first example of kinesin-II-dependent regulated movement of proteins into cilia or flagella, this motor protein has been shown to be involved in regulated movement of membrane vesicles on cytoplasmic microtubules. In Xenopus melanophores, pigment granule dispersion is driven by kinesin II and cytoplasmic dynein is implicated in granule aggregation (Nilsson and Wallin, 1997Go; Tuma et al., 1998Go). Recent work has shown that regulation is at the level of interaction of kinesin II with microtubules. The motor protein is constitutively present on the granules but can bind to microtubules only upon a change in its phosphorylation state (Reese and Haimo, 2000Go). If Chlamydomonas used a similar mechanism for regulating CALK movement, and kinesin II and CALK were pre-associated in the cell body, it is likely that cell body CALK would have behaved as a much larger entity in the sucrose gradients. It will be interesting to learn more about the gamete-activation-induced events that mediate its kinesin-II-dependent movement.

The observations reported here on the role of kinesin II in regulation of CALK localization during gamete activation are part of an emerging concept that microtubule motors are important players in signal transduction (Gundersen and Cook, 1999Go; Verhey and Rapoport, 2001Go). Recently, we showed that kinesin II is essential for activation of signal transduction during fertilization in Chlamydomonas (Pan and Snell, 2000bGo). In other systems, kinesin and kinesin-related proteins have been shown to interact directly or indirectly with signal-transducing proteins, including protein kinases. In some cases, the interaction influences the activity of the motor protein and, in others, the motor protein is crucial for the delivery of signaling molecules to their sites of action (Bowman et al., 2000Go; Day et al., 2000Go; Donelan et al., 2002Go; Morfini et al., 2002Go; Nagata et al., 1998Go; Setou et al., 2002Go; Verhey et al., 2001Go). The relationships documented here between kinesin II and CALK represent a useful system to learn more about the mechanisms that regulate the spatial and temporal localization of proteins in cilia and flagella during signal transduction.


    Acknowledgments
 
We thank our UT-Southwestern colleague F. Grinnell for helpful discussions and for careful reading of the manuscript. This work was supported by a grant from the National Institutes of Health (GM25661) to W.J.S.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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