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Article |
A CDK-related kinase regulates the length and assembly of flagella in Chlamydomonas
Correspondence to Paul A. Lefebvre: pete{at}umn.edu
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Little is known about how cells regulate the size of their organelles. In this study, we find that proper flagellar length control in Chlamydomonas reinhardtii requires the activity of a new member of the cyclin-dependent kinase (CDK) family, which is encoded by the LF2 (long flagella 2) gene. This novel CDK contains all of the important residues that are essential for kinase activity but lacks the cyclin-binding motif PSTAIRE. Analysis of genetic lesions in a series of lf2 mutant alleles and site-directed mutagenesis of LF2p reveals that improper flagellar length and defective flagellar assembly correlate with the extent of disruption of conserved kinase structures or residues by mutations. LF2p appears to interact with both LF1p and LF3p in the cytoplasm, as indicated by immunofluorescence localization, sucrose density gradients, cell fractionation, and yeast two-hybrid experiments. We propose that LF2p is the catalytic subunit of a regulatory kinase complex that controls flagellar length and flagellar assembly.
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Introduction |
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Flagella are dynamic structures that undergo continuous assembly and disassembly, mainly at their distal ends (Marshall and Rosenbaum, 2001; Song and Dentler; 2001). The steady-state length of flagella is likely to be the result of equilibrium between flagellar assembly and disassembly. A wealth of experimental evidence indicates that flagellar assembly and maintenance require intraflagellar transport (IFT), a kinesin/dynein-based transport system that involves at least two protein complexes of >17 polypeptides (Kozminski et al., 1993; Cole et al., 1998). IFT particles have been observed to associate with flagellar proteins and preassembled complexes (Qin et al., 2004) and to move at defined rates up and down the flagella (Kozminski et al., 1993; Iomini et al., 2001; Dentler, 2005). Recent studies indicate that IFT is involved in the transport of signaling molecules (Qin et al., 2005; Wang et al., 2006) and in Hedgehog signaling in mouse primary cilia (Huangfu et al., 2003). The compartmentalization of IFT particles can also be modulated in response to flagellar adhesion during mating in C. reinhardtii (Wang et al., 2006).
Because IFT is essential for flagellar assembly, it is a likely target of regulation for controlling the length of flagella. One model for length control proposes that the length of flagella is governed by intrinsic properties of IFT that determine the extent of flagellar assembly by balancing rates of assembly and disassembly (Marshall and Rosenbaum, 2001). Genetic studies demonstrate that flagellar length is regulated by specific protein products (McVittie, 1972; Barsel et al., 1988; Asleson and Lefebvre, 1998). There are four genetic loci (LF1 (long flagella 1), LF2, LF3, and LF4) at which mutations result in abnormally long flagella, often two to three times the normal length (McVittie, 1972; Barsel et al., 1988; Asleson and Lefebvre, 1998). The lf1 mutant has very long flagella and regrows flagella very slowly after deflagellation. Five mutant alleles of LF2 have been identified, and they cause varying degrees of excessive flagellar length and defective flagellar regeneration. Four previously described lf3 mutant alleles cause the assembly of long flagella, but they can regenerate flagella normally. Recently, we described two new null mutations at LF3 that confer a distinct unequal length flagella phenotype; the two flagella are different in lengths on most mutant cells (Tam et al., 2003). The null mutants also regenerate flagella very slowly and have prominent swellings at the distal ends of their flagella that are filled with IFT-like particles. About a dozen lf4 mutants, which are isolated after DNA insertional mutagenesis, have very long flagella but can regrow flagella with WT kinetics after deflagellation. The gene products of three of these LF genes have been identified. LF1 and LF3 encode novel proteins of unknown function (Tam et al., 2003; Nguyen et al., 2005). LF4 encodes a MAPK (Berman et al., 2003), providing the first evidence that protein kinase pathways are involved in flagellar length control. Recent studies also implicate glycogen synthase kinase 3, an aurora kinase, and an NIMA-related kinase in the regulation of flagellar assembly and disassembly (Pan et al., 2004; Wilson and Lefebvre, 2004; Bradley and Quarmby, 2005).
Similar mechanisms for regulating the length of cilia/ flagella may exist in other organisms. For example, in Leishmania, the length of flagella can be shortened or increased by the overexpression or deletion of a MAPK (Bengs et al., 2005). In sea urchin blastula, cilia at the apical tuft are two to three times longer than cilia present on the rest of the embryo (Burns, 1973). In mammals, motile cilia within the same organ can be different in length depending on their location (Clary-Meinesz et al., 1997), and the length of primary cilia varies with the diameter of bile ducts (Huang et al., 2006). Perturbation of ciliary length has been shown to correlate with human diseases such as primary ciliary dyskinesia (Niggemann et al., 1992). Recently, it has been shown that mice with a particular form of juvenile cystic kidney disease have kidneys with abnormally long primary cilia (Smith et al., 2006).
In this study, we cloned LF2 and identified its gene product as a new member of the cyclin-dependent kinase (CDK) family. CDKs have attracted intense research interest because many of them play essential roles in cell cycle progression (Morgan, 1997). In addition, CDK5 performs multiple important functions in terminally differentiated neuronal cells (Smith and Tsai, 2002). In the present study, we identify a new function for this class of kinases in regulating the size and development of an organelle. Although a close homologue of LF2p has not been identified in other organisms, several CDK-related kinases of unknown functions are highly expressed in the testis, in which germ cells are differentiating into flagellated sperm cells (Besset et al., 1999; Wohlbold et al., 2006), raising the possibility that these kinases may be the counterparts of LF2p in regulating flagellar length in higher organisms. The current study will inspire a new direction for exploring additional roles of CDKs in nondividing cells.
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1 µm (Fig. 1 D) even hours after flagellar excision.
LF2 encodes a CDK-related kinase
The tagged lf2-6 mutant enabled us to identify genomic DNA clones that rescued the mutant phenotype upon transformation. A 7.5-kb XhoI–XhoI fragment containing a predicted CDK gene was able to complement the mutant phenotype upon transformation into lf2-6 cells (Fig. 2 A).
By combining EST sequences with RT-PCR and 5' rapid amplification of cDNA ends (RACE) analysis, we were able to obtain a complete 2,244-bp cDNA sequence for LF2 (genomic and cDNA sequences are available from GenBank/EMBL/DDBJ under accession nos. DQ994241 and DQ994242). When cloned into a C. reinhardtii gene expression cassette, the 1.1-kb coding region of this putative cDNA rescued the lf2-6 mutation by transformation.
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LF2p contains all of the aa known to be critical for the catalytic activity of CDK kinases, including the glycine-rich loop (GXGXXG) for ATP binding in subdomain I and the invariant lysine (position 41) for phosphotransfer in subdomain II (Fig. 2 B). In CDK2, phosphorylation of threonine at position 14 and tyrosine at position 15 within the glycine-rich loop inhibits its activity, whereas phosphorylation of threonine at position 160 within subdomain VIII activates the kinase (Morgan, 1997). LF2p lacks the threonine and tyrosine residues in the glycine-rich loop but retains the second threonine at position 168 (Fig. 2 B). Another feature of many CDKs is the 
helix (subdomain III) containing the PSTAIRE motif required for cyclin binding. Cyclins expressed at various phases of the cell cycle regulate the level and activity of CDKs (Morgan, 1997). In LF2p, a unique sequence, PDVVVRE, replaces the PSTAIRE motif, indicating that LF2p probably does not interact with cyclins and, therefore, is classified as a CDK-related kinase.
RNA analysis of LF2
Expression of the LF2 transcript in WT and various lf mutant cells was analyzed on RNA blots with a DNA hybridization probe from the predicted LF2 coding region (Fig. 2 C). A 2.4-kb RNA was detected in WT cells (CC-620) as well as in long flagella mutants lf1-1, lf2-2, lf3-2, and lf4. The LF2 transcript was not observed in the insertional mutant lf2-6, strongly suggesting that lf2-6 is a null mutant. Interestingly, the LF2 transcript level was substantially reduced in lf3-5, a null mutant of LF3, and in two double lf mutants, lf1-1 lf3-2 and lf2-1 lf3-2, all with unequal length flagella. This observation shows that accumulation of the LF2 RNA could be affected by null mutations in LF3 or by double mutations of LF1, LF2, or LF3.
During flagellar regeneration, the transcript levels for flagellar proteins increase rapidly and transiently (Lefebvre et al., 1980). We examined the transcript level of LF2 at various times after flagellar amputation in WT cells. In contrast to flagellar gene transcripts such as PF20, whose level increased during flagellar regeneration, the amount of LF2 RNA appeared to decrease early during regeneration (Fig. 2 D), suggesting that LF2p is unlikely to be a flagellar protein.
Expression of epitope-tagged LF2p
Attempts to generate antibodies to bacterially expressed LF2 protein or to synthetic peptides were unsuccessful. Therefore, we tagged the cDNA construct with the HA epitope so that LF2p could be detected with a commercially available HA antibody. The HA-tagged construct rescued the lf2 mutants as efficiently as the untagged version. When total proteins from several rescued strains were analyzed on Western blots using an HA antibody, a cluster of at least three protein bands migrating at 
42 kD was detected only in cells rescued with the HA-tagged construct (Fig. 3 A, top and bottom).
The amount of the higher mol wt isoforms decreased, whereas the level of the smallest mol wt isoform increased when cell extracts containing the HA-tagged proteins were incubated at 37°C (Fig. 3 B, no PPI). However, this size change was inhibited if phosphatase inhibitors were included during the preparation of cell extracts (Fig. 3 B, +PPI). These results may be explained by the presence of phosphatase activity in cell extracts and because the higher mol wt forms of LF2p are phosphorylated forms of LF2p.
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Because LF2 plays a regulatory role in flagellar assembly, we examined the distribution of LF2p in the cell body and in the flagella. The majority of LF2p was detected in the cell bodies, although a very small amount was consistently observed in flagella (Fig. 3 D). The presence of LF2p in these flagellar samples could not be explained by the presence of contaminating cell bodies in these samples because very little staining with antibodies to a chloroplast protein, OEE1 (Mayfield et al., 1987), was observed with these samples (Fig. 3 D). The amount of LF2p in flagella isolated from 3.6 x 107 cells was about the same as the amount contained in 105 cell bodies, indicating that 0.3% of LF2p was distributed in flagella (Fig. 3 D). Some of the flagellar form of LF2p was detergent insoluble and remained with the axonemal fraction after 1% NP-40 extraction. It is noteworthy that there was no difference in the relative amount of the different LF2p isoforms in flagella versus cell bodies.
LF2p localization similar to LF3p
Previous studies indicated that the protein products of LF1 and LF3 are predominantly localized inside the cell, probably in protein complexes (Tam et al., 2003; Nguyen et al., 2005). When cells expressing HA-LF2p or HA-LF3p were analyzed in parallel by immunofluorescence, similar punctate staining of both proteins was observed inside the cell bodies (Fig. 4 A).
No flagellar staining was detectable in either case.
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Interaction of LF2p with other LF proteins in the yeast two-hybrid system
We used a GAL4 yeast two-hybrid system to test for direct interactions among LF proteins. A 1.1-kb cDNA of LF1 and full-length coding regions of LF2, LF3, and LF4 were cloned into yeast vectors, and their interaction was determined by the level of ß-galactosidase activity (Table I).
LF2p interacted specifically with LF3p and LF1p. Interaction between LF1p and LF3p was also detected. In contrast, LF4p did not interact with any of the three LF proteins.
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In lf2-3, two nucleotide changes were found: one in the 5' splice site (GT to AT) and one in the 3' splice site (AG to AC) of intron 1. As a consequence, intron 1 was retained in the mature RNA, and 39 aa were inserted in subdomain I right after the glycine-rich loop. The insertion in lf2-3 is expected to disrupt the structure of the protein and to affect its activity. The lf2-3 mutant strain has a severe phenotype: >50% of cells have abnormally long flagella (Fig. 6 A), and many cells did not regenerate flagella until 2–3 h after deflagellation (Fig. 6 B). In lf2-5, a nonsense mutation changing the codon TGG to TGA was found. The mutant protein is predicted to contain 233 aa lacking the last kinase subdomain XI and the C-terminal tail. The function of subdomain XI and the C-terminal tail is not known, but the lf2-5 truncated protein appears to retain partial function, as lf2-5 has a moderate flagellar length defect and can regenerate flagella with nearly normal kinetics (Barsel et al., 1988).
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These results clearly demonstrate the importance of the kinase activity of LF2p for maintaining proper flagellar length. In contrast, the G21V and K41R constructs were able to support flagellar assembly in the null mutant background. One possible explanation for this result is that LF2p supports flagellar growth by a function independent of its kinase activity, such as facilitating the assembly of a protein complex. Alternatively, the mutated constructs may have residual kinase activity that is sufficient to support flagellar assembly but not proper length control. To distinguish these two possibilities, we combined both G21V and K41R mutations in the same LF2 construct to further decrease the kinase activity of the protein without affecting the LF2 protein in other ways. When transformed into lf2-6 cells, the double mutant construct was unable to rescue the null mutant. We conclude from these results that the single mutant constructs allow flagellar assembly because they retain partial kinase activity.
The protein products from six K41R and G21V transformant lines were examined by Western blot analysis. In all samples, only a single band was observed at the position of the fastest migrating isoform of LF2p (K41R transformants are shown in Fig. 6 C). The higher mol wt isoforms, which are the presumed phosphorylated proteins (Fig. 3, B and C), were missing, suggesting that LF2p kinase activity may be required directly or indirectly to phosphorylate LF2p.
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Discussion |
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Nine CDK proteins have been annotated in the C. reinhardtii genome (Bisova et al., 2005). LF2p, which is annotated as CDKI1, appears to have evolved to perform specific functions in flagellar assembly. Although no orthologue of LF2p can be identified in other organisms, it is most similar to the mammalian CDK-related kinase PNQALRE in sequence identity and overall structural organization. PNQALRE was first proposed to be a CDK-activating kinase that phosphorylates other CDKs (Liu et al., 2004). However, a more recent study reported that the CDK-activating activity is caused by the association of PNQALRE with CDK7 (Wohlbold et al., 2006). Interestingly, PNQALRE mRNA is most abundant in testes (Wohlbold et al., 2006), the site of assembly of sperm tail axonemes, raising the possibility that PNQALRE may play a similar role to LF2p in ciliogenesis.
LF2p kinase activity is required in vivo
Some proteins with conserved kinase domains do not require kinase activity to function (Yoshikawa et al., 2001). We used site-directed mutagenesis to test the role of LF2 kinase activity in vivo. A change at the second glycine in the highly conserved ATP-binding domain or a change in the invariant lysine in subdomain II often produces inactive protein kinases. Both mutant proteins fail to maintain flagellar length control, indicating that the kinase activity of LF2p is required to enforce flagellar length. Importantly, these mutations do not completely abolish the function of LF2p because the transformed cells were able to assemble flagella. It is possible that these mutations do not totally eliminate the catalytic activity of LF2p. There is a precedent that even mutating the invariant lysine could leave residual catalytic activity in some kinases (Robinson et al., 1996), and there is at least one example of an active kinase that lacks this conserved lysine (Xu et al., 2000). When both G21V and K41R mutations were introduced in a single construct to further reduce the kinase activity of LF2p, the double mutant construct became completely nonfunctional in rescue of the mutant phenotype. Because these mutations should only affect the phosphotransfer reaction but not the structure of the protein, the kinase activity of LF2p is the only activity required to carry out its functions. In addition, it appears that a higher level of kinase activity is required for flagellar length control, whereas a lower level of kinase activity is sufficient to support flagellar growth. We are developing in vitro kinase assays to test the predictions from these experiments.
Most CDKs are phosphorylated by other kinases, and a few can phosphorylate themselves (Chao et al., 2007). For LF2p, there are at least three isoforms. The higher mol wt forms may be phosphorylated proteins, as they could be converted to the lowest mol wt form by a phosphatase-like activity in cell extracts and were focused at a lower pH on 2D gels. Interestingly, only the smallest isoform of the protein was detected in strains expressing the low activity K41R and G21V constructs, raising the possibility that LF2p may autophosphorylate or that there is a feedback loop that regulates LF2p phosphorylation.
A protein kinase complex involving LF1, LF2, and LF3 proteins
Previous genetic studies indicate that the gene products of LF1, LF2, and LF3 may work together to regulate flagellar assembly (Barsel et al., 1988; Tam et al., 2003). Double mutants of lf1 and any of the hypomorphic alleles of LF2 or LF3 produce a synthetic stumpy flagella or unequal length flagella phenotype (Barsel et al., 1988; Tam et al., 2003). Although the length defect of lf mutants can be complemented rapidly when these cells fuse with WT cells to form temporary dikaryons during mating, all pairwise crosses of lf1, lf2, and lf3 alleles fail to restore their flagellar length control in dikaryons (Barsel et al., 1988), indicating that some common structure or process is defective in these mutants. The resemblance of null or hypomorphic mutants in LF2 and LF3 also lends support to the idea that these genes work in similar pathways.
During flagellar regeneration, the level of RNA transcript for all three LF genes did not increase but rather decreased transiently during the first 30 min after deflagellation (Tam et al., 2003; Nguyen et al., 2005; this study). In addition, the RNA transcript of LF2 was diminished in the lf3-null mutant and in double mutants of lf1 lf3 and lf2 lf3, and a similar reduction in the RNA transcript of LF3 has also been observed in the null mutant of lf2 and in double lf mutants (Tam et al., 2003). These almost identical accumulation patterns of LF transcripts suggest that the expression of these three genes is coordinated.
At the protein level, the LF1, LF2, and LF3 proteins were localized by immunofluorescence to similar cytoplasmic foci, and these proteins also cosedimented on sucrose density gradients, suggesting that they may form a protein complex. Using yeast two-hybrid assays, we demonstrated the specific interaction of these proteins in vivo. Based on all of these results, we propose that LF1, LF2, and LF3 proteins work together as a protein complex in the cytoplasm, which we call the length regulatory complex (LRC) because of its role in regulating flagellar length. We hypothesize that LF2p is the catalytic subunit of the LRC, with LF1p and LF3p being the accessory proteins for activation or recruitment. Many CDKs are inactive as a monomer and require binding to cyclins for activation. All cyclins contain a conserved region of 100 aa known as the cyclin box that binds the PSTAIRE helix of CDKs (Lees and Harlow, 1993). Noncyclin binding partners also exist for CDKs. One class of noncyclin regulatory proteins is distinct from cyclins in their primary sequence but have a similar tertiary structure to cyclins. For example, p35/p25 binds and regulates the activity of CDK5, a kinase with important functions in neuronal cells (Tarricone et al., 2001). Other noncyclin regulatory proteins that bind CDKs have no structural similarity to cyclins: for example, the Ringo/Speedy proteins that interact with CDK1 and CDK2 (Nebreda, 2006), PIF-1B and PIF2 that bind to the N-terminal extension of the CDK-related kinase PFTAIRE (Rascle et al., 2003), and MAT1, a RING finger protein that can stabilize and alter the substrate specificity of a CDK7–cyclin H complex (Devault et al., 1995; Yankulov and Bentley, 1997). LF1p and LF3p are novel proteins that are distinct from cyclins, and they are likely to be new examples of noncyclin binding partners for CDKs.
Does LF4p interact with the LRC?
Previously, our laboratory identified a MAPK, LF4p, as a regulator of flagellar length (Berman et al., 2003). LF4p, unlike LF2p, does not affect flagellar assembly. Null lf4 mutants can assemble flagella both during the cell cycle and after deflagellation (Asleson and Lefebvre, 1998). In addition, cell fractionation studies show that LF4p is enriched in flagella (Berman et al., 2003), but very little of LF1, LF2, and LF3 proteins are located in the flagella (Tam et al., 2003; Nguyen et al., 2005; this study). We do not know how LF4p localizes inside the cell because antibodies to this protein do not work for immunolocalization, but the cytoplasmic form of the LF4 protein sedimented in lighter fractions in sucrose density gradients than the other LF proteins. Previously, it was shown that lf4 mutations can suppress the lf1 regeneration phenotype in lf1 lf4 double mutants, and lf4 mutations can suppress the stumpy flagella phenotype of lf1-1 lf2-3 double mutants to produce long flagella (Asleson and Lefebvre, 1998). However, we are doubtful that LF4p works directly downstream from the LRC because the lf4 mutation cannot suppress the null lf2 or lf3 mutant phenotype (unpublished data). Our yeast two-hybrid experiments also did not show any direct interaction of LF4p with LRC proteins. It is more likely that the LF4 kinase pathway and LRC pathway work in parallel to regulate flagellar length, with some cross talk between them.
Targets of the LRC
CDKs have diverse roles and many phosphorylation targets (Ubersax et al., 2003). The complex phenotypes of lf mutants suggest that the LRC may also have multiple targets. One obvious level of regulation is RNA accumulation. We observed a decreased level of transcript accumulation for the LF2 and LF3 genes in the null mutants or double mutants of LF1, LF2, and LF3 (Tam et al., 2003; this study). Many CDKs have been shown to be part of the transcriptional protein complex, to phosphorylate the C-terminal domain of the RNA polymerase large subunit, or to be part of RNA splicing complexes (for review see Loyer et al., 2005). It will be important to determine whether the LRC regulates these cellular functions.
Another possible target for the LRC is the IFT machinery. lf mutants, especially the null mutants, show an overaccumulation of IFT particles at the distal ends of their flagella, the major location for flagellar assembly and disassembly. A similar abnormal accumulation of IFT particles at flagellar tips occurs in mutants defective in specific IFT components (Pazour et al., 1998; Piperno et al., 1998; Hou et al., 2004; Pedersen et al., 2005). Moreover, some IFT mutants show unequal length flagella (Pazour et al., 1998; Hou et al., 2004), leading to the hypothesis that LF1, LF2, and LF3 proteins may regulate IFT (Tam et al., 2003). Dentler (2005) measured the rate of IFT particle movements on various lf mutant flagella and did not detect any major difference in the kinetics of retrograde or anterograde transport in lf mutants. Recently, Pan and Snell (2005) demonstrated that the cargo-carrying capacity of IFT particles could be modified. When flagella were induced to resorb by chemical treatment, a large amount of cargo-free IFT particles entered into the shortening flagella and carried disassembled flagellar components back to the cell. It is possible that the LRC regulates qualitative properties of IFT such as cargo loading, the unloading capacity of IFT particles, or the activation/inactivation switch between the anterograde/retrograde motors that occurs at the distal end of flagella.
Because the LRC is localized mainly in the cytoplasm, a satisfactory model must explain how IFT is modified in the cytoplasm to affect its function in flagella. The identity of the LRC as a protein kinase complex suggests that phosphorylation may be the mechanism. Little is known about how the IFT particles are assembled and transported inside the cytoplasm before they enter the flagella. Based on the similarity of domain structures between IFT proteins and the protein components of intracellular coated vesicles, it has been postulated that the IFT machinery may have originated as specialized membrane vesicles (Jekely and Arendt, 2006). There are many examples of CDKs or CDK-related kinases that phosphorylate intracellular membrane components (Lowe et al., 1998; Smith and Tsai, 2002; Kano et al., 2004; Palmer et al., 2005). It is not difficult to envision the use of a specialized protein kinase complex, the LRC, to regulate a specialized transport system for cilia and flagella.
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Materials and methods |
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Cloning of LF2
DNA from lf2-6 was cloned into a 
-bacteriophage vector using the Lambda Fix II/XhoI partial fill-in vector kit (Stratagene) according to the manufacturer's instructions and was screened with 32P-labeled pUC119 DNA to identify clones containing genomic sequences flanking the integrated NIT1 plasmid. A fragment from one of the phage clones was used to identify eight clones from a bacterial artificial chromosome (BAC) library of C. reinhardtii genomic DNA (available from the Clemson University Genomics Institute). Crude DNA from BAC clones was prepared by alkaline lysis. A fragment from one of the BAC clones was used as a hybridization probe to identify a 20-kb genomic clone, 2-4, from a WT C. reinhardtii -phage library. To test for the presence of the LF2 gene in these clones, BAC or -DNA was cotransformed with pArg7.8 (Debuchy et al., 1989) into arg7 strains containing lf2-3 or lf2-6 mutations as described previously (Tam and Lefebvre, 1993).
The sequence of a 17-kb region around LF2 was determined by sequencing plasmid subclones of 2-4 using universal sequencing primers and gene-specific primers (Advanced Genetics Analysis Center, University of Minnesota). The complete cDNA sequence was obtained and verified using three different approaches: RT-PCR and 5' RACE products, two ESTs available from the Chlamydomonas Genome project, and PCR products amplified from a gametic cDNA library (provided by B. Snell, University of Texas Southwestern Medical Center, Dallas, TX). PCR, 5' RACE, RNA analysis, and quantitation were performed as described previously (Tam et al., 2003). The genetic lesions in lf2 mutants were determined by direct sequencing of genomic PCR and RT-PCR products from the mutant alleles. No sequence change could be found in lf2-4 (CC-2288), and it no longer displayed any mutant phenotype, suggesting that the mutation may have reverted.
Construction of LF2 cDNA constructs
The coding region of LF2 was amplified from a gametic cDNA library with a primer with an NdeI site (5'-CCGCATATGCCGTCGACGCTTCAAGGC-3') and a primer with an EcoRI site (5'-GACTGAATTCTCACACGAGCGGCAATGACG-3') using PfuUltra polymerase (Stratagene) and was cloned into pPCR2.1 (Invitrogen). A resulting clone was digested with NdeI–EcoRI, and the 1.1-kb insert was cloned into a psaD promoter/3' untranslated region cassette (Fischer and Rochaix, 2001) to generate the cDNA clone psaD-LF2. To add the HA epitope to psaD-LF2, the HA cassette was excised from p3XHA (Tam et al., 2003) and cloned into a unique PmlI site 16 aa before the translational stop codon. Lysine 41 and glycine 21 were mutagenized to an arginine and a valine, respectively, according to protocols from the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene). All clones were sequenced to ensure that no error was introduced during the amplification or cloning processes.
Flagellar regeneration, light, and EM microscopy
Cells were induced to shed their flagella by pH shock and were grown under bright light with shaking or stirring. Cells were taken at different times, fixed with an equal volume of 2% glutaraldehyde, and examined with differential interference contrast (DIC) microscopy optics using a microscope (Diaplan; Leica) with a 100x NA 1.25 objective (Leitz). Images of cells were captured using a video camera (CCD 72; Dage-MTI, Inc.) and Image 1.59 software (Scion). Flagellar length was measured with Image J version 1.31 software (National Institutes of Health). Thin-section EM of whole cells was performed as described previously using a microscope (1200EXII; JEOL; Tam et al., 2003). All DIC and EM images were assembled using Photoshop CS2 software (Adobe).
Protein sample preparation, Western blotting, and immunofluorescence
To determine the distribution of LF2p in flagella, cells were harvested by centrifugation, resuspended in 10 mM Hepes and 5 mM MgSO4, and deflagellated by pH shock. Cell bodies were collected by low speed centrifugation. The supernatant fraction containing flagella was underlaid with 25% sucrose and centrifuged to remove the remaining cell bodies. Purified flagella were collected by centrifugation at 23,000 g. Half of the flagella was further extracted twice with 1% NP-40 in buffer to prepare axonemes. Protein concentration was determined using the Protein Assay kit (Bio-Rad Laboratories).
Whole-cell or cell body protein samples were prepared by boiling 5 x 107 cells in 0.5 ml SDS-PAGE buffer (62.5 mM Tris, pH 6.8, 5% 2-mercaptoethanol, 2% SDS, 10% glycerol, and 0.05% bromophenol blue). Flagella were boiled in SDS-PAGE buffer to a final concentration of 2 mg/ml. Soluble proteins were extracted from cells by two to three cycles of freezing/thawing and were analyzed on sucrose density gradients as described previously (Tam et al., 2003). Phosphatase inhibitors (50 mM NaF, 25 mM ß-glycerophosphate, and 1 mM sodium orthovanadate) were added to the extraction buffer except when noted otherwise.
All protein samples were size fractionated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Primary antibodies used were a rat HA antibody (3F10; 1:1,200; Roche Biochemicals), a rabbit anti-OEE1 (1:3,000), and a rabbit antibody to LF4p (Berman et al., 2003). Secondary antibodies are an anti–rat HRP at 1:8,000 and an anti–rabbit HRP at 1:24,000 dilutions (Sigma-Aldrich). Blots were analyzed sequentially with different antibodies without stripping. ECL reagents (GE Healthcare) were used for detection.
Protein samples for 2D gel electrophoresis analysis were prepared by freezing and thawing cells in a buffer containing 40 mM Tris, pH 7.5, 1 mM EDTA, 1 mM sodium orthovanadate, and protease inhibitor (P-8340; Sigma-Aldrich). 80 µg (in 5 µl) of the soluble proteins was mixed with 10 µl of 8-M urea and 4% Triton X-100 for 1 h at RT, and 120 µl of rehydration buffer (8 M urea, 4% CHAPS, 20 mM DTT, 0.2% biolytes 3–10, and 0.001% bromophenol blue) was then added. Samples were applied to 7-cm immobilized pH gradient strips, pH 3–10 or 5–8 (Bio-Rad Laboratories), and were focused using a Protean IEF cell (Bio-Rad Laboratories) and the default slow ramp method with active rehydration for 12 h. The second dimension was run on 8% polyacrylamide gels (10 x 10 cm). Proteins were transferred to PVDF membranes and analyzed with the HA antibody.
Immunofluorescence was performed on methanol-fixed cells using the HA antibody at a 1:400 dilution and an AlexaFluor488 fluorochrome–conjugated secondary antibody at a 1:500 dilution (Invitrogen) according to procedures described previously (Tam et al., 2003). Images were captured on a CCD camera (CoolCam; Cool Camera Co.) and assembled using Photoshop CS2 (Adobe).
Yeast two-hybrid assay
The interaction of LF proteins was studied according to procedures detailed in the Yeast Protocols Handbook (CLONTECH Laboratories, Inc.). The full-length coding region of LF2, LF3, and LF4 and a partial LF1 cDNA encoding aa 1–330 were cloned into plasmid vectors pGBKT7 (GAL4 DNA-binding domain) and pGADT7 (GAL4 activation domain) and tested in the yeast strain SFY526. All LF constructs were also tested alone or in combination with control plasmids containing murine p53 or SV40 large T-antigen to ensure that they did not activate the reporter gene without a specific interacting partner. Expression of the fusion proteins in transformant yeast strains was confirmed by Western analysis using anti-Myc and -HA antibodies. The interaction was quantified by liquid ß-galactosidase assay using O-nitrophenyl ß-D-galactopyranoside as the substrate and was expressed in Miller units.
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Acknowledgments |
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This work was supported by the National Institutes of Health grant GM34437 to P.A. Lefebvre.
Submitted: 5 October 2006
Accepted: 6 February 2007
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References |
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