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Vol. 20, Issue 13, 3055-3063, July 1, 2009
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*Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455; Laboratory for 3D Fine Structure, Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309; Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322; and Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
Submitted April 8, 2009;
Accepted April 29, 2009
Monitoring Editor: Erika Holzbaur
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Smith and Yang, 2004). Several lines of evidence have indicated that the outer and inner arm dyneins are functionally distinct, differing in their subunit composition and organizational arrangement in the axoneme. In Chlamydomonas reinhardtii, a model genetic organism for cilia/flagellar studies, the inner dynein arms are composed of at least seven different dynein subspecies precisely organized in a 96-nm repeat pattern along with the outer dynein arms, radial spokes, and dynein regulatory complex (DRC) (Figure 1, Goodenough and Heuser, 1985a,b; Mastronarde et al., 1992; Porter et al., 1996; Porter and Sale, 2000; Nicastro et al., 2006; Wirschell et al., 2007; Bui et al., 2008; King and Kamiya, 2009). Genetic and phenotypic analyses have shown that the inner arm dyneins are responsible for control of the size and shape of the axonemal bend (Brokaw and Kamiya, 1987; Brokaw, 1994, 2008; Kamiya, 2002; King and Kamiya, 2009). However, we do not know how each dynein isoform is localized to its unique position in the 96-nm repeat structure or how the activity of each isoform is regulated.
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; Kamiya, 2002; Wirschell et al., 2007; King and Kamiya, 2009). I1 dynein is the only two-headed inner arm dynein and is located near the base of radial spoke 1, where it forms a trilobed structure at the proximal end of the axonemal 96-nm repeat (Figure 1). I1 is composed of two heavy chains (HCs), 1
and 1β; three intermediate chains, IC140, IC138, and IC97; and several light chains, including LC7a, LC7b, LC8, Tctex1, and Tctex2b (Goodenough and Heuser, 1985a,b; Piperno et al., 1990; Smith and Sale, 1991, 1992; Porter et al., 1992; Myster et al., 1997, 1999; Harrison et al., 1998; Perrone et al., 1998, 2000; Yang and Sale, 1998; DiBella et al., 2004a,b; Hendrickson et al., 2004; Wirschell et al., 2009).
Mutations that disrupt specific I1 subunits, or specific domains of those subunits, often result in assembly of incomplete or partial I1 dynein complexes, useful for revealing protein interactions, structural domains, and regulatory functions. For example, the bop5-1 mutant expresses a truncated IC138 and assembles all of the I1 dynein subunits with the exception of LC7b and FAP120, a recently identified I1-dynein associated protein, revealing an interaction between the C terminus of IC138, LC7b, and FAP120 (Hendrickson et al., 2004; Ikeda et al., 2009; this study). Likewise, mutant strains expressing truncated dynein HCs that lack the motor domains of either the 1 or 1β HC still assemble the remaining I1 dynein subunits (Myster et al., 1999; Perrone et al., 2000). Electron microscopy (EM) of isolated axonemes revealed the location of the globular motor domains within the trilobed structure of the I1 dynein and thereby indirectly suggested the location of the IC/LC complex at the base of I1 dynein (Myster et al., 1999; Perrone et al., 2000). One goal is to test this model for organization of the IC/LC domain in the axoneme (Figure 1).
Diverse evidence indicates that I1 dynein plays a key role in regulation of microtubule sliding by a mechanism involving phosphorylation of IC138 (reviewed in Porter and Sale, 2000; Smith and Yang, 2004; Wirschell et al., 2007). Unlike other axonemal dyneins, the isolated I1 complex does not efficiently translocate microtubules in in vitro motility assays, possibly indicating a novel regulatory function (Smith and Sale, 1991; Kagami and Kamiya, 1992; Kotani et al., 2007; Kikushima and Kamiya, 2008). Mutations in I1 assembly or IC138 phosphorylation result in altered axonemal bending and disrupted phototaxis, suggesting an important role in these processes (Brokaw and Kamiya, 1987; King and Dutcher, 1997; Hennessey et al., 2002; Okita et al., 2005). In addition, mutations in I1 subunits suppress paralysis in a central pair mutant, indicating a functional link between I1 activity and the central pair apparatus/radial spoke mechanism for control of microtubule sliding (Porter et al., 1992; Smith, 2002). In vitro functional assays using isolated axonemes also have revealed an essential role for I1 dynein in the regulation of microtubule sliding through a mechanism that seems to involve the radial spokes and reversible phosphorylation of IC138 (Habermacher and Sale, 1997; King and Dutcher, 1997; Yang and Sale, 2000; Hendrickson et al., 2004). Thus, we predict that assembly of IC138 is required for regulation of microtubule sliding by the radial spokes.
Here, we focus on IC138 and a null allele, bop5-2, obtained by insertional mutagenesis, that lacks IC138. Most I1 dynein subunits are present in bop5-2 axonemes, but IC138, IC97, FAP120, and presumably LC7b are missing. We propose that these proteins form a regulatory unit, referred to as the "IC138 subcomplex." Analysis of bop5-2 axonemes by EM and computer image averaging revealed a defect at the base of the I1 dynein, thus confirming the location of the IC138 subcomplex in the 96-nm axoneme repeat. Microtubule sliding velocities are also reduced in bop5-2 axonemes. Transformation with the wild-type IC138 gene restores assembly of the IC138 subcomplex and rescues microtubule sliding. We also analyzed microtubule sliding in a double mutant lacking the radial spoke heads (pf17) and IC138. As expected, treatment with protein kinase inhibitors increased sliding velocities in pf17 axonemes, but not in pf17 bop5-2 double mutants. Thus, the IC138 subcomplex is required for regulation of microtubule sliding by the central pair/radial spoke/phosphorylation pathway, but not for I1 assembly or targeting in the axoneme.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cells were maintained on Tris acetate phosphate (TAP) medium (Harris, 1989). Cultures were resuspended in minimal medium or 10 mM HEPES, pH 7.6, and bubbled overnight to facilitate flagellar assembly and analysis of motility.
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) and the selectable marker pSI103 containing the aphVIII gene (Sizova et al., 2001) and plating cells on TAP medium containing 10 µg/ml paromomycin. Because transformation with IC138 did not clearly rescue the motility defects, whole cell extracts of the transformants were screened on Western blots probed with the IC138 antibody. These and other experiments revealed the presence of a second, closely linked mbo-like mutation in the bop5-2 strain in addition to the IC138 deletion. Dominance tests were performed by mating bop5-2, which also contains an ac17 mutation, to an arg7-2 strain and selecting for diploid cells on minimal medium. All diploids were mating type minus and displayed wild-type motility.
Southern Blot and Polymerase Chain Reaction (PCR) Analyses
Isolation of genomic DNA, restriction enzyme digests, agarose gels, and Southern blots were performed as described previously (Perrone et al., 2000, 2003; Rupp et al., 2001, 2003). PCR primers were designed using the sequence of the IC138 gene (GenBank accession AY743342); the JGI Chlamydomonas genome database, versions 1.0, 2.0, and 3.0 (http://genome.jgi-psf.org//Chlre3/Chlre3.home.html); and the MacVector software package (MacVector, Cary, NC).
Isolation of Axonemes and Dynein Purification
Flagella were isolated by pH shock or dibucaine treatment and demembranated using 0.1–0.5% IGEPAL CA-630 (Sigma-Aldrich, St. Louis, MO) as described previously (Witman, 1986). Axonemes were resuspended in HMEEN (10 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM EGTA, 0.1 mM EDTA, and 30 mM NaCl) plus 1 mM dithiothreitol and 0.1 µg/ml protease inhibitors (leupeptin, aprotinin, and pepstatin). Dynein extraction, dialysis, and sucrose gradient centrifugation were performed as described previously (Myster et al., 1997, 1999; Perrone et al., 1998, 2000).
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western Blot Analysis
Samples were analyzed on 5–15% polyacrylamide, 0–2.4 M glycerol gradient gels. Protein was transferred to Immobilon P (Millipore, Billerica, MA), and total protein on the blot was visualized with Blot FastStain (Millipore Bioscience Research Reagents, Temecula, CA). The following antibodies were used at the indicated dilutions: 1 DHC, 1:1000 (Myster et al., 1997); IC140, 1:10,000 (Yang and Sale, 1998); IC138, 1:20,000 (Hendrickson et al., 2004); IC97, 1:10,000 (Wirschell et al., 2009); IC69, 1:10:000 (Sigma-Aldrich); FAP120, 1:10,000 (Ikeda et al., 2009); move backwards only (MBO) 2, 1:10,000 (Tam and Lefebvre, 2002); tctex1, 1:50 (Harrison et al., 1998); and tctex2b, 1:50 (DiBella et al., 2004b). Immunoreactive bands were detected using alkaline phosphatase-conjugated secondary antibodies and a chemiluminescent detection system (Tropix, Bedford, MA).
EM and Image Analysis
Axonemes were prepared for EM (Porter et al., 1992), and the methods for digitization and image averaging of thin sections were as described previously (Mastronarde et al., 1992; O'Toole et al., 1995).
Analysis of Flagellar Motility and Microtubule Sliding
The motility phenotypes of freely swimming cells were monitored using an Axioscope (Carl Zeiss, Thornwood, NY) equipped with phase contrast optics and a halogen light source (Porter et al., 1992; Myster et al., 1997, 1999). Selected fields were recorded using a Rolera-MGi EMCCD camera (Q-Imaging, Tucson, AZ) and analyzed using the MetaMorph software package, version 7.1.7.0 (Molecular Devices, Sunnyvale, CA).
Microtubule sliding velocity was measured using the method of Okagaki and Kamiya (1986) and as described previously (Howard et al., 1994; Habermacher and Sale, 1996, 1997; Hendrickson et al., 2004). Briefly, isolated flagella were resuspended in buffer without protease inhibitors, demembranated with buffer containing 0.5% Nonidet-P-40, and added to perfusion chambers. Microtubule sliding was initiated by the addition of buffer containing 1 mM ATP and 3 µg/ml subtilisin A type VIII protease (Sigma-Aldrich). Sliding was recorded using an Axiovert 35 microscope (Carl Zeiss) equipped with dark field optics and a silicon intensified camera (VE-1000; Dage-MTI, Michigan City, IN). The video images were converted to a digital format using LabVIEW 7.1 software (National Instruments, Austin, TX). Sliding velocity was determined manually by measuring microtubule displacement on tracings calibrated with a micrometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Perrone et al., 1998, 2000; Rupp et al., 2001; Rupp and Porter, 2003). Southern blots of genomic DNA isolated from >50 mutant strains were hybridized with a 9-kb fragment containing the complete IC138 gene. One strain, 6F5, was associated with a significant rearrangement of the IC138 gene (Supplemental Figure 1). PCR of wild-type and 6F5 DNA indicated that 
20 kb of genomic DNA has been deleted, including >90% of the IC138 transcription unit (Figure 2). To confirm that the mutant motility phenotype is linked to the insertion of the NIT1 plasmid and associated deletion, we backcrossed the 6F5 strain, now known as bop5-2, to a nit1 strain with wild-type motility (L8). Analysis of tetrad progeny showed that the motility defects cosegregated with the ability to grow on ammonium-free medium and the absence of IC138 (see Materials and Methods).
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, 1999; Perrone et al., 1998, 2000). To determine whether the loss of IC138 has a similar phenotype, we analyzed axonemes from wild-type and mutant strains on Western blots probed with antibodies to dynein subunits (Figure 3). Consistent with previous reports, the 1 HC, IC140, and IC138 are missing or reduced in the IC140 mutant ida7-1, and IC138 is truncated in bop5-1 (Perrone et al., 1998; Hendrickson et al., 2004). However, although bop5-2 axonemes lack IC138, both the 1 HC and IC140 are present at wild-type levels (Figure 3B). Recent studies have identified two other I1 dynein-associated proteins, IC97 (Wirschell et al. 2009) and the axoneme polypeptide FAP120 (Ikeda et al., 2009). Western blots probed with antibodies to IC97 and FAP120 revealed that these two proteins are missing in bop5-2 axonemes (Figure 3C). Because LC7b is a subunit of both I1 and outer arm dyneins (DiBella et al., 2004a), we did not analyze its assembly in bop5-2. However, previous studies of bop5-1 have shown that the I1 dynein lacks LC7b when IC138 is truncated (Hendrickson et al., 2004). Thus, it is likely that I1 dynein lacks LC7b when IC138 is missing (Figure 3A).
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20–21 S (Figure 4A); but in bop5-2 extracts, the I1 dynein is partially dissociated (Figure 4B). The 1 HC and IC140 cosediment across a broad region, with a peak at 15–16 S, whereas two LCs, Tctex1 and Tctex2b, sediment more slowly near the top of the gradient. Thus, loss of the IC138 subcomplex does not impact the assembly of I1 dynein into the axoneme, but it does affect the stability of the I1 complex in high salt extracts.
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). However, in initial efforts to rescue the bop5-2 motility defect with IC138, we failed to recover transformants with wild-type motility. These observations suggested that there must be a second mutation in bop5-2 that affects motility even though IC138 has reassembled into the axoneme (see below). We therefore analyzed whole cell extracts from 74 transformants on Western blots probed with an antibody specific for IC138 and identified three strains that expressed wild-type levels of the IC138 polypeptide in their cytoplasm. Western blots of isolated axonemes confirmed that re-expression of IC138 was accompanied by the reassembly of IC138, IC97, and FAP120 (Figure 3C). These results demonstrate that the assembly of IC97 and FAP120 is dependent on the presence of IC138 and that IC138, IC97, LC7b, and FAP120 form a subcomplex within the I1-dynein.
Localization of the IC138 Subcomplex within the Structure of the I1 Dynein
Previous work has shown that the I1 dynein forms a trilobed structure located at the proximal end of the 96-nm axoneme repeat (Goodenough and Heuser, 1985a,b; Piperno and Ramanis, 1991; Mastronarde et al., 1992; Nicastro et al., 2006; Figure 1). We also analyzed several strains in which constructs encoding the amino-terminal portions of the two I1 HCs were used to restore the assembly of I1 dyneins lacking one or the other I1 motor domain (Myster et al., 1999; Perrone et al., 2000). Analysis of isolated axonemes by thin section EM and image averaging identified the position of the two motor domains within the structure of the I1 dynein. These studies suggested that the multiple ICs and LCs are located within the third lobe of the I1 dynein, at a strategic position between the radial spokes and the outer dynein arms (Figure 1).
To directly determine the location of the I1 dynein ICs, we prepared isolated axonemes from wild-type, bop5-2, and IC138 rescued strains for thin section EM. Longitudinal sections with clear views of the 96-nm repeat were processed by computer image averaging (O'Toole et al., 1995). Comparison of all three strains indicated that some of the densities associated with the I1 dynein are reduced in bop5-2 axonemes and restored in axonemes from the IC138 rescued strain (bop5-2::IC138) (Figure 5). Difference plots demonstrated that the defect in the assembly of the IC138 subcomplex is associated with a statistically significant decrease in the density of the third lobe of I1 dynein. This third lobe corresponds to the base of the I1 dynein, which is also responsible for interaction of I1 with the doublet microtubule. Such a position is also consistent with the hypothesis that IC138 is a target for regulated phosphorylation by the radial spokes due to its proximity to radial spoke 1 (Porter and Sale, 2000; Smith and Yang, 2004; Gaillard et al., 2006; Wirschell et al., 2007). The position of the IC138 subcomplex also may facilitate interactions between the inner and outer dynein arms to coordinate their activity (reviewed in Brokaw, 1994; Kamiya, 2002; King and Kamiya, 2009).
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). mbo mutants are associated with defects in the assembly of six axonemal polypeptides (Segal et al., 1984). To determine whether there might be any mbo-related deficiencies in the bop5-2 strain, we probed Western blots of isolated axonemes with an antibody against MBO2p (Tam and Lefebvre, 2002). MBO2p is missing or reduced in axonemes from both the bop5-2 strain and the IC138 rescued strain (Figure 3C), even though MBO2p could be detected in whole cell extracts (VanderWaal and Porter, unpublished results). These observations are consistent with the presence of a second, mbo-like mutation that affects motility in the bop5-2 strain (see Discussion). Given this complexity, we needed an alternative method to analyze the function of the IC138 subcomplex.
To test the hypothesis that the IC138 subcomplex plays a regulatory role in control of microtubule sliding, we used a microtubule sliding disintegration assay to measure sliding velocities in isolated axonemes (Okagaki and Kamiya, 1986). This assay has proven to be a reliable method to assess dynein activity, or regulation of dynein activity, in axonemes that are paralyzed or otherwise impaired for motility (Witman et al., 1978; Smith and Sale, 2001; Smith, 2002). Our prediction was that IC138 plays a fundamental role in I1 dynein function; that its assembly is required for normal microtubule sliding; and that sliding velocities would be reduced in bop5-2 axonemes, similar to the reduced sliding velocities characteristic of I1 dynein mutants (Smith and Sale, 1991; Habermacher and Sale, 1997).
As described previously, in 1 mM MgATP, microtubule sliding is very rapid in isolated wild-type axonemes (Figure 6A) (Howard et al., 1994; Habermacher and Sale, 1997; Smith, 2002). As predicted, microtubules slide at greatly reduced velocities in bop5-2 axonemes. Moreover, microtubule sliding is increased when bop5-2 cells are transformed with IC138 (Figure 6A). The same result was observed in all of the bop5-2::IC138 transformants. Treatment with the kinase inhibitor protein kinase inhibitor (PKI) had no effect on sliding velocities in axonemes from wild type or bop5-2 and bop5-2 transformants (data not shown). The results indicate that assembly of the IC138 subcomplex is required for I1 dynein activity and normal microtubule sliding.
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; Smith and Yang, 2004; Wirschell et al., 2007). The regulatory pathway was revealed by functional and pharmacological analysis of microtubule sliding in paralyzed axonemes from central pair or radial spoke mutants (Habermacher and Sale, 1996, 1997; Yang and Sale, 2000; Smith and Sale, 2001; Hendrickson et al., 2004; Howard et al., 2004). For example, dynein-driven microtubule is globally inhibited in isolated, paralyzed axonemes from radial spoke mutants such as pf14 or pf17, and normal microtubule sliding velocity can be rescued by pretreating the axonemes with kinase inhibitors. Rescue of microtubule sliding requires assembly of the I1 dynein, indicating that I1 plays an essential role in this pathway (Habermacher and Sale, 1997; Yang and Sale, 2000). The mechanism of inhibition and the rescue of microtubule sliding correlate with phosphorylation and dephosphorylation of IC138 (Habermacher and Sale, 1997; Porter and Sale, 2000; Smith and Yang, 2004; Wirschell et al., 2007). Because IC138 is the primary phosphoprotein in the I1 dynein (Habermacher and Sale, 1997; King and Dutcher, 1997; Yang and Sale, 2000; Hendrickson et al., 2004), the results suggest that IC138 is the key substrate required for phosphoregulation of microtubule sliding.
To further test the regulatory role of the IC138 subcomplex, we crossed bop5-2 with the paralyzed radial spoke mutant pf17 to recover the double mutant pf17 bop5-2. We also transformed pf17 bop5-2 with the IC138 gene to recover several IC138 rescued strains (pf17 bop5-2::IC138). Expression of IC138 in the transformants was confirmed by PCR and Western blotting (our unpublished data). We then measured microtubule sliding velocities of disintegrating axonemes in the absence or presence of the kinase inhibitor PKI. We predicted that rescue of microtubule sliding in pf mutants with PKI would require the assembly of IC138. As described previously (Porter and Sale, 2000; Smith and Yang, 2004; Wirschell et al., 2007), microtubule sliding is greatly reduced in axonemes from pf17, and the addition of PKI restores microtubule sliding to wild-type levels (Figure 6B). Similarly, sliding velocities are greatly reduced in the double mutant, pf17 bop5-2 and the transformant pf17 bop5-2::IC138. However, PKI treatment only increases sliding velocities in pf17 bop5-2::IC138 (Figure 6B). Thus, assembly of the IC138 subcomplex (IC138, IC97, FAP120, and LC7b) is necessary for regulation of I1-dynein mediated microtubule sliding by the radial spoke–phosphorylation pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wilkerson et al., 1995), and mutations in IC140 are associated with defects in assembly of the I1 dynein (Perrone et al., 1998; Figure 3B). However, IC138 is part of a distinct subcomplex, which is not required for either I1 dynein assembly or targeting into the axoneme.
The IC138 Subcomplex: IC138 Is Closely Associated with LC7b, IC97, and FAP120
Previous studies of bop5-1 have shown that the I1 dynein lacks LC7b when IC138 is truncated (Hendrickson et al., 2004; Figure 3A). Because LC7b is also an outer dynein subunit (DiBella et al., 2004a), we did not directly analyze its presence in bop5-2. However, we can reasonably infer that the I1 dynein lacks LC7b when IC138 is missing.
IC97 is a novel I1 subunit that shares homology with axonemal proteins in several organisms, including the murine lung adenoma susceptibility 1 protein. Several biochemical assays indicate that IC97 interacts directly with tubulin subunits (Wirschell et al., 2009). We show here that assembly of IC97 into the axoneme depends on the presence of IC138, because it is missing in bop5-2 axonemes but restored in the IC138 rescued strain (Figure 3C).
IC138 also influences the assembly of FAP120, a novel ankyrin-related protein recently identified as an I1-associated protein (Ikeda et al., 2009). We show here that FAP120 is missing in bop5-2 and restored in the bop5-2::IC138 rescued strain, which confirms the close association between this polypeptide and IC138 (Figure 3C). However, the interaction between FAP120 and IC138 may be mediated indirectly through LC7b, because FAP120 is also missing in bop5-1, which assembles a truncated version of IC138 but lacks LC7b (Ikeda et al., 2009; Figure 3A).
Together, these observations demonstrate that IC138, LC7b, IC97, and FAP120 form a distinct subcomplex on the A-microtubule (Figure 7A). The presence of multiple subcomplexes within the I1 dynein is consistent with the phenotype of a tctex2b mutation, which blocks assembly of Tctex2b but does not prevent assembly of other I1 subunits into the axoneme (DiBella et al., 2004b).
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The IC138 Subcomplex Is Required for I1 Dynein-mediated Microtubule Sliding
The phenotypes of bop5-2 and the IC138 rescued strains indicate that there are two closely linked mutations that affect flagellar motility in bop5-2, one mutation associated with the loss of the IC138 subcomplex, and a second mutation associated with defects in assembly of MBO2p. Transformation with a wild-type copy of IC138 altered motility but did not correct the MBO2p-associated defects (Figures 3C and Supplemental Figure S2). The locus of the mbo mutation is unknown but currently under investigation.
Given this complexity, and to more directly assess the effect of the bop5-2 mutation on I1 activity, we measured microtubule sliding velocities during sliding disintegration in vitro, an assay useful for assessing dynein activity in axonemes that are otherwise paralyzed or defective in motility (Witman et al., 1978; Okagaki and Kamiya, 1986; Smith and Sale, 1991). Interestingly, loss of the IC138 subcomplex was correlated with a decrease in sliding velocity similar in magnitude to that observed with the loss of the entire I1 dynein complex (Habermacher and Sale, 1997). Reassembly of the IC138 subcomplex restored sliding velocities to wild-type levels (Figure 6A). Thus, the IC138 subcomplex is required to couple the activity of I1 motor domains to microtubule sliding.
Defects in radial spokes disrupt the signaling pathway that regulates I1-dynein activity, resulting in hyperphosphorylated forms of IC138 and decreased sliding velocities (Habermacher and Sale, 1997; King and Dutcher, 1997; Yang and Sale, 2000; Hendrickson et al., 2004). Inhibition of dynein activity can be overcome by treatment with kinase inhibitors; axoneme-associated phosphatases are thought to dephosphorylate IC138 and increase sliding velocities to wild-type levels (Porter and Sale, 2000; Gaillard et al., 2006; Wirschell et al., 2008). To demonstrate that IC138 is the critical substrate for the axonemal phosphatases, we analyzed microtubule sliding of bop5-2 in a radial spoke mutant (Figure 6B). As predicted, treatment with kinase inhibitors only increased microtubule sliding when the IC138 subcomplex was present, consistent with a model in which assembly of I1 dynein, IC138, and possibly other subcomplex proteins, is required for regulation by the radial spoke–phosphorylation pathway (Figure 7B).
Within the IC138 subcomplex, FAP120 and LC7b do not seem to be required for regulation of I1 activity by the radial spoke pathway. bop5-1 axonemes lack LC7b and FAP120, but microtubule sliding in the double mutant pf17 bop5-1 can be rescued with kinase inhibitors, indicating that LC7b and FAP120 are not necessary for regulation of microtubule sliding by the radial spokes (Hendrickson et al., 2004; Ikeda et al., 2009). However, the bop5-1 strain does display altered motility and can partially suppress the motility defects observed in pf10 (Dutcher et al., 1988), which indicates that LC7b and FAP120 must play some role in modifying I1 activity. The precise functions of LC7b and FAP120 await further study but probably include a role for I1 dynein in control of axonemal bending not revealed by microtubule sliding assays.
Although IC138 is the only phosphoprotein in the I1 dynein and the primary target for regulation by the radial spoke–phosphorylation pathway, recent studies suggest that changes in the phosphorylation state of IC138 must be communicated through other axoneme components to effectively regulate motility. Indeed, in vitro microtubule gliding assays of the isolated I1 complex have failed to detect any changes in motor activity in response to kinase or phosphatase treatment (Sakakibara, personal communication). Moreover, microtubule sliding assays with I1 dyneins lacking one or the other motor domain have indicated that the 1β DHC is the primary motor domain that responds to changes in the phosphorylation state of IC138 (Fox, Tritschler, Porter, and Sale, unpublished data; Toba et al., 2008). Our recent studies on IC97 further suggest that this subunit plays a critical role in communicating changes in the phosphorylation state of IC138 to other components within the axoneme (Wirschell et al., 2009). A better understanding of the mechanism by which the IC138 subcomplex regulates I1 activity and microtubule sliding will require additional high resolution structural analysis of wild-type and mutant axonemes (Nicastro et al., 2006; Bui et al., 2008), biophysical studies of I1 dyneins with altered subunit composition (Kotani et al., 2007), and identification and mutation of key phosphoresidues in IC138.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Mary E. Porter (porte001{at}umn.edu)
Abbreviations used: DRC, dynein regulatory complex; FAP, flagellar-associated protein; HC, heavy chain; IC, intermediate chain; LC, light chain; MBO, move backwards only; PKI, protein kinase inhibitor; TAP, Tris acetate phosphate.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brokaw, C. J. (2008). Thinking about flagellar oscillation. Cell Motil. Cytoskeleton in press.
Brokaw, C. J., and Kamiya, R. (1987). Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil. Cytoskeleton 8, 68–75.[CrossRef][Medline]
Bui, K. H., Sakakibara, H., Movassagh, T., Oiwa, K., and Ishikawa, T. (2008). Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella. J. Cell Biol 183, 923–932.
DiBella, L. M., Sakato, M., Patel-King, R. S., Pazour, G. J., and King, S. M. (2004a). The LC7 light chains of Chlamydomonas flagellar dyneins interact with components required for both motor assembly and regulation. Mol. Biol. Cell 10, 4633–4646.
DiBella, L. M., Smith, E. F., Patel-King, R. S., Wakabayashi, K., and King, S. M. (2004b). A novel Tctex2-related light chain is required for stability of inner dynein arm I1 and motor function in the Chlamydomonas flagellum. J. Biol. Chem 279, 21666–21676.
Dutcher, S. K., Gibbons, W., and Inwood, W. B. (1988). A genetic analysis of suppressors of the PF10 mutation in Chlamydomonas reinhardtii. Genetics 120, 965–976.
Gaillard, A. R., Fox, L. A., Rhea, J. M., Craige, B., and Sale, W. S. (2006). Disruption of the A-kinase anchoring domain in flagellar radial spoke protein 3 results in unregulated axonemal cAMP-dependent protein kinase activity and abnormal flagellar motility. Mol. Biol. Cell 17, 2626–2635.
Goodenough, U. W., and Heuser, J. E. (1985a). Outer and inner dynein arms of cilia and flagella. Cell 41, 341–342.[Medline]
Goodenough, U. W., and Heuser, J. E. (1985b). Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella. J. Cell Biol 100, 2008–2018.
Habermacher, G., and Sale, W. S. (1996). Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas. J. Cell Sci 109, 1899–1907.[Abstract]
Habermacher, G., and Sale, W. S. (1997). Regulation of flagellar dynein by phosphorylation of a 138-kD inner arm dynein intermediate chain. J. Cell Biol 136, 167–176.
Harris, E. H. (1989). The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use, San Diego, CA: Academic Press.
Harrison, A., Olds-Clarke, P., and King, S. M. (1998). Identification of the t complex-encoded cytoplasmic dynein light chain tctex1 in inner arm I1 supports the involvement of flagellar dyneins in meiotic drive. J. Cell Biol 140, 1137–1147.
Hendrickson, T. W., Perrone, C. A., Griffin, P., Wuichet, K., Mueller, J., Yang, P., Porter, M. E., and Sale, W. S. (2004). IC138 is a WD-repeat dynein intermediate chain required for light chain assembly and regulation of flagellar bending. Mol. Biol. Cell 15, 5431–5442.
Hennessey, T. M., Kim, D. Y., Oberski, D. J., Hard, R., Rankin, S. A., and Pennock, D. G. (2002). Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila. Cell Motil. Cytoskeleton 53, 281–288.[CrossRef][Medline]
Howard, D. R., Habermacher, G., Glass, D. B., Smith, E. F., and Sale, W. S. (1994). Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase. J. Cell Biol 127, 1683–1692.
Ikeda, K., Yamamoto, R., Wirschell, M., Yagi, T., Bower, R., Porter, M. E., Sale, W. S., and Kamiya, R. (2009). A novel ankyrin-repeat protein interacts with the regulatory proteins of inner arm dynein f (I1) of Chlamydomonas reinhardtii. Cell Motil. Cytoskeleton 66, 10.1002/cm.20324.
Kagami, O., and Kamiya, R. (1992). Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein. J. Cell Sci 103, 653–664.[Abstract]
Kamiya, R. (2002). Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants. Int. Rev. Cytol 219, 115–155.[Medline]
Kikushima, K., and Kamiya, R. (2008). Clockwise translocation of microtubules by flagellar inner-arm dyneins in vitro. Biophys J 94, 4014–4019.[CrossRef][Medline]
King, S. J., and Dutcher, S. K. (1997). Phosphoregulation of an inner dynein arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant strains. J. Cell Biol 136, 177–191.
King, S. M., and Kamiya, R. (2009). Axonemal dyneins: assembly, structure, and force generation. In: The Chlamydomonas Sourcebook: Cell Motility and Behavior, Vol. 3, ed. G. B. Witman, Oxford: Academic Press, 131–208.
Kotani, N., Sakakibara, H., Burgess, S. A., Kojima, H., and Oiwa, K. (2007). Mechanical properties of inner-arm dynein-f (dynein I1) studied with in vitro motility assays. In: Biophys. J, 93, . 886–894.[CrossRef][Medline]
Mastronarde, D. N., O'Toole, E. T., McDonald, K. L., McIntosh, J. R., and Porter, M. E. (1992). Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas. J. Cell Biol 118, 1145–1162.
Mitchell, D. R., and Kang, Y. (1991). Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene. J. Cell Biol 113, 835–842.
Myster, S. H., Knott, J. A., O'Toole, E., and Porter, M. E. (1997). The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex. Mol. Biol. Cell 8, 607–620.[Abstract]
Myster, S. H., Knott, J. A., Wysocki, K. M., O'Toole, E., and Porter, M. E. (1999). Domains in the 1-alpha dynein heavy chain required for inner arm assembly and flagellar motility in Chlamydomonas. J. Cell Biol 146, 801–818.
Nicastro, D., Schwartz, C., Pierson, J., Gaudette, R., Porter, M. E., and McIntosh, J. R. (2006). The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313, 944–948.
Okagaki, T., and Kamiya, R. (1986). Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms. J. Cell Biol 103, 1895–1902.
Okita, N., Isogai, N., Hirono, M., Kamiya, R., and Yoshimura, K. (2005). Phototactic activity in Chlamydomonas ‘non-phototactic’ mutants deficient in Ca2+-dependent control of flagellar dominance or in inner-arm dynein. J. Cell Sci 118, 529–537.
O'Toole, E., Mastronarde, D., McIntosh, J. R., and Porter, M. E. (1995). Computer-assisted analysis of flagellar structure. Methods Cell Biol 47, 183–191.[Medline]
Perrone, C. A., Myster, S. H., Bower, R., O'Toole, E. T., and Porter, M. E. (2000). Insights into the structural organization of the I1 inner arm dynein from a domain analysis of the 1-beta dynein heavy chain. Mol. Biol. Cell 11, 2297–2313.
Perrone, C. A., Tritschler, D., Taulman, P., Bower, R., Yoder, B. K., and Porter, M. E. (2003). A novel Dynein light intermediate chain colocalizes with the retrograde motor for intraflagellar transport at sites of axoneme assembly in Chlamydomonas and mammalian cells. Mol. Biol. Cell 14, 2041–2056.
Perrone, C. A., Yang, P., O'Toole, E., Sale, W. S., and Porter, M. E. (1998). The Chlamydomonas> IDA7 locus encodes a 140-kDa dynein intermediate chain required to assemble the I1 inner arm complex. Mol. Biol. Cell 9, 3351–3365.
Piperno, G., and Ramanis, Z. (1991). The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms. J. Cell Biol 112, 701–709.
Piperno, G., Ramanis, Z., Smith, E. F., and Sale, W. S. (1990). Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and location in the axoneme. J. Cell Biol 110, 379–389.
Porter, M. E., Knott, J. A., Myster, S. H., and Farlow, S. J. (1996). The dynein gene family in Chlamydomonas reinhardtii. Genetics 144, 569–585.[Abstract]
Porter, M. E., Power, J., and Dutcher, S. K. (1992). Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms. J. Cell Biol 118, 1163–1176.
Porter, M. E., and Sale, W. S. (2000). The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility. J. Cell Biol 151, F37–F42.[CrossRef][Medline]
Rupp, G., O'Toole, E., and Porter, M. E. (2001). The Chlamydomonas PF6 locus encodes a large alanine/proline-rich polypeptide that is required for assembly of a central pair projection and regulates flagellar motility. Mol. Biol. Cell 12, 739–751.
Rupp, G., and Porter, M. E. (2003). A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product. J. Cell Biol 162, 47–57.
Segal, R. A., Huang, B., Ramanis, Z., and Luck, D. J. (1984). Mutant strains of Chlamydomonas reinhardtii that move backwards only. J. Cell Biol 98, 2026–2034.
Sizova, I., Fuhrmann, M., and Hegemann, P. (2001). A streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277, 221–229.[CrossRef][Medline]
Smith, E. F. (2002). Regulation of flagellar dynein by the axonemal central apparatus. Cell Motil. Cytoskeleton 52, 33–42.[CrossRef][Medline]
Smith, E. F., and Sale, W. S. (1991). Microtubule binding and translocation by inner dynein arm subtype I1. Cell Motil. Cytoskeleton 18, 258–268.[CrossRef][Medline]
Smith, E. F., and Sale, W. S. (1992). Regulation of dynein-driven microtubule sliding by the radial spokes in flagella. Science 257, 1557–1559.
Smith, E. F., and Yang, P. (2004). The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil. Cytoskeleton 57, 8–17.[CrossRef][Medline]
Tam, L. W., and Lefebvre, P. A. (2002). The Chlamydomonas MBO2 locus encodes a conserved coiled-coil protein important for flagellar waveform conversion. Cell Motil. Cytoskeleton 51, 197–212.[CrossRef][Medline]
Toba, S., Fox, L. A., Sakakibara, H., Porter, M. E., Sale, W. S., and Oiwa, K. (2008). Distinct roles of 1 and 1β heavy chains of the I1 inner arm dynein of Chlamydomonas flagella. Mol. Biol. Cell 19, (Suppl. abstract 1787/B249. (CD-ROM).
Wilkerson, C. G., King, S. M., Koutoulis, A., Pazour, G. J., and Witman, G. B. (1995). The 78,000 M(r) intermediate chain of Chlamydomonas outer arm dynein is a WD-repeat protein required for arm assembly. J. Cell Biol 129, 169–178.
Wirschell, M., Hendrickson, T., and Sale, W. S. (2007). Keeping an eye on I 1, I1 dynein as a model for flagellar dynein assembly and regulation. Cell Motil. Cytoskeleton 64, 569–579.[CrossRef][Medline]
Wirschell, M., Yang, C., Yang, P., Fox, L., Yanigasawa, H., Kamiya, R., Witman, G. B., Porter, M. E., and Sale, W. S. (2009). IC97 is a novel intermediate chain of I1 dynein that interacts with tubulin and regulates interdoublet sliding. Mol. Biol. Cell 20, 3044–3054.
Witman, G. B. (1986). Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol 134, 280–290.[Medline]
Witman, G. B., Plummer, J., and Sander, G. (1978). Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components. J. Cell Biol 76, 729–747.
Yang, P., and Sale, W. S. (1998). The Mr 140,000 Intermediate chain of Chlamydomonas flagellar inner arm dynein is a WD-repeat protein implicated in dynein arm anchoring. Mol. Biol. Cell 9, 3335–3349.
Yang, P., and Sale, W. S. (2000). Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity. J. Biol. Chem 275, 18905–18912.
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