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. 2009 Jul 1;20(13):3055–3063. doi: 10.1091/mbc.E09-04-0277

IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility

Raqual Bower *, Kristyn VanderWaal *, Eileen O'Toole †, Laura Fox ‡, Catherine Perrone *, Joshua Mueller *, Maureen Wirschell ‡, R Kamiya §, Winfield S Sale ‡, Mary E Porter *,✉
Editor: Erika Holzbaur
PMCID: PMC2704157  PMID: 19420135

Abstract

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

INTRODUCTION

The dynein motors form the inner and outer rows of arm structures attached to the doublet microtubules of cilia and flagella (Porter and Sale, 2000; 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.

Figure 1.

Figure 1.

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Proposed organizational structure of I1 dynein within 96-nm axoneme repeat. The schematic diagram shows the proposed location of the IC/LC complex based on previous studies of I1 dynein motor domain mutants (Myster et al., 1998; Perrone et al., 2000; modified from Porter and Sale, 2000).

To address general questions of assembly and regulation of dynein, we have taken advantage of motility mutants in Chlamydomonas and focused on the structural and functional properties of the I1 inner arm dynein, also known as dynein f (reviewed in Porter and Sale, 2000; 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), 1α 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 1α 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.

MATERIALS AND METHODS

Strains, Culture Conditions, and Genetic Analysis

Strains used in this study are summarized in Table 1. The bop2-5 strain (6F5) was generated by transformation of the A54e18 strain (nit1, ac17, mt plus) with the plasmid pMN56 containing the nitrate reductase gene NIT1 (Myster et al., 1997). 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.

Table 1.

Strains used in this study

Strain name Motility Axoneme phenotype References
Control strains
    137c (nit1 nit2) (CC-125) Fast forward Wild type Harris (1989)
    A54 e18 (nit1-1 ac17 sr1) (CC-2929) Fast forward Wild type
    L5 (nit1, apm1-19, mt+) (CC-4263) Fast forward Wild type Tam and Lefebvre (1993)
    L8 (nit1, apm1-19, mt−) (CC-4264) Fast forward Wild type Tam and Lefebvre (1993)
    arg7-8 (CC-1826) Fast forward Wild type Harris (1989)
    mbo2-1 (CC-2377) Move backwards Missing mbo proteins Segal et al. (1984)
    mbo3-1 (CC-3670) Mixed motility Reduced mbo proteins Segal et al. (1984)
I1 related strains
    pf9-3 (CC-3913) Slow forward Missing I1 dynein Myster et al. (1997)
    bop5-1 (CC-4080) Slow forward Truncated IC138, loss of LC7b, FAP120 Dutcher et al. (1988); Hendrickson et al. (2004); Ikeda et al. (2009)
    bop5-2 (CC-4284) Mixed motility Missing IC138 subcomplex, MBO2p This study
    bop5-2::IC138 (CC-4285) Mixed motility, mostly backwards Missing MBO2p This study
    pf17 (CC-262) Paralyzed Missing radial spokes Harris (1989)
    pf17 bop5-2 Paralyzed Missing radial spokes and IC138 subcomplex This study
    pf17 bop5-2::IC138 Paralyzed Missing radial spokes This study
Diploid strains
    bop5-2, ac17, ARG7 Fast forward Not tested This study
    BOP5, AC17, arg7-8
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To determine whether the motility phenotype was linked to the NIT1 plasmid, bop5-2 was crossed to L8 (nit1-305, apm1-19, mt minus), and the resulting progeny of seven tetrads and additional random progeny were analyzed for their motility phenotypes and their ability to grow on selective media lacking ammonium. IC138 rescued strains were generated by transformation of bop5-2 with a plasmid containing the wild-type IC138 gene (Hendrickson et al., 2004) 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: 1α 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.

RESULTS

Identification of a Null Mutation in IC138

To identify new alleles at the IC138/BOP5 locus, we screened a collection of motility mutants generated by insertional mutagenesis with the nitrate reductase gene NIT1. This collection has been used previously to identify mutations in other I1 dynein subunits, central pair proteins, and the DRC (Myster et al., 1997; 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).

Figure 2.

Figure 2.

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Deletion of the IC138 gene in the bop5-2 strain. Shown here is a schematic diagram of the intron-exon structure of the IC138 gene in wild-type and the deleted region in bop5-2. PCR with gene-specific primers demonstrated that only the 5′ end and first exon of the IC138 gene is retained in bop5-2. Additional PCR reactions and Southern blots showed that the deletion extends ∼15 kb beyond the 3′ end of the IC138 gene.

Loss of IC138 Disrupts the Assembly of a Subset of I1 Dynein Subunits

Null mutations in other I1 dynein subunits, such as the two dynein HCs and IC140, typically result in the failure to assemble the I1 dynein complex into the flagellar axoneme (Myster et al., 1997, 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 1α 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 1α 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).

Figure 3.

Figure 3.

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Identification of an IC138 subcomplex within the I1 dynein. (A) Schematic diagrams of the IC138 subcomplex in I1 dyneins from wild-type, bop5-1, bop5-2, and IC138 rescued (bop5-2::IC138) strains. The other I1 dynein LCs are not shown here. (B) Western blot of isolated axonemes from wild-type and mutant cells. Note the presence of IC140 and the 1α HC in the bop5-2 axonemes, but the absence of IC138. As reported previously, IC138 is truncated in bop5-1 and shifted by hyperphosphorylation in mia1 and mia2. The outer arm subunit IC69 serves as a loading control. (C) Western blot of isolated axonemes from wild-type, bop5-2, and IC138 rescued cells. IC138, IC97 and FAP120 are missing in bop5-2 axonemes, but all three polypeptides are restored in axonemes from the IC138 rescued strain. Note that the MBO2 protein is not part of the IC138 subcomplex, because it missing in axonemes from both bop5-2 and the IC138 rescued strain.

To further characterize the effect of the loss of IC138 on I1 dynein, we prepared dynein extracts by incubation of isolated axonemes with 0.6 M NaCl and then fractionated the extracts on sucrose density gradients. Western blots of sucrose gradient fractions were probed with a series of antibodies against I1 dynein subunits. In wild-type extracts, all of the I1 dynein subunits cosediment at ∼20–21 S (Figure 4A); but in bop5-2 extracts, the I1 dynein is partially dissociated (Figure 4B). The 1α 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.

Figure 4.

Figure 4.

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Dissociation of the I1 dynein complex in bop5-2 dynein extracts. (A) Western blot of sucrose gradient fractions from a wild-type dynein extract probed with antibodies against several I1 dynein subunits. The I1 dynein subunits cosediment and peak at >20 S. FAP120 does not copurify with I1 dynein after salt extraction of wild-type axonemes (Ikeda et al., 2009) and thus was not analyzed here. (B) Western blot of sucrose gradient fractions from a bop5-2 dynein extract probed with antibodies against I1 dynein subunits. IC140 and the 1α DHC cosediment at >12 S, but the I1 dynein LCs Tctex1 and Tctex2b dissociate and sediment near the top of the sucrose gradient.

Rescue of IC138 Assembly Defects

We previously transformed the bop5-1 mutation with a wild-type copy of the IC138 gene and observed rescue of the slow swimming motility phenotype and reassembly of the full-length IC138 polypeptide (Hendrickson et al., 2004). 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).

Figure 5.

Figure 5.

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Defects in I1 structure in bop5-2 axonemes. Top row, averages of the 96-nm axoneme repeat from wild-type, bop5-2, and IC138 rescued (bop5-2::IC138) cells, based on six, six, and nine individual axonemes and 61, 63, and 93 repeating units, respectively. The proximal end of the repeat is on the left, the outer arms (OA) are shown on the top, and the two radial spokes (S1 and S2) are shown on the bottom. The I1 dynein is the trilobed structure at the proximal end of the repeat. The density of the third lobe near the base of S1 is reduced in bop5-2 and restored in the IC138 rescued (bop5::IC138) strain. Bottom row, diagram of densities within the 96-nm repeat and difference plots showing a statistically significant difference in the third lobe of the I1 dynein in bop5-2 (see arrow).

The IC138 Subcomplex Alters Flagellar Motility and Dynein-driven Microtubule Sliding

Although transformation with IC138 restores assembly of I1 dynein, we did not observe complete rescue of the motility defects. Both the original bop5-2 mutant and IC138 rescued cells swim poorly, with a high percentage of cells that move backward or spin in place (Supplemental Figure S2). The motility phenotypes are similar to that described previously for the move backward only (mbo) mutations (Segal et al., 1984). 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.

Figure 6.

Figure 6.

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IC138 is required for regulated microtubule sliding. (A) Microtubule sliding disintegration assays indicate that bop5-2 axonemes display slow microtubule sliding velocities relative to wild type. Sliding velocities increase to wild-type levels in the IC138 rescued strains (bop5-2::IC138). (B) Pretreatment of radial spoke mutants with kinase inhibitors increases microtubule sliding velocities only in presence of the IC138 subcomplex (pf17 and pf17 bop5-2::IC138). Kinase inhibitors have no effect in the absence of the IC138 subcomplex (pf17 bop5-2). Microtubule sliding velocities are expressed as micrometers per second. The average microtubule sliding velocity was calculated from three independent experiments, each with a sample size of at least 70 axonemes. Values shown are means and standard deviations.

The IC138 Subcomplex Is Required for Regulation of Microtubule Sliding by the Radial Spoke–Phosphorylation Pathway

Diverse evidence indicates that assembly of I1 dynein is required for regulation of microtubule sliding by a regulatory pathway that involves the central pair apparatus, radial spokes, and axonemal kinases and phosphatases (reviewed in Porter and Sale, 2000; 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.

DISCUSSION

IC138 Is Not Required for Assembly of the I1 Dynein

To better understand the role of IC138 in the control of dynein activity, we identified a null mutation, bop5-2, associated with the deletion of >90% of the IC138 gene. Surprisingly, loss of IC138 did not impact assembly of I1 dynein into the axoneme; both I1 HCs, IC140, Tctex1, and Tctex2b are still present (Figures 3 and 4). Mutations in either IC1 or IC2 usually block assembly of the outer arm dynein (Mitchell and Kang, 1991; 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).

Figure 7.

Figure 7.

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Model of the IC138 subcomplex and its role in regulating I1 dynein activity. (A) Shown here is a schematic diagram of the I1-dynein subunits on the A-tubule of the outer doublet. IC138 forms a regulatory subcomplex with LC7b, FAP120, and IC97. Attachment of the IC138 subcomplex to the outer doublet may be facilitated by IC97, which also interacts with tubulin and LC8 (Wirschell et al., 2009). Attachment of the remaining I1 subunits to the A-tubule may be facilitated by IC140 (Perrone et al., 1998; Yang et al., 1998). The precise locations of the other LC subunits are unknown. (B) Diagram showing the role of the IC138 subcomplex in regulating I1 dynein activity in response to signals from the radial spoke complex. In wild-type and IC138 rescued cells, the I1 dynein is active and microtubule sliding is fast. Radial spoke mutations result in hyperphosphorylation of IC138, inhibition of I1 activity, and reduced microtubule sliding velocities. Pretreatment of axonemes with kinase inhibitors results in dephosphorylation of IC138 by endogenous phosphatases, stimulation of I1 activity, and increased microtubule sliding. In the absence of the IC138 subcomplex, the I1 dynein is inactive under all conditions.

The IC138 Subcomplex Is Located at the Base of the I1 Dynein

Our previous studies of I1 dynein HC mutants identified the positions of the two motor domains, and by implication, suggested the position of the IC/LC complex (Figure 1). Here, we show directly by EM image averaging of bop5-2 axonemes that the IC138 subcomplex is located at the base of the I1 dynein, between radial spoke 1 and the outer dynein arms (Figures 5 and 7A). However, the N-terminal regions of the dynein HCs, IC140, and other I1 LCs are probably still present in this region. More detailed insight into the unique position of each subunit and their respective interactions within the IC138 subcomplex will require cryoelectron tomography of I1 mutant axonemes with less severe structural defects (e.g., bop5-1, ida7::IDA7 5a, tctex2b, fla14-3), as well as the development of alignment and averaging procedures that can accurately determine differences in three dimensions. Even so, the analysis of bop5-2 clearly locates IC138, which is the only known I1 phosphoprotein, in a position to be regulated by the radial spoke phosphorylation machinery.

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.

Supplementary Material

[Supplemental Materials]
E09-04-0277_index.html (841B, html)

ACKNOWLEDGMENTS

We thank Steven Myster (Beckman Corporation) for isolation of the bop5-2 mutant, Shawna McDonald for assistance with identification of the IC138 mutation, Tal Kramer (Emory University) for isolation of the pf17 bop5-2 double mutant, Douglas Tritschler (University of Minnesota) for transformation of the pf17 bop5-2 strain, and Kazuho Ikeda (Tokyo University) for the FAP120 antibody. These studies were supported by National Institutes of Health grants GM-55667 (to M.E.P.) and GM-051173 (to W.S.S.); a grant from the Ministry of Education, Culture, Sports, Science and Technology (to R. K.); and a National Research Service Award postdoctoral fellowship GM-075446 (to M. W.). E.T.O. was supported by National Institutes of Health Biotechnology Resource grant RR00592 to A. Hoenger. K. V. was supported in part by predoctoral fellowship 0715799Z from the American Heart Association Greater Midwest Affiliate, and Grant-in-Aid 20828 from the University of Minnesota Graduate School to M.E.P.

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.

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-04-0277) on May 6, 2009.

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