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Mol Biol Cell. 2004 October; 15(10): 4633–4646.
PMCID: PMC519155

The LC7 Light Chains of Chlamydomonas Flagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation

Tim Stearns, Monitoring Editor

Abstract

Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced ~20% in axonemes isolated from strains lacking inner arm I1 and are ~80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles ~30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm γ heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins.

INTRODUCTION

Dyneins are large multisubunit complexes that function as microtubule-based molecular motors in eukaryotic cells (reviewed in (King, 2002 blue right-pointing triangle; Vale, 2003 blue right-pointing triangle). Ciliary and flagellar dyneins comprise the inner and outer arms and constitute a heterogeneous motor population that seems to have multiple force-generating roles. In these structures, the axonemal microtubules provide a scaffold for an extensive network of additional proteins that collectively regulate the waveform, beat frequency, and ultimately motility of these organelles. Therefore, defining the precise composition of these motor enzyme complexes and the mechanisms by which they are assembled and regulated is of major importance to understanding flagellar motility.

Overall, dynein molecular motors are built around two basic designs (for recent review, see Sakato and King, 2004 blue right-pointing triangle). Cytoplasmic dynein and the outer arm and inner arm I1 axonemal dyneins contain two to three heavy chain motor units (~520 kDa each; HCs) that belong to the ancient AAA+ family of ATPases (Neuwald et al., 1999 blue right-pointing triangle; King, 2000 blue right-pointing triangle). For example, Chlamydomonas outer arm dynein contains three distinct HCs (α, β, and γ) that exhibit different enzymatic and motor properties (for review, see DiBella and King, 2001 blue right-pointing triangle). Dyneins also contain WD-repeat intermediate chains (ICs) that are associated with the N-terminal regions of the HCs and function in cargo and/or ATP-independent microtubule binding. Two general classes of light chains (LCs) are also part of the dynein complex. Several LC components so far found only in outer arm axonemal dynein possess putative regulatory regions (e.g., EF-hand and thioredoxin-like domains), associate directly with the HCs and likely influence motor function (Harrison et al., 2002 blue right-pointing triangle; Sakato and King, 2003 blue right-pointing triangle). In addition, a leucine-rich repeat LC interacts directly with the γ outer arm dynein HC motor domain and also may play a regulatory role (Benashski et al., 1999 blue right-pointing triangle; Wu et al., 2000 blue right-pointing triangle).

Members of the second LC class associate with the ICs and are located within the cargo-binding region. LCs from this latter group belong to three distinct protein families and in cytoplasm, incorporation of discrete isoforms is thought to define specific motor subtypes that may recognize particular intracellular cargoes (DiBella et al., 2001 blue right-pointing triangle). Within flagella, the highly conserved LC8 protein associates with the outer dynein arms, inner arm I1, and the radial spokes, and also is involved in intraflagellar transport (King and Patel-King, 1995b blue right-pointing triangle; Harrison et al., 1998 blue right-pointing triangle; Pazour et al., 1998 blue right-pointing triangle; Yang et al., 2001 blue right-pointing triangle). LCs from the Tctex1/Tctex2 family were originally identified as putative distorters/sterility factors involved in the non-Mendelian transmission of murine t-haplotypes (Lader et al., 1989 blue right-pointing triangle; Huw et al., 1995 blue right-pointing triangle) and were subsequently found to be dynein LCs (King et al., 1996 blue right-pointing triangle; Patel-King et al., 1997 blue right-pointing triangle). In Chlamydomonas, LC2 (Tctex2a) is necessary for assembly of the outer arm motor unit onto the axoneme (Patel-King et al., 1997 blue right-pointing triangle; Pazour et al., 1999 blue right-pointing triangle), and Tctex2b is a component of inner arm I1 that contributes to the stability and motor function of that enzyme (DiBella et al., 2004 blue right-pointing triangle). Tctex1 is an integral component of inner arm I1 (Harrison et al., 1998 blue right-pointing triangle) and is also present in cytoplasmic dynein (King et al., 1996 blue right-pointing triangle) where it is involved in attachment of specific cargoes (Tai et al., 1999 blue right-pointing triangle).

The third LC family, LC7/Roadblock, was initially identified through molecular cloning of LC7 (henceforth termed LC7a) from the Chlamydomonas outer arm and isolation of Drosophila mutants defective in the roadblock gene (Bowman et al., 1999 blue right-pointing triangle). In flies, roadblock mutant larvae suffer from posterior, sluggish motility that eventually leads to complete paralysis. These mutants also exhibit mitotic defects, along with severe axonal loss and nerve degeneration as a result of cargo accumulation, which is consistent with defects in retrograde axonal cargo transport. Recent studies have identified two mammalian roadblock (Robl) isoforms (Robl1 and Robl2) and indicate that both LCs can bind to cytoplasmic dynein ICs (Susalka et al., 2002 blue right-pointing triangle) and form both homo- and heterodimers (Nikulina et al., 2004 blue right-pointing triangle). In addition, the levels of Robl LCs may be modulated in response to specific cellular states. For example, one member of this LC class expressed in rat visual cortex is down-regulated in response to light stimuli (Ye et al., 2000 blue right-pointing triangle). In Chlamydomonas, LC7a is an integral component of the outer arm and lack of this protein in the oda15 null mutant results in arm assembly defects and aberrant flagellar motility (Pazour and Witman, 2000 blue right-pointing triangle).

To further define the role of LC7/Roadblock LCs in dynein function, we have identified and analyzed an additional member of this family in Chlamydomonas. This LC, termed LC7b, shares 48% and 47% identity with the Drosophila Roadblock cytoplasmic dynein light chain and Chlamydomonas LC7a, respectively, and we demonstrate that LC7a and LC7b are both components of Chlamydomonas inner arm I1 and outer arm dynein. We find that association of LC7b is mediated, at least in part, by LC7a. Furthermore, zero-length cross-linking reveals that LC7b interacts directly with a component of the outer arm docking complex, the outer arm β heavy chain-associated thioredoxin, LC3, and with the IC138 IC that is required for phosphorylation-dependent control of inner arm I1 (Habermacher and Sale, 1996 blue right-pointing triangle, 1997 blue right-pointing triangle; Yang and Sale, 2000 blue right-pointing triangle). These results provide the first evidence for how outer arm dynein is attached to the trimeric docking complex and suggest that members of this LC family play a role in both dynein-cargo attachment and motor regulation.

MATERIALS AND METHODS

Strains and Media

Wild-type (cc124) Chlamydomonas reinhardtii and the following mutant strains were used in this study: oda1, oda9, ida1, ida4, pf14, pf18 (obtained from Chlamydomonas Genetics Center, Duke University), oda15 (strain 3167.2; Pazour and Witman, 2000 blue right-pointing triangle) and pf28pf30 (Piperno et al., 1990 blue right-pointing triangle; WS4 isolate obtained from Dr. Winfield Sale, Emory University School of Medicine). All cells were grown in R (M medium plus 0.0075 M sodium acetate), or Tris-Acetate-Phosphate (TAP) media (Harris, 1989 blue right-pointing triangle).

Purification of Axonemes and Dynein

Wild-type and mutant strains of Chlamydomonas reinhardtii were deflagellated with dibucaine by using standard methods (King et al., 1986 blue right-pointing triangle) and demembranated with 1% IGEPAL CA-630 (catalog no. I-3021; Sigma-Aldrich, St. Louis, MO; replaces Nonidet P-40). For dynein purification, isolated axonemes were first treated with 0.6 M potassium acetate and then extracted with 0.6 M NaCl (King et al., 1986 blue right-pointing triangle; Nakamura et al., 1997 blue right-pointing triangle). NaCl-extracted proteins were fractionated using either 5–20% sucrose density gradients or by anion exchange chromatography (see below). Samples were routinely electrophoresed in 5–15% polyacrylamide gradient gels and stained with Coomassie Brilliant Blue (CBB), or transferred to nitrocellulose for immunoblotting.

Anion Exchange Chromatography

To separate the different species of axonemal dynein, the 0.6 M NaCl axonemal extract was dialyzed against buffer A (20 mM Tris, pH 7.5, 60 mM KCl, 0.5 mM EDTA, 0.1 mM Tween 20, 1 mM dithiothreitol, 1 mM phenylmethyl-sulfonyl fluoride), and applied to an anion exchange column (Mono Q HR5/5, Pfizer, New York, NY) using a Biologics chromatography work station (Bio-Rad, Hercules, CA). Proteins were eluted at a flow rate of 0.5 ml/min with a linear salt gradient of 0–50% buffer B (buffer A with 1 M KCl) and collected into 0.3-ml fractions. To initially identify fractions pertinent to this study, samples were electrophoresed in 8% polyacrylamide gels, before silver staining for protein detection (Merril et al., 1981 blue right-pointing triangle).

Molecular Analysis of LC7b

The entire coding region for Chlamydomonas LC7b was obtained by polymerase chain reaction (PCR) using wild-type Chlamydomonas first strand cDNA as the template. Both the forward primer 5′-GCGCTCTAGAATGTCGGATATCGAGTC-3′ and reverse primer 3′-GCGCGAATTCTTATGTGGAGGCCGCGTTGGG-5′ were designed based on the entire coding sequence derived from the Chlamydomonas expressed sequence tag AV387124 and incorporate an XbaI site at the 5′ end and an EcoRI site at the 3′ end, respectively. After restriction digestion, the gel-purified product was subcloned into pBluescript SK-(Stratagene, La Jolla, CA) across XbaI/EcoRI restriction sites. The sequenced clone was used to probe a Southern blot of Chlamydomonas genomic DNA, and a northern blot of Chlamydomonas RNA from nondeflagellated cells and from cells 30-min postdeflagellation that were actively regenerating flagella. The full-length cDNA also was used to isolate an ~20-kb genomic fragment containing the full-length LC7B gene from a λDashII (Stratagene) genomic DNA library previously generated from wild-type strain 1132D-(R. S.Patel-King and S. M.King, unpublished data).

Preparation of Recombinant Protein and Antibody

The full-length LC7b cDNA was subcloned into the pMAL-c2 vector (New England Biolabs, Beverly, MA) across the XmnI and EcoRI restriction sites. This resulted in the LC7b protein fused to the C terminus of maltose binding protein (MBP) via a 19-residue linker containing a factor Xa cleavage site. The overexpressed fusion protein was purified by amylose affinity chromatography. The full-length fusion protein was used as the immunogen to generate rabbit antiserum CT116. Nitrocellulose membrane containing recombinant LC7b was used to affinity-purify the antibody (Olmsted, 1986 blue right-pointing triangle). A MBP-LC7a fusion protein and the LC7a-specific antibody R7178 were described previously (Bowman et al., 1999 blue right-pointing triangle). Additional antibodies used in this study include 1869A (IC2) and 1878A (IC1) (King et al., 1986 blue right-pointing triangle), R4058 (LC8) (King and Patel-King, 1995b blue right-pointing triangle), R4930 (LC3) (Patel-King et al., 1996 blue right-pointing triangle), R5391 (LC2) (Patel-King et al., 1997 blue right-pointing triangle), R5932 (LC1) (Benashski et al., 1999 blue right-pointing triangle), αDC2 (DC2 kindly provided by Drs. Ritsu Kamiya, University of Tokyo, Tokyo, Japan; and Ken-ichi Wakabayashi, University of Connecticut Health Center, Farmington, CT) (Wakabayashi et al., 2001 blue right-pointing triangle), 12γB(γ HC) (King et al., 1985 blue right-pointing triangle), and αIC138 (Gri5-2; IC138) and αIC140 (IC140) (Yang and Sale, 1998 blue right-pointing triangle) (both kindly provided by Dr. Winfield Sale, Emory University School of Medicine, Atlanta, GA).

Native Gel Electrophoresis of MBP-LC Fusion Proteins

To examine the oligomeric state of LC7a and LC7b in solution, the MBP-LC fusion proteins were electrophoresed in gels of different acrylamide concentrations (at pH 8.8). The retardation coefficient (KR) was determined from the negative slope of 100 [log (Rf × 100)]. The standard proteins used were jack bean urease (545-kDa hexamer; 272-kDa trimer), bovine serum albumin (132-kDa dimer; 66-kDa monomer), ovalbumin (45 kDa), bovine carbonic anhydrase (29 kDa), and α-lactalbumin (14.2 kDa). A plot of log KR vs. log Mr for these proteins was used as a standard curve from which the native molecular masses of the MBP fusion proteins could be determined.

Chemical Cross-linking of Purified Dynein with the Zero-Length Cross-Linker 1-Ethyl-3-[3-dimethylaminopropyl]-carbodiimide (EDC)

To investigate protein–protein interactions involving LC7b, purified dynein was treated with 0–20 mM EDC dissolved in 100 mM HEPES, pH 7.4. Samples were incubated at room temperature for 60 min, and the reactions were terminated by addition of β-mercaptoethanol in HMEK (30 mM HEPES, pH 7.5, 5 mM MgSO4, 0.5 mM EDTA, 25 mM KCl) to a final concentration of 0.1 M (Benashski and King, 2000 blue right-pointing triangle).

Densitometric Analysis of Wild-Type and Mutant Axonemes

Various concentrations of wild-type and mutant axonemes were electrophoresed in 5–15% polyacrylamide gradient gels and either stained with Coomassie Blue or transferred to nitrocellulose. Blots were probed with CT116, 1878A (vs. IC1 of the outer arm; King et al., 1986 blue right-pointing triangle) or αIC140 (vs. IC140 of inner arm I1; Yang and Sale, 1998 blue right-pointing triangle). Signals were developed using a chemiluminescent substrate. Densitometry was performed on several different axonemal amounts for each sample to ensure that signals were within the linear response range, by using an Alpha Innotech Imaging System and AlphaEase Software (Athens, GA). Mutant axoneme amounts were normalized to wild-type by using the Coomassie Blue-stained α/β tubulin dimer bands.

Electron Microscopy of oda15 Axonemes

Samples were prepared for electron microscopy by using the method of Schroeter et al. (1994 blue right-pointing triangle). Axoneme samples were fixed with 2.5% glutaraldehyde, 1% tannic acid (#1764; Mallinckrodt, St. Louis, MO) in 100 mM sodium cacodylate, pH 7.4. Intact pellets were then transferred into a postfix solution (100 mM cacodylate, pH 7.4, 1% osmium tetroxide, 0.8% potassium ferricyanide) and subsequently washed in cacodylate buffer. Fixed samples were incubated in 1% tannic acid, 100 mM cacodylate, pH 7.4; stained en bloc with 0.5% aqueous uranyl acetate; and dehydrated in an ethanol series in propylene oxide. Samples were transitioned into plastic using increasing concentrations of Polybed (Polysciences, Warrington, PA) in propylene oxide. Blocks were polymerized at 60°C for 2–3 d, sectioned, and poststained with 2% aqueous uranyl acetate for 30 min and lead citrate (Sato, 1967 blue right-pointing triangle). Sections were examined in a Philips CM10 transmission electron microscope operating at an accelerating voltage of 60 kV.

RESULTS

Identification and Molecular Analysis of a Novel LC7/Roadblock Dynein Light Chain

To identify additional dynein components in Chlamydomonas flagella, we searched the Chlamydomonas expressed sequence tag (EST) and nonredundant databases by using LC7a from outer arm dynein (accession no. AF140239) as the initial query sequence. The search identified Chlamydomonas EST AV387124, which is 1362 base pairs in length and contains a single open reading frame encoding a 100-residue protein with a calculated molecular weight of 11,147 Da and a pI of 5.56. This protein (here termed LC7b) exhibits 47% sequence identity (63% similarity) with LC7a. A phylogenetic tree illustrating the relationship between LC7b and other members of this LC family is shown in Figure 1a. A secondary structure prediction made using Predictprotein (Rost and Sander, 1993 blue right-pointing triangle) suggests that LC7b contains α-helices in the N-terminal and central sections of the polypeptide, with two short β strands between the helices and three additional strands in the C-terminal regions (Figure 1b). This predicted arrangement of secondary structural elements is consistent with similar analyses for other LC7/Roadblock family members (Koonin and Aravind, 2000 blue right-pointing triangle; Nikulina et al., 2004 blue right-pointing triangle) and is clearly distinct from the LC8 (Liang et al., 1999 blue right-pointing triangle) and Tctex1/Tctex2 (Wu et al., 2001 blue right-pointing triangle) LC families.

Figure 1.
Molecular cloning and phylogenetic analysis of LC7b. (a) Phylogenetic analysis of the LC7/Roadlock family of LCs. Distances were calculated using ClustalW. Chlamydomonas LC7b (GenBank accession no. AV387124) shares 57% identity with the Drosophila cytoplasmic ...

A Southern blot of Chlamydomonas wild-type genomic DNA digested with SmaI and BamHI produced single bands when probed with the full-length LC7b cDNA, suggesting that this protein is encoded by a single gene (Figure 1c). A single band of ~1.36 kb was readily detected in a northern blot of RNA isolated from cells that were actively regenerating flagella (Figure 1d); this band was barely detectable in RNA from nondeflagellated cells, suggesting that LC7b is a flagellar protein. The LC7b gene was cloned using the cDNA construct as the initial probe and found to contain six exons (Figure 1e). Restriction fragment length polymorphism (RFLP) analysis revealed that the LC7B gene is located on linkage group XII/XIII, between TUB1 and TUB2.

LC7b Is Present in Flagellar Axonemes

The entire LC7b coding region was subcloned into the pMAL-c2 vector and expressed as an N-terminal fusion with MBP. The full-length fusion protein was used as the antigen to generate rabbit polyclonal antiserum CT116. After separation from MBP, immobilized LC7b protein was used to affinity purify the LC7b antibody. Western blotting against recombinant outer dynein arm LCs revealed that CT116 reacted only with its target antigen, and specifically did not recognize LC7a (Figure 2a) or any other axonemal dynein components (Figure 2b).

Figure 2.
Antibody specificity and localization of LC7b in axonemes. (a) MBP fusions to LC7a and LC7b were digested with Factor Xa, and the LCs separated from MBP by SDS-PAGE, stained with Coomassie Blue (top), or transferred to nitrocellulose (bottom). Blots were ...

To determine whether LC7b is indeed an integral dynein LC, axoneme samples isolated from wild-type and mutant strains lacking various flagellar structures were probed with the CT116 antibody (Figure 2c). LC7b was detected in wild-type axonemes and similar levels were observed in mutants lacking a subset of inner arms I2/3 (ida4), radial spokes (pf14), and central pair components (pf18). The levels of LC7b were reduced by ~20% in axonemes from mutants lacking inner arm I1 (ida1), and by ~80% in the absence of the outer arms and the outer arm docking complex (oda1) or outer arms alone (oda9) (Table 1). The LC7a-null mutant, oda15, also showed a significant decrease. The most dramatic reduction (of >90%) was seen in a mutant (pf28pf30) lacking both the outer arms and inner arm I1. The decreased amounts of LC7b in mutants lacking individual dyneins and the severely diminished levels observed in the pf28pf30 double mutant imply that this protein associates with both the outer arms and inner arm I1. This is consistent with the arrangement of four outer arms and one copy of inner arm I1 in the 96-nm axonemal repeat.

Table 1.
Dynein subunit content

LC7a and LC7b are Both Components of the Outer Dynein Arm and Inner Arm I1

To confirm the location of LC7b, axonemes were treated with 0.6 M NaCl, which removes dynein components, and the soluble extracts were fractionated in 5–20% sucrose density gradients. In ida1 extracts, which lack inner arm subtype I1, the majority of LC7b copurified with the trimeric outer dynein arm (Figure 3a); minor pools of LC7b also were observed sedimenting higher in the gradient and likely represent dissociation products. In addition, when the same extract was fractionated under conditions that cause the outer arm to partially dissociate (i.e., the absence of Mg2+ and an increase in hydrostatic pressure; Nakamura et al., 1997 blue right-pointing triangle), LC7b comigrated with the αβ HC dimer (Figure 3b). The location of DC2 from the outer arm docking complex also is shown in Figure 3, a and b, and illustrates that significant levels of this protein copurify with LC7b only in the presence of Mg2+. Furthermore, in oda9 axonemal extracts, the majority of LC7b cofractionated with components of inner arm I1 (Figure 4a), whereas in wild-type, it copurified with both sets of arms (Figure 4b). To ensure that LC7b associated with each dynein, wild-type and oda9 high salt extracts also were fractionated by anion exchange chromatography. As predicted, LC7b copurified with inner arm I1 in oda9 extracts (Figure 4c) and with both outer arm dynein and inner arm I1 in wild-type extracts (Figure 4d).

Figure 3.
LC7b is a component of the outer dynein arm. (a) A 0.6 M NaCl extract of ida1 axonemes was loaded onto a 5–20% sucrose gradient either in the presence of Mg2+ (a) or without Mg2+ (b). After sedimentation under low (a) or high (b) hydrostatic pressure, ...
Figure 4.
LC7b copurifies with both the outer arms and inner arm I1. In both a and b, 0.6 M NaCl axonemal extracts were loaded onto 5–20% sucrose gradients. After sedimentation, fractions were electrophoresed in 5–15% acrylamide gels and either ...

Previously, we demonstrated that LC7a was greatly reduced in axonemes prepared from mutants lacking outer dynein arms, although some protein could still be detected (Bowman et al., 1999 blue right-pointing triangle). Western blot analysis of wild-type and oda1 axonemes using the LC7a-specific R7178 antibody (Figure 2a) confirmed that this protein is present in outer armless samples, although at lower levels (Figure 5a). Furthermore, we observed a significant LC7a signal in 18S dynein fractions from oda1 extracts that had been fractionated in a sucrose density gradient (Figure 5b). These observations strongly suggest that the outer arm protein LC7a is also a component of inner arm I1.

Figure 5.
Outer dynein arm LC7a is also a component of inner arm I1. (a) Approximately 150 μg of wild-type (cc124) and oda1 axonemes and (b) oda1 purified 18S dynein were electrophoresed in 5–15% gradient gels then either stained with CBB (top) ...

The oligomeric state of the LC7 proteins was determined by native gel electrophoresis. Although the MBP-LacZ control (calculated molecular mass of 50.8 kDa) migrated as a monomer of Mr 56,200, the LC7a (54.4 kDa) and LC7b (50.8 kDa) fusion proteins migrated with Mr 104,800 and Mr 94,500, respectively. These results suggest that both proteins can form dimers in solution.

LC7b Interacts Directly with the Outer Arm Docking Complex and the Thioredoxin, LC3

To identify outer arm dynein components that interact with LC7b, dynein purified from the ida1 mutant was treated with the zero-length cross-linker EDC and cross-linked products detected by immunoblotting (King et al., 1991 blue right-pointing triangle). A Coomassie Blue-stained gel of ida1 outer arm dynein revealed that incubation with 20 mM EDC generated many cross-linked products as evidenced by a smear in the gel lane and the accumulation of material with very high Mr (Figure 6a). When an EDC-treated sample was probed with CT116, two major cross-linked products (Mr 94,700 and Mr 27,400) and a minor product (Mr 19,200) containing LC7b were detected (Figure 6b). The Mr of the largest product suggested that LC7b (11.1 kDa) had formed a complex with either an outer arm IC (IC1, 76.6 kDa; IC2, 63.5 kDa) or DC2 (62.2 kDa) from the docking complex. Immunoblotting using monoclonal antibodies specific for IC1 (1878A) and IC2 (1869A) revealed that LC7b had not cross-linked to either subunit (Figure 6, f and g). However, when the EDC-treated sample was probed for DC2, a product of identical Mr to that detected by the CT116 antibody was observed, strongly suggesting that DC2 and LC7b were part of the same cross-linked complex (Figure 6, c and e). Based on the size of the Mr 27,400 band, samples were probed with antibodies against all outer arm LCs and the docking complex DC3. Only the LC3 polyclonal antibody R4930 detected a band of similar size to that seen with the LC7b antibody, suggesting that these two LCs were in direct contact (Figure 6, d and h). In addition, the Mr 19,200 band was not detected by any antibody (including R7178 vs. LC7a) other than CT116, and because it also is present in cross-linked purified inner arm I1 dynein (see below), it likely represents an LC7b dimer. Immunoblot analysis of EDC-cross-linked samples using the R7178 antibody against LC7a did not reveal any major products; after prolonged exposure, several very minor bands (Mr of ~85,000–120,000) of unknown composition were detected.

Figure 6.
LC7b cross-links to DC2 from the outer arm docking complex and the LC3 thioredoxin. Purified outer arm dynein from ida1 axonemes was cross-linked with 0 or 20 mM EDC. Samples were electrophoresed in 5–15% gradient gels and then either stained ...

LC7b Also Associates with IC138 from Inner Arm I1

In a similar manner to that described above, inner arm I1 purified from the oda1 mutant was treated with EDC (Figure 7a) and probed with CT116. A major, reactive band of Mr 133,000 was detected, suggesting that LC7b had formed a complex with an inner arm IC (Figure 7b). An immunoblot of EDC-treated oda1 dynein using an antibody against IC140 revealed no cross-linked products of the appropriate Mr (our unpublished data). However, when a similar sample was probed for IC138, the same cross-linked product was detected (Figure 7, c and d). This product was not present in untreated dynein, indicating that it resulted from the zero-length cross-linking reaction and that LC7b and IC138 interact directly (Figure 7, b and c). An additional minor band of Mr 17,900 containing LC7b also was present in the cross-linked samples. Immunoblots using antibodies specific for Tctex1, Tctex2b, LC7a, and LC8 did not identify any similar sized cross-linked products, indicating that the Mr 17,900 band did not contain any of these LCs (our unpublished data). The simplest interpretation is that this band represents an LC7b dimer as also was observed in cross-linked outer arm dynein.

Figure 7.
LC7b cross-links to IC138 from inner arm I1. Purified inner arm I1 dynein from oda1 axonemes was chemically cross-linked with 0 or 20 mM EDC. Samples were electrophoresed in 5–15% gradient gels and then either stained with CBB or transferred to ...

LC7a Is Required for the Stable Association of LC7b, the γ Heavy Chain, and DC2 with the Outer Arm

To further investigate the putative interaction between LC7b and DC2 detected by EDC cross-linking, we took advantage of the mutant oda15, which is defective for LC7a and exhibits a slow, jerky swimming (Pazour and Witman, 2000 blue right-pointing triangle). Immunoblot analysis using the R7178 antibody confirmed that LC7a is completely missing in this strain, although considerable amounts of other outer arm dynein polypeptides (e.g., the γ HC-associated LC1 protein) are present (Figure 8a). This observation suggested that oda15 still retains significant numbers of outer dynein arms. To assess this, we examined oda15 axonemal cross-sections and found widely varying numbers of outer arms, ranging from a full complement to none (mean 3.2), associated with the doublet microtubules (Figure 8b). This observation is consistent with the quantitative densitometry analysis (Table 1), which indicates that ~20% of outer arm IC1 is retained in oda15 axonemes. Longitudinal sections revealed that, in regions containing outer arms, runs of motors were assembled with a 24-nm repeat as in wild-type. However, gaps occurred in the arrangement at apparently random intervals (Figure 8c), and many microtubule segments seemed completely devoid of attached dyneins. IC140 levels also were reduced to approximately one-third that seen in wild-type strains (Table 1), suggesting that inner arm I1 assembly is also compromised in the oda15 mutant.

Figure 8.
Axonemes from oda15 contain randomly placed outer dynein arms. (a) Approximately 50 μg of wild-type (cc124) and oda15 (LC7a null mutant) axonemes were electrophoresed in 5–15% gradient mini gels then either stained with Coomassie Blue ...

Dyneins were extracted from oda15 axonemes and sedimented in a sucrose density gradient under conditions that normally preserve association of the outer arm with the trimeric docking complex (Takada et al., 1992 blue right-pointing triangle; Figure 3). Electrophoretic and immunoblot analysis revealed that LC2, LC8, and both ICs from the outer arm cosedimented at ~18S in fractions 6–9 (Figure 9). However LC1 and the γ HC cosedimented at ~12S, as also occurs when Mg2+ is lacking in the gradient. As in wild-type samples, the peak of IC140 from inner arm I1 occurred slightly higher in the oda15 sucrose gradient than the outer arm, suggesting that at least some of this dynein also assembled in the mutant. Interestingly however, LC7b did not comigrate with either the outer arms or inner arm I1, but rather completely dissociated and was found at the very top of the gradient. Furthermore, we observed that the docking complex protein DC2 also was released and sedimented at ~7S, rather than remaining associated with the outer arm (Figure 3). Consequently, we conclude that LC7a is required for the stable association of LC7b, the docking complex, and the γ HC subcomplex with the remainder of the outer arm dynein.

Figure 9.
LC7a provides stability to the outer dynein arm. Sucrose gradient (containing Mg2+) fractionation of an oda15 high salt extract performed under conditions identical to those used for Figure 3a. Samples were electrophoresed in 5–15% gradient gels ...

DISCUSSION

Chlamydomonas Contains Two Members of the LC7/Roadblock Family

Previously, we identified LC7 (here termed LC7a) within the outer dynein arm as a founding member of the LC7/Road-block class of dynein light chains (Bowman et al., 1999 blue right-pointing triangle). Here, we describe a second member of this LC family, termed LC7b, found in Chlamydomonas that is ~50% identical to both LC7a and the Drosophila cytoplasmic dynein LC, roadblock. The LC7/Roadblock dynein LCs belong to an ancient superfamily involved in the regulation of NTPases (Koonin and Aravind, 2000 blue right-pointing triangle). One member of this family, MglB from Myxococcus xanthus, seems to modulate the GTPase MglA, which is involved in the regulation of gliding motility (Hartzell and Kaiser, 1991a blue right-pointing triangle; 1991b blue right-pointing triangle). Mammals also contain two members of this family that are differentially expressed (e.g., Robl1 but not Robl2 has been detected in brain). Furthermore, levels of Robl are dramatically altered in response to light stimuli in the rat visual cortex (Ye et al., 2000 blue right-pointing triangle), and the balance between Robl1 and Robl2 in human hepatocellular carcinoma tissue is shifted compared with healthy liver tissue (Jiang et al., 2001 blue right-pointing triangle).

LC7a and LC7b Associate with Both the Outer Dynein Arm and Inner Arm I1

Four outer dynein arms and one inner arm I1 normally assemble within a 96-nm repeat along the axonemal microtubules. Consistent with this arrangement, we found that LC7b levels are ~80% below wild type in mutants completely lacking the outer arms or outer arms and docking complex (oda1, oda9) and ~20% reduced in the absence of inner arm I1 (ida1) (Table 1). LC7b is almost completely absent from the axoneme when both classes of motor are missing (pf28pf30), and furthermore, this protein copurifies with both dyneins in sucrose density gradients and following anion exchange chromatography. These data indicate that this LC associates with both classes of dynein arm and further implies that it cannot assemble in the absence of other dynein components. This latter property is a common feature of many dynein proteins and is consistent with their preassembly into subcomplexes within the Chlamydomonas cytoplasm (Fowkes and Mitchell, 1998 blue right-pointing triangle).

Previously, we identified LC7a within Chlamydomonas outer arm dynein, and found that this protein was greatly reduced, but not absent, in mutant axonemes prepared from strains lacking that dynein (Bowman et al., 1999 blue right-pointing triangle). In this report, we demonstrate that LC7a is also an integral component of inner arm I1. Consequently, these two dyneins contain the same two members of this LC class. In cytoplasmic dynein, Robl1 and Robl2 are capable of forming both homo- and heterodimers (Susalka et al., 2002 blue right-pointing triangle; Nikulina et al., 2004 blue right-pointing triangle), suggesting that they assemble in pairs within the dynein motor unit. We observed that both LC7a and LC7b fusion proteins also form dimers in solution, and our cross-linking data further suggests that at least LC7b forms homodimers within both axonemal dyneins. Thus, the LC7/Roadblock LCs follow the general characteristic that LC components within the dynein IC/LC complex exist as dimers, e.g., the inner arm proteins Tctex1 (Harrison et al., 1998 blue right-pointing triangle; Wu et al., 2001 blue right-pointing triangle) and Tctex2b (DiBella et al., 2004 blue right-pointing triangle); outer arm LC6 (Pfister et al., 1982 blue right-pointing triangle; King and Patel-King, 1995b blue right-pointing triangle), and LC8, which is present in both dyneins (Pfister et al., 1982 blue right-pointing triangle; King and Patel-King, 1995b blue right-pointing triangle; Liang et al., 1999 blue right-pointing triangle) (Figure 10). The one apparent exception to this rule is the LC2 (Tctex2a) component of the outer arm, which is present at a stoichiometry of one per dynein (Pfister et al., 1982 blue right-pointing triangle; Patel-King et al., 1997 blue right-pointing triangle). However, we have recently identified an additional outer arm component related to Tctex1 that may act as its partner (L.M.DiBella and S. M.King, unpublished data).

Figure 10.
Protein interaction maps of the outer dynein arm and inner arm I1. Diagrams illustrating intradynein associations within the outer arm and inner arm I1 are shown. Experimentally determined interactions are indicated by solid connections and dashed lines ...

LC7a Is Not Absolutely Required for Outer Dynein Arm Assembly

Many components (including some LCs) of both the outer dynein arm and inner arm I1 are essential for assembly of the motor unit onto the axoneme, e.g., the 1α and 1β HCs from inner arm I1 (Kamiya et al., 1991 blue right-pointing triangle; Porter et al., 1992 blue right-pointing triangle; Myster et al., 1997 blue right-pointing triangle; Perrone et al., 2000 blue right-pointing triangle); IC140 from the I1 dynein (Perrone et al., 1998 blue right-pointing triangle; Yang and Sale, 1998 blue right-pointing triangle); the outer arm β and γ HCs (Mitchell and Rosenbaum, 1985 blue right-pointing triangle; Kamiya, 1988 blue right-pointing triangle; Sakakibara et al., 1993 blue right-pointing triangle); IC1 and IC2 from the outer arm (Kamiya, 1988 blue right-pointing triangle; King et al., 1995 blue right-pointing triangle; Wilkerson et al., 1995 blue right-pointing triangle); LC8, a LC required for IFT that also is found in the outer arm, inner arm I1, and radial spokes (King and Patel-King, 1995b blue right-pointing triangle; Pazour et al., 1998 blue right-pointing triangle); and the outer arm LC, LC2 (Pazour et al., 1999 blue right-pointing triangle). However, LC7a is apparently not absolutely required for this process, because the LC7a-null mutant oda15 can assemble ~20% of its outer arms and ~35% of the I1 class of inner arm. Analysis of purified dyneins from this strain indicated that the outer arms were unstable, because LC7b, the γ HC, and DC2 dissociated from the motor unit after extraction even when purified in the presence of Mg2+ under conditions that maintains their integrity within wild-type dynein. Furthermore, LC7b also completely dissociated, suggesting that it requires LC7a to form stable interactions with both dyneins.

Does LC7b Mediate Attachment of the Outer Arm to the Docking Complex?

The trimeric docking complex (DC1-DC3) is essential for correct placement of the outer dynein arm onto the axoneme (Koutoulis et al., 1997 blue right-pointing triangle; Wakabayashi et al., 2001 blue right-pointing triangle; Takada et al., 2002 blue right-pointing triangle), although a fraction of dyneins can assemble in the absence of DC3 (Casey et al., 2003 blue right-pointing triangle). Our cross-linking data reveal that DC2 and LC7b are in direct contact in purified outer arm dynein. This finding, together with the observation that both proteins dissociate from dyneins extracted from oda15 axonemes suggest that these proteins interact in the flagellum. This is the first evidence that links a component of the docking complex to the outer arm. Although the interaction of LC7b and DC2 represents the only identified connection between these two structures (Figure 10), this does not rule out the possibility of additional interactions between other dynein and docking complex components. The instability of the γ HC in this mutant raises the possibility that it also interacts with LC7b. However, in ida1 sucrose gradients purified under conditions that cause the γ HC and DC2 to dissociate from the remaining components of the outer arm, LC7b copurifies with the αβ HC dimer complex. EDC cross-linking revealed that LC7b is also in direct contact with the β HC-associated thioredoxin LC3. This observation raises the intriguing possibility that LC3 and the DC3 docking complex protein that binds Ca2+ in a redox-sensitive manner (Casey et al., 2003 blue right-pointing triangle), are in close proximity.

A protein interaction map for the outer dynein arm is shown in Figure 10 (top) and illustrates the complexity of associations that exist within this dynein motor. In addition to direct associations with LC3 and DC2, additional potential linkages involving LC7a and other subunits within the cargo-binding region are indicated. For example, as the Robl1 and Robl2 cytoplasmic dynein LCs both bind to the IC74 IC in a region distinct from the LC8 and Tctex1 binding sites (Susalka et al., 2002 blue right-pointing triangle), IC1 and IC2 from the axonemal outer arm are also potential LC7a and LC7b interacting partners.

LC7b Interacts with a Regulatory Subunit of Inner Arm I1

Within inner arm I1, we found that LC7b cross-links to IC138; however, because LC7b also detaches from inner arm I1 in the absence of LC7a, it must again form additional associations within this dynein (Figure 10). Both cAMP-dependent protein kinase (Hasegawa et al., 1987 blue right-pointing triangle; Howard et al., 1994 blue right-pointing triangle) and casein kinase 1 (Yang and Sale, 2000 blue right-pointing triangle) have been implicated in the regulation of microtubule sliding through inner arm I1 (Smith and Sale, 1992 blue right-pointing triangle). Moreover, IC138 is the only known phosphorylated subunit within this dynein; implicating it as a major regulatory component involved in this process (Habermacher and Sale, 1997 blue right-pointing triangle). Interaction of LC7b with IC138 places it in an intriguing position within this dynein that may transduce regulatory signals.

In conclusion, we have identified a novel member of the LC7/Roadblock family in Chlamydomonas. We show that both LC7a and LC7b associate with outer arms and I1 inner arms and that LC7a mediates a stable association between several dynein components, including LC7b within both dyneins. These LCs participate in multiple interactions within the outer arm and their relationship with the docking complex seems to play a major role in the assembly and stability of this dynein motor. In addition, within inner arm I1, LC7b associates with a major regulatory subunit in a manner that is stabilized by LC7a.

Acknowledgments

We thank Dr. Winfield Sale (Emory University School of Medicine) for Chlamydomonas strain pf28pf30 and for the polyclonal antibodies to IC140, and IC138; Drs. Ritsu Kamiya (University of Tokyo) and Ken-ichi Wakabayashi (University of Connecticut Health Center) for the polyclonal αDC2 antibody; and Drs. Carolyn Silflow and Paul Lefebvre (University of Minnesota, St. Paul, MN) for mapping the LC7B gene. We also thank Dr. Zhengyu Peng, Li Luo and Tobin Cammett (University of Connecticut Health Center) for Eshcerichia coli strain MM294 and for technical support and John Aghajanian for help with electron microscopy. This study was supported by grant GM-51293 (to S.M.K.) from the National Institutes of Health. S.M.K. is an investigator of the Patrick and Catherine Weldon Donaghue Medical Research Foundation.

Notes

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E04–06–0461. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–06–0461.

Abbreviations used: CBB, Coomassie Brilliant Blue; DC, docking complex; EDC, 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide; HC, heavy chain; IC, intermediate chain; LC, light chain; Robl, roadblock.

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