IC97 Is a Novel Intermediate Chain of I1 Dynein That Interacts with Tubulin and Regulates Interdoublet Sliding

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Molecular biology of the cell (Impact Factor: 4.47). 06/2009; 20(13):3044-54. DOI: 10.1091/mbc.E09-04-0276
Source: PubMed


Our goal is to understand the assembly and regulation of flagellar dyneins, particularly the Chlamydomonas inner arm dynein called I1 dynein. Here, we focus on the uncharacterized I1-dynein IC IC97. The IC97 gene encodes a novel IC without notable structural domains. IC97 shares homology with the murine lung adenoma susceptibility 1 (Las1) protein--a candidate tumor suppressor gene implicated in lung tumorigenesis. Multiple, independent biochemical assays determined that IC97 interacts with both alpha- and beta-tubulin subunits within the axoneme. I1-dynein assembly mutants suggest that IC97 interacts with both the IC138 and IC140 subunits within the I1-dynein motor complex and that IC97 is part of a regulatory complex that contains IC138. Microtubule sliding assays, using axonemes containing I1 dynein but devoid of IC97, show reduced microtubule sliding velocities that are not rescued by kinase inhibitors, revealing a critical role for IC97 in I1-dynein function and control of dynein-driven motility.

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Available from: Maureen Wirschell
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    • "Since neither IC138 nor IC140 was identified by mass-spec analysis in the IC97 crosslinked product and the IC97 antibody fails to recognize the crosslinked products of either IC138 or IC140, it is unlikely that IC97 is the binding partner for IC138 and IC140 in these crosslinking reactions [15]. However, IC97 and one of the intermediate chains of the outer dynein arm have been shown to interact directly with αand β-tubulin, which are the most abundant proteins in the axoneme [15] [33]. Moreover, since the intermediate chains are at the base of the dynein complex in the cargo-binding region, which is attached to the A-tubule of the doublet microtubule, we hypothesized that the binding partner in the 200-kDa crosslinked product is tubulin. "
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    ABSTRACT: Dyneins are minus end directed microtubule motors that play a critical role in ciliary and flagellar movement. Ciliary dyneins, also known as axonemal dyneins, are characterized based on their location on the axoneme, either as outer dynein arms or inner dynein arms. The I1 dynein is the best-characterized subspecies of the inner dynein arms; however the interactions between many of the components of the I1 complex and the axoneme are not well defined. In an effort to elucidate the interactions in which the I1 components are involved, we performed zero-length crosslinking on axonemes and studied the crosslinked products formed by the I1 intermediate chains, IC138 and IC140. Our data indicate that IC138 and IC140 bind directly to microtubules. Mass-spectrometry analysis of the crosslinked product identified both α- and β-tubulin as the IC138 and IC140 binding partners. This was further confirmed by crosslinking experiments carried out on purified I1 fractions bound to Taxol-stabilized microtubules. Furthermore, the interaction between IC140 and tubulin is lost when IC138 is absent. Our studies support previous findings that intermediate chains play critical roles in the assembly, axonemal targeting and regulation of the I1 dynein complex.
    Preview · Article · Sep 2013 · Biochimica et Biophysica Acta
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    • "Axoneme samples were run on 5–15% polyacrylamide gradient gels and transferred to Immobilon P (Millipore, Billerica, MA) as described previously (Bower et al., 2009). Antibodies were used at the following dilutions (vol/vol): IC138, 1:10,000 (Hendrickson et al., 2004); IC97, 1:10,000 (Wirschell et al., 2009); IC140, 1:10,000 (Yang and Sale, 1998); FAP120, 1:10,000 (Ikeda et al., 2009); IC69, 1:50,000 (Sigma-Aldrich, St. Louis, MO). Signal was detected with alkalinephosphatase–conjugated secondary antibodies and the Tropix detection system (Bower et al., 2009). "
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    ABSTRACT: I1 dynein, or dynein f, is a highly conserved inner arm isoform that plays a key role in the regulation of flagellar motility. To understand how the IC138 IC/LC subcomplex modulates I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles. bop5-3, bop5-4, and bop5-5 are null alleles, whereas bop5-6 is an intron mutation that reduces IC138 expression. I1 dynein assembles into the axoneme, but the IC138 IC/LC subcomplex is missing. bop5 strains, like other I1 mutants, swim forward with reduced swimming velocities and display an impaired reversal response during photoshock. Unlike mutants lacking the entire I1 dynein, however, bop5 strains exhibit normal phototaxis. bop5 defects are rescued by transformation with the wild-type IC138 gene. Analysis of flagellar waveforms reveals that loss of the IC138 subcomplex reduces shear amplitude, sliding velocities, and the speed of bend propagation in vivo, consistent with the reduction in microtubule sliding velocities observed in vitro. The results indicate that the IC138 IC/LC subcomplex is necessary to generate an efficient waveform for optimal motility, but it is not essential for phototaxis. These findings have significant implications for the mechanisms by which IC/LC complexes regulate dynein motor activity independent of effects on cargo binding or complex stability.
    Full-text · Article · Jun 2011 · Molecular biology of the cell
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    • "Microtubule sliding velocities were measured using the method of Okagaki and Kamiya ( Okagaki and Kamiya, 1986 ) and as previously described ( Howard et al. , 1994 ; Habermacher and Sale, 1996 , 1997 ; Hendrickson et al. , 2004 ; Bower et al. , 2009 ; Gokhale et al. , 2009 ; Wirschell et al. , 2009 ). Briefl y, isolated fl agella were resuspended in buffer without protease inhibitors and demembranated with buffer containing 0.5% Nonidet P-40 in 10 mM HEPES, pH 7.4; 5 mM MgSO 4 ; 1 mM DTT; 0.5 mM EDTA; 1% polyethylene glycol (20,000 MW); and 25 mM potassium acetate. "
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    ABSTRACT: The Chlamydomonas I1 dynein is a two-headed inner dynein arm important for the regulation of flagellar bending. Here we took advantage of mutant strains lacking either the 1α or 1β motor domain to distinguish the functional role of each motor domain. Single- particle electronic microscopic analysis confirmed that both the I1α and I1β complexes are single headed with similar ringlike, motor domain structures. Despite similarity in structure, however, the I1β complex has severalfold higher ATPase activity and microtubule gliding motility compared to the I1α complex. Moreover, in vivo measurement of microtubule sliding in axonemes revealed that the loss of the 1β motor results in a more severe impairment in motility and failure in regulation of microtubule sliding by the I1 dynein phosphoregulatory mechanism. The data indicate that each I1 motor domain is distinct in function: The I1β motor domain is an effective motor required for wild-type microtubule sliding, whereas the I1α motor domain may be responsible for local restraint of microtubule sliding.
    Full-text · Article · Feb 2011 · Molecular biology of the cell
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