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. 2009 Jul;20(13):3044-54.
doi: 10.1091/mbc.e09-04-0276. Epub 2009 May 6.

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

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Free PMC article

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

Maureen Wirschell et al. Mol Biol Cell. .
Free PMC article

Erratum in

  • Mol Biol Cell. 2009 Aug;20(15):3617

Abstract

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.

Figures

Figure 1.
Figure 1.
Chlamydomonas IC97 is homologous to murine Las1. Alignment of the Chlamydomonas IC97 sequence and its murine orthologue, Las1, shows a high degree of conservation throughout the two proteins. Chlamydomonas IC97 is listed under accession FJ156241 and Las1 under accession AAQ93498.1. The alignment was performed using the ClustalW server at http://www.ebi.ac.uk/Tools/clustalw2/index.html. IC97 and Las1 are 26% identical and 41% similar overall (expect value 9 e−19).
Figure 2.
Figure 2.
Antibodies to C_850038 recognize the IC97 subunit of I1 dynein. (A) The IC97 antibody was used to probe Western blots of axonemes derived from wild-type and several dynein mutants. The antibody recognizes a band, with an Mr of ∼90,000 that is present in wild type (WT) and dynein mutants that are defective in the outer dynein arm (oda2), or inner arm subtypes a, c, and d (ida4). This band is specifically missing in I1-dynein mutants (ida1, ida3, and ida7) that fail to assemble I1 dynein in the axoneme. The lower panel contains the Coomassie-stained gel showing protein loads. (B) Western blots of FPLC fractions from oda2 dynein extracts were probed with the IC97 antibody (top). The band recognized by the antibody cofractionates with the I1-dynein complex in the dynein-f peak (detected with antibodies to the IC138 subunit; bottom) confirming that the antibody specifically recognizes the IC97 I1-dynein subunit (Porter et al., 1992; Pazour et al., 1998; Yang and Sale, 1998; Kamiya et al., 1991; King and Dutcher, 1997; Myster et al., 1997; Harrison et al., 1998; Bowman et al., 1999; Pazour and Witman, 2000; Perrone et al., 1998, ; DiBella et al., 2004b,c; Hendrickson et al., 2004).
Figure 3.
Figure 3.
IC97 interacts with both α- and β-tubulin. (A) Western blots of wild-type axonemes that were treated with EDC were probed with the IC97 antibody. A prominent cross-linked product of ∼140-kDa is formed (arrow; uncross-linked IC97 is marked by the arrowhead). (B) The IC97 antibody was used to immunoprecipitate the cross-linked product (arrow; uncross-linked IC97 is marked by the arrowhead). The image is a representative silver-stained gel of the immune complexes showing that the IC97 antibody pulls down both uncross-linked IC97 (arrowhead) and the EDC-generated cross-linked product (arrow). The band corresponding to the cross-linked product was excised from an identical SYPRO Ruby-stained gel and components identified by tandem mass spectrometry. The cross-linked product represents a mixture of IC97 cross-linked to a-tubulin and IC97 cross-linked to β-tubulin. (C) Western blots of the IC97-immune complexes derived from EDC-treated axonemes containing HA-tagged α1-tubulin (Kozminski et al., 1993) were probed with the IC97 and HA antibodies. The IC97 antibody detects both uncross-linked (arrowhead) and cross-linked IC97 (arrow). The HA antibody detects only the cross-linked product indicating that it contains IC97 and HA-tagged α1-tubulin. (D) FPLC-purified I1 dynein was bound to Taxol-stabilized microtubules (assembled from purified tubulin) and then EDC cross-linked. The 140-kDa IC97-cross-linked product is formed indicating that IC97 cross-links to tubulin in vitro. (E) Blot overlays using biotinylated tubulin (left) reveal a tubulin-interacting band at the expected size for IC97 in FPLC-purified I1 dynein fractions (asterisk). Also evident is tubulin binding to IC140/IC138 (diamond) of I1 dynein and IC1 (circle) of the outer dynein arm (King et al., 1991). The IC2 component of the outer dynein arm is observed in the Coomassie-stained gel (right), but it is not detected in the blot overlay. The left lanes (αβ + e) are purified outer dynein arm fractions; the right lanes (f) are purified I1-dynein fractions.
Figure 4.
Figure 4.
IC97 is not required for I1-dynein assembly or anchoring in the axoneme. (A) Western blots of partial I1-dynein assembly mutants bop5-1, ida7-1::IC140 5A, and bop5-2. IC97 assembles into axonemes of both ida7-1::IC140 5A and bop5-1 but does not localize to the axoneme when IC138, FAP120, and LC7b are missing (bop5-2). Bottom, Coomassie-stained gel of the same samples. (B) FPLC fractions of dynein extracts from a double mutant ida7-1::IC140 5A oda9 demonstrate that IC97, while assembled in the axoneme, does not cofractionate with I1 dynein, but rather is detected in earlier fractions. (C) Western blots of axonemes from a new LC8 mutant, fla14-3 that expresses a larger LC8 protein (top, left; Yang, Yang, Wirschell, and Davis, unpublished data), demonstrates that IC97 and FAP120 fail to assemble in the axoneme (bottom). The rest of the I1-dynein complex seems to assemble in the absence of IC97/FAP120 (represented by IC140; top, right).
Figure 5.
Figure 5.
IC97 is required for regulation of microtubule sliding. (A) Western blots of isolated axonemes from pf17 and fla14-3 were probed with an antibody to the IC138 I1-dynein subunit. Axonemes were untreated or treated with CIP. IC138 is extensively phosphorylated (hyperphosphorylated) in radial spoke mutants such as pf17 and in the fla14-3 mutant (compare the untreated vs. treated bands; Hendrickson et al., 2004). (B) Microtubule sliding velocities were measured in wild-type, pf17, and fla14-3 axonemes (bars indicate means + SD). Sliding rates were reduced in fla14-3 axonemes relative to wild type—a result consistent with other radial spoke mutant axonemes (pf17). In contrast to pf17 axonemes, the sliding rates of fla14-3 axonemes do not increase upon addition of the kinase inhibitors protein kinase inhibitor (PKI) or DRB indicating that IC97 is required for PKI/DRB-mediated rescue of microtubule sliding. (C) In vitro phosphorylation of pf17 or fla14-3 axonemes by using [γ-32P]ATP was performed in the presence or absence of CK1-specific inhibitors. In pf17 and fla14-3 axonemes, when axonemes are pretreated with kinase inhibitors (CKI-7 or DRB), radioactive phosphate incorporation into IC138 is reduced, indicating that IC138 is largely dephosphorylated.
Figure 6.
Figure 6.
Models for I1-dynein structural interactions and for control of I1-dynein activity. (A) The I1-dynein interaction map showing the IC97 interactions determined in this study. IC97 interacts with IC140 based on analysis of the ida7-1::IC140 5A mutant; IC97 interacts directly with IC138 based on analysis of the bop5-2 mutant; biochemical data indicate a direct interaction between IC97 and α- and β-tubulin; and IC97 may interact with the LC8 dimer (dashed line) based on our analysis of the fla14-3 mutant, although we hypothesize that IC97 does not directly interact with LC8. In addition, FAP120 may interact with IC97 and the C terminus of IC138 (based on the bop5-1 and fla14-3 mutants). Interactions involving the LC subunits have not been fully determined, other than the LC7b-IC138 interaction (Dibella et al., 2004a; Hendrickson et al., 2004). (B) A model for regulation of I1-dynein activity showing the requirement of IC97 for I1-dynein activity is shown. In the presence of IC97, such as in pf17 axonemes, I1-dynein can be dephosphorylated (extrinsically with kinase inhibitors) and subsequently activated. However in the absence of IC97 (such as in fla14-3), I1 dynein remains inactive even though IC138 dephosphorylation occurs.

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