Molecular Biology of the Cell
Vol. 20, 3044–3054, July 1, 2009
IC97 Is a Novel Intermediate Chain of I1 Dynein That
Interacts with Tubulin and Regulates Interdoublet Sliding
Maureen Wirschell,* Chun Yang,†Pinfen Yang,†Laura Fox,*
Haru-aki Yanagisawa,‡Ritsu Kamiya,‡George B. Witman,§Mary E. Porter,?
and Winfield S. Sale*
*Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322;†Department of
Biology, Marquette University, Milwaukee, WI 53201;‡Department of Biological Sciences, Graduate School of
Science, University of Tokyo, Tokyo 113-0033, Japan;§Department of Cell Biology, University of
Massachusetts Medical School, Worcester, MA 01655; and?Department of Genetics, Cell Biology and
Development, University of Minnesota, Minneapolis, MN 55455
Submitted April 8, 2009; Revised April 23, 2009; Accepted April 29, 2009
Monitoring Editor: Erika Holzbaur
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 ?- and ?-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.
Dyneins are minus-end–directed microtubule motors im-
portant for a variety of cellular functions, including mem-
brane-bound organelle transport, assembly and orientation
of the mitotic spindle, nuclear migration, assembly of the
Golgi apparatus, and ciliary and flagellar motility. In the
ciliary/flagellar axoneme, the outer and inner dynein arms
convert the energy derived from ATP hydrolysis into mi-
crotubule sliding, which in turn drives flagellar beating
and bending. Analysis using the model genetic organism
Chlamydomonas reinhardtii has revealed that the inner arm
dynein system is responsible for generation of the flagellar
waveform—the size and shape of the flagellar bend (Brokaw
and Kamiya, 1987; Kamiya, 2002; King and Kamiya, 2009).
The inner dynein arms, of which there are at least seven
isoforms (King and Kamiya, 2009), are heterogeneous in
composition and structural arrangement on the axoneme,
binding to the axoneme in precise locations to form part of
a 96-nm repeating module along each doublet microtubule
(Goodenough and Heuser, 1985; Piperno et al., 1990; Burgess
et al., 1991; Mastronarde et al., 1992; Porter et al., 1992;
Nicastro et al., 2006; Bui et al., 2008). The two-headed I1
dynein isoform, also called dynein f, is distributed uniformly
along the length of the axoneme as a triad structure located
proximal to each radial spoke S1 (Nicastro et al., 2006; Porter
and Sale, 2000; Wirschell et al., 2007).
I1 dynein is among the best characterized inner arm iso-
forms, containing two heavy chains (?-HC and ?-HC), three
intermediate chains (IC138, IC140, and IC97—also termed
IC110), and five known light chains (LC8, LC7a, LC7b,
TcTex1, and TcTex2b) (Table 1; (Piperno et al., 1990; Myster
et al., 1997; 1999; Harrison et al., 1998; Pazour et al., 1998;
Perrone et al., 1998, 2000; Yang and Sale, 1998; DiBella et al.,
2004a,b; Hendrickson et al., 2004). Recently, a new I1-dy-
nein–associated protein, flagellar-associated protein (FAP)120,
has been identified that interacts with IC138, LC7b, and IC97
(Ikeda et al., 2008; Bower et al., 2009). Multiple lines of evidence
demonstrate that I1 dynein is an unusual dynein motor that
plays a critical regulatory role in the axoneme (Smith and Sale,
1991; Kotani et al., 2007; Wirschell et al., 2007). The I1-dynein
complex has been implicated as a target of the regulatory
signals that control flagellar motility (Porter et al., 1992; Porter
and Sale, 2000; Smith and Yang, 2004; Wirschell et al., 2007).
Flagella that are lacking I1 dynein exhibit defective flagellar
waveform and phototaxis, indicating that I1 dynein plays a
role in these processes (Brokaw and Kamiya, 1987; Brokaw,
1994; King and Dutcher, 1997; Okita et al., 2005).
In Chlamydomonas, four independent loci, when defective,
result in a specific failure to assemble the I1-dynein complex
in the axoneme (DiBella and King, 2001). Three of these loci
encode I1-dynein subunits; ida1, ida2, and ida7 mutants are
defectiveinthe ?-HC,?-HC,andIC140,respectively(Myster et
al., 1997; Perrone et al., 1998, 2000). Of particular use are novel
This article was published online ahead of print in MBC in Press
on May 6, 2009.
Address correspondence to: Winfield S. Sale (win@cellbio.
Abbreviations used: bop, bypass of paralysis; FAP, flagellar associ-
ated protein; HC, heavy chain; IC, intermediate chain; LC, light
chain; PKI, protein kinase inhibitor.
3044© 2009 by The American Society for Cell Biology
I1-dynein complexes (Perrone et al., 1998; Hendrickson et al.,
2004; Bower et al., 2009). These novel mutations allow for
detailed analysis of I1-dynein subunit interactions and func-
tion. For example, in the bypass of paralysis (bop)5-1 mutant,
expressing a C-terminal truncation of IC138, I1 dynein assem-
bles but lacks LC7b (Hendrickson et al., 2004) and FAP120
(Ikeda et al., 2008; Bower et al., 2009), indicating that IC138,
LC7b, and FAP120 interact. IC138 is a phosphoprotein in I1
dynein and is a key substrate for the regulatory mechanisms
that control flagellar motility (reviewed in Porter and Sale,
2000; Wirschell et al., 2007).
To date, all of the known I1-dynein subunits, except IC97,
have been cloned and characterized. To better understand
I1-dynein function in flagellar motility and how I1 dynein is
assembled, we determined the identity of the IC97 subunit.
IC97 is a novel IC; it does not encode WD-repeat motifs like
other known dynein ICs and is highly conserved, sharing
homology with proteins in a number of organisms that
assemble motile cilia/flagella, notably, the Las1 protein im-
plicated in pulmonary carcinoma in mice (Zhang et al., 2003).
Within the axoneme, IC97 interacts with both ?- and ?-tu-
bulin subunits; and within I1 dynein, IC97 interacts with
both IC140 and IC138 at the base of the dynein complex.
IC97 forms part of a regulatory subcomplex of I1-dynein
proteins that includes IC138, LC7b, and FAP120 (Ikeda et
al., 2008) that is now referred to as the IC138 subcomplex
(Bower et al., 2009).
Analysis of mutants that assemble partial I1-dynein com-
plexes reveal that both IC97 and IC138, although not neces-
sary for I1-dynein assembly, are required for regulation of I1
dynein. One such mutant, a new allele at the LC8 locus,
fla14-3, assembles I1-dynein complexes that are devoid of
IC97. fla14-3 mutant axonemes show reduced microtubule
sliding velocities that are not rescued by kinase inhibitors
even though IC138 seems to become dephosphorylated,
demonstrating that IC97 plays a critical role in regulation of
MATERIALS AND METHODS
Strains and Culture Conditions
Chlamydomonas strains used in this study are summarized in Table 2. Cells
were grown in Tris-acetate-phosphate medium or L-medium, with aeration
on a 14:10-h light:dark cycle (Harris, 1989; Harris, 2009).
Cloning and Sequencing of IC97 Sequences
The cloning of IC97 was based on tandem mass spectrometry (MS/MS)
identification of peptides derived from band-purified IC97 protein. Gene
model C_850038 in the JGI Chlamydomonas genome database version 2.0
(http://shake.jgi-psf.org/chlre2/chlre2.home.html) encodes the IC97 gene
(Supplemental Figure S1) and was used to design primers (Integrated DNA
Technologies, Coraville, IA) for polymerase chain reaction (PCR) amplifica-
tion of IC97 cDNA sequences (cDNA library 7 provided by Greg Pazour
(University of Massachusetts, Amherst, MA). PCR products were cloned into
pGEM-T-Easy (Promega, Madison, WI) or pCR2.1/GW8/TOPO cloning vec-
tors (Invitrogen, Carlsbad, CA) and sequenced to verify intron–exon bound-
aries (DNA Sequencing Facility, Iowa State University, Ames, IA). The full-
length cDNA is 2.973 kb.
An IC97 cDNA probe containing base pairs 1343–2685 (Supplemental Fig-
ure S1B) was used to screen a BAC library (Clemson University Genomics
Institute, Clemson, SC) and a 9.791-kb genomic EcoR1-HindIII fragment
containing the IC97 gene was identified and subcloned into pBlueScript
SK-vector (plasmid IC97-H). IC97 is found in the flagellar proteome as FAP94
(Pazour et al., 2005) and is published under accession FJ156240 and FJ156241
for the gene and cDNA sequences, respectively.
Table 1. I1-dynein components and mutants
SubunitMol. wt.Gene/mutantProperties References
ida1 (pf9, pf30)
Lacks inner arm I1, slow swimmingKamiya et al. (1991)
Myster et al. (1997)
Porter et al. (1992)
Kamiya et al. (1991)
Perrone et al. (2000)
Perrone et al. (1998)
Yang et al. (1998)
King et al. (1997)
Lacks inner arm I1, slow swimming
Lacks inner arm I1, slow swimming
Partial I1 assembly, regulatory phosphoprotein part of
Hendrickson et al. (2004)
Bower et al. (2009)
Porter et al. (1992)
IC97 (IC110)90 (110)Non-WD repeat protein,
homology to Las1/Casc1 proteins, part of IC138
Bower et al. (2009)
Pazour et al. (1998) LC8 10
Required for flagellar assembly, part of I1 dynein,
radial spokes and outer dynein arm
Yang et al. (2001)
Pazour and Witman (2000) LC7a14
Required for outer arm assembly, slow swimming,
associates with I1 dynein and may interact with
DiBella et al. (2004a)
Bowman et al. (1999)
Matsuo et al. (2008)
Dibella et al. (2004a)LC7b11LC7/Robl family member, interacts with IC138,
interacts with LC3 and DC2 of ODA
Hendrickson et al. (2004)
Harrison et al. (1998)Tctex113Dimeric protein, potential cargo binding activity, also
found in cytoplasmic dynein
Not required for I1 assembly, stabilizes I1 dynein
Not required for I1 dynein assembly, ankryn repeat
protein, part of IC138 subcomplex
DiBella et al. 2004a
Ikeda et al. (2009)
I1-Dynein Assembly and Regulation
Vol. 20, July 1, 20093045
Sequence alignments were performed using LaserGene SeqMan (DNAStar,
Madison, WI). The LaserGene EditSeq module was used for translations and
to estimate the predicted mass of the IC97 protein. Primers were designed
using Primer3 (www.genome.wi.mit.edu/cgi-bin/primer/primer3) (Rozen
and Skaletsky, 2000). The BLAST program (Altschul et al., 1990) was used to
search for homologous sequences. The COILS program (www.ch.embnet.
org/software/COILS_form.html) was used to predict coiled-coil regions in
IC97 and its orthologues. ClustalW, version 1.82, was used for alignment and
comparison of the murine Las1 protein (AAQ93498.1) and IC97 (http://
Isolation of Axonemes, Dynein Purification, and
Flagella were isolated as described previously (Witman, 1986) and demem-
branated using NP-40 (Calbiochem, San Diego, CA). Axonemes were
resuspended in HMDEKP (30 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM
dithiothreitol ?DTT?, 0.5 mM EDTA, 25 mM KCl, and 1 mM phenylmeth-
ylsulfonyl fluoride) plus the protease inhibitors aprotinin and leupeptin
(Sigma-Aldrich, St. Louis MO). Dyneins were extracted in HMDEKP plus
0.6 M KCl and fractionated by fast-performance liquid chromatography
(FPLC) on a MonoQ column (GE Healthcare, Piscataway, NJ) as described
previously (Kagami and Kamiya, 1995).
Chemical cross-linking was carried out using 1-ethyl-3-?3-dimethylamin-
opropyl? carbodiimide hydrochloride (EDC) by treating microtubule-bound
I1-dynein fractions or treating isolated axonemes with EDC for 30–60 min at
room temperature, respectively (Benashski and King, 2000; Wirschell et al.,
2008). All cross-linking reactions were performed in HMEK buffer (30 mM
HEPES, pH 7.4, 5 mM MgO4, 0.5 mM EDTA, and 25 mM KCl) supplemented
with protease inhibitors. Reactions were neutralized with a 20-fold molar
excess of ?-mercaptoethanol. Cross-linked axonemes were collected by
centrifugation and resuspended to 2 mg/ml with sample buffer. SDS-
PAGE was performed by standard techniques using Precision Plus dual-
color protein standards to estimate relative mobility (Mr) (Bio-Rad Labo-
ratories, Hercules, CA).
IC138 hyperphosphorylation was determined by comparing untreated and
phosphatase-treated axonemes (calf intestinal phosphatase ?CIP?) by SDS-
PAGE. Briefly, axonemes were treated with buffer or CIP for 30 min at room
temperature and then directly fixed for SDS-PAGE analysis.
IC138 phosphorylation status was determined by in vitro phosphorylation
of isolated axonemes using radioactive ??-32P?ATP (PerkinElmer Life and
Analytical Sciences, Boston, MA) (Yang and Sale, 2000). pf17 and fla14-3
axonemes were resuspended to 5 mg/ml in CK reaction buffer (25 mM Tris,
pH 8.0, 0.05 mM EDTA, 0.1% ?-mercaptoethanol, 3.5 mM magnesium acetate,
0.01% Brij 35, 10 mM NaCl, and 50 ?M sodium orthovanadate) and incubated
at 30°C for 30 min. ??-32P?ATP was added to a final concentration of 40 ?M,
and axonemes were incubated at 30°C for another 5 min. Samples were
immediately fixed with sample buffer for analysis on 5% SDS-PAGE, trans-
ferred to nitrocellulose, and subsequently detected by autoradiography. The
migration of IC138 was confirmed by Western blot of unlabeled axonemes run
on the same gel.
MS/MS analyses of band-purified IC97 and the IC97 cross-linked product
were performed at University of Massachusetts Proteomic Mass Spectrometry
Laboratory by Dr. John Leszyk. MS/MS spectra were used to search the JGI
Chlamydomonas Genome database, version 2.0.
Reconstitution of I1-Dynein Complexes onto Isolated
FPLC-purified I1 dynein and IC97 fractions derived from the double mutant
ida7-1::IC140 5A oda9 where dialyzed and then combined with isolated pf28
pf30 ssh1 axonemes. The reactions were incubated for 15 min at room tem-
perature in HMDEKP buffer. Axonemes and supernatant fractions were col-
lected by centrifugation and reconstitution of I1-dynein components analyzed
by Western blot.
Antibody Production and Western Blot Analyses
A restriction fragment containing nucleotides 1737-2492 of the IC97 cDNA
(Supplemental Figure S1B) was digested from clone pMW173.1 with
BamH1 and EcoR1 and subcloned into the pet28b expression vector
(Novagen, San Diego, CA) to create plasmid pMW185.1 (Supplemental
Table 2. Chlamydomonas strains used in this study
Strain DefectMotility References
CC-4077 (ida7-1::IC140 5A)c
ida7-1::IC140 5A oda9
Harris (1989, 2009)
Yang et al., unpublished
Perrone et al. (1998)
Radial spokes, I1 dynein, retrograde IFT
Truncated IC140; lacks outer dynein
Partial I1 dynein assembly; IC138 point
Partial I1 dynein assembly; null IC138
Lacks I1 dynein; 1?HC mutant
Very slow swimming,
Dutcher et al. (1988); Hendrickson et al.
Bower et al. (2009); Dutcher et al. (1988)
Kamiya et al. (1991); Myster et al.
Kamiya et al. (1991); Perrone et al.
Kamiya et al. (1991)
Lacks I1 dynein; 1?HC mutant
Lacks I1 dynein; gene product
Lacks inner arm isoforms a, c, and d
Lacks I1 dynein; IC140 mutant
Kamiya et al. (1991)
Perrone et al. (1998); Yang and Sale
McVittie (1972); Huang et al. (1981)
Kozminski et al. (1993)
Ledizet and Piperno (1986); Freshour et
CC-262 (pf17 mt-)
pf28 pf30 ssh1d
Lacks radial spoke head; RSP1 mutant
Lacks outer dynein arm; Lacks I1
dynein; suppressor of short mutation
aYang, Yang, Wirschell, and Davis (unpublished) determined that the pf5 mutation in strain CC1331 is an allele at the FLA14 locus (fla114-3)
that encodes the LC8 protein. Other strains denoted as pf5 in the Chlamydomonas Stock Center contain a mutation that is tightly linked to the
FLA14 locus and thus are not fla14-3/pf5 alleles.
bAssembly defects in fla14-3 include an effect on the retrograde IFT motor as evidenced by assembly of half to full-length flagella; no defects
in assembly of the outer dynein arm are observed; I1-dynein assembly defects are restricted to loss of IC97 and FAP120 specifically; and the
radial spokes are reduced resulting in the paralyzed flagellar phenotype.
cThe ida7-1::IC140 5A strain was first described in Perrone et al. (1998).
dStrain pf28 pf30 ssh1 is a triple mutant lacking both the outer dynein arm and I1 dynein; the ssh1 mutation allows for wild-type length flagella
in the double dynein mutant background (LeDizet and Piperno, 1995; Freshour et al., 2006).
M. Wirschell et al.
Molecular Biology of the Cell3046
Figure S1B). The expression construct was transformed into Escherichia coli
strain BL21(DE3)pLysS (Stratagene, La Jolla, CA) and expression induced
with 1 mM isopropyl ?-d-1-thiogalactopyranoside. The His-tagged fusion
protein contains amino acids 443-693 of the IC97 protein sequence. Insoluble
inclusion bodies containing the His-tagged IC97 fusion protein were solubi-
lized in 6 M urea in 1? binding buffer and passed over a nickel column for
purification according to the manufacturer’s instructions (Novagen). The
eluted fusion protein was used as an antigen for production of an IC97-
specific antiserum (Spring Valley Laboratories, Woodbine, MD). Western
blots were probed with the following antibodies: IC97 antiserum (W2-T),
1:10,000; IC138 antiserum, 1:20,000 (Hendrickson et al., 2004); IC140 anti-
serum, 1:10,000 (Yang and Sale, 1998); R4058 against LC8, 1:1000 (King and
Patel-King, 1995); and anti-hemagglutinin (HA) high-affinity rat monoclonal
antibody, 1:500 (Roche Diagnostics, Mannheim, Germany). Images were ac-
quired by exposing blots to x-ray film and converted to digital format by
scanning and editing in Adobe Photoshop CS2 (Adobe Systems, Mountain
View, CA). Figures were prepared using CorelDraw Graphics Suite 13 (Corel,
Mountain View, CA).
Immunoprecipitation of IC97-interacting Proteins
Immunoprecipitation was performed as described previously (King et al.,
1991). Briefly, EDC-treated wild-type axonemes were solubilized with SDS-
sample buffer and diluted 20-fold with Tris-buffered saline (TBS) buffer (10
mM Tris, pH 7.5, and 150 mM NaCl). The diluted axonemes were precleared
with protein A beads (Sigma-Aldrich) for 2–3 h, and then incubated with
protein A beads bound with the IC97 antiserum. The immune complexes
were collected, washed with TBS buffer plus 1% NP-40 and 0.05% Tween 20
and fixed with SDS-sample buffer. The immunoprecipitated were separated
by SDS-PAGE and analyzed by Western blot and protein stain.
Microtubule Binding Assays and Tubulin Blot Overlays
Purified tubulin (Cytoskeleton, Denver, CO) was polymerized at 37°C in the
presence of 10 ?M Taxol (Cytoskeleton), and microtubules were collected by
centrifugation. Taxol-stabilized microtubules were incubated with FPLC-pu-
rified I1-dynein fractions containing 10 ?M Taxol for 30 min at room temper-
ature and the I1-dynein-bound microtubules and then collected by centrifu-
gation. The I1-dynein–bound microtubule pellets were cross-linked as
described above and then fixed for SDS-PAGE. Tubulin blot overlays were
performed as described previously (Yanagisawa and Kamiya, 2004).
Microtubule Sliding Assay
In vitro microtubule sliding assays were performed based on the methods of
Okagaki and Kamiya (1986), with modifications (Kagami and Kamiya, 1992;
Howard et al., 1994; Habermacher and Sale, 1997). Briefly, axonemes from
strains CC1331 (fla14-3), pf17, and CC124 were resuspended in motility buffer
(10 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, 25 mM
potassium acetate, and 1% polyethylene glycol 20,000), loaded into a perfu-
sion chamber, and sliding events were initiated by perfusion of motility buffer
containing 1 mM ATP and subtilisin A, type VIII protease (Sigma-Aldrich).
Sliding events were observed by darkfield microscopy and recorded by SIT
camera and converted to a digital image for analysis by using LabView 7.1
software (National Instruments, Austin, TX). Microtubule displacement was
measured manually from tracings calibrated with a micrometer and used to
calculate sliding velocities.
Identification of the IC97 Subunit of I1 Dynein
To identify the IC97 subunit, I1-dynein, purified by FPLC
was resolved on SDS-PAGE, and proteins were stained with
Coomassie (data not shown). The band corresponding to the
IC97 subunit was excised, digested with trypsin, and pep-
tides were separated by liquid chromatography. MS/MS
analyses of the resulting peptides were used to search the
Chlamydomonas genome database and identified a candidate
gene model (C_850038 in JGI version 2). In total, 17 individ-
ual peptides were identified that span the gene model (Sup-
plemental Figure S1A). The gene model prediction is influ-
enced by the presence of gaps in the genomic sequence. As
a result, three peptides seem to reside in predicted introns
and one peptide resides in the predicted 5? untranslated
region (UTR). Gene model C_850038 (FAP94) was identified
in the KCl fraction of the flagellar proteome (Pazour et al.,
2005), a result consistent with a dynein protein.
Cloning IC97 Sequences
To verify the exact sequence of the protein encoded by gene
model C_850038, we designed primers based on predicted
exons and the peptides identified by MS/MS. Sequences
from a wild-type Chlamydomonas cDNA library were ampli-
fied by PCR and sequenced to confirm the complete open
reading frame (ORF) plus 412-base pairs of 5? and 282-base
pairs of 3? UTRs. The ORF predicts a 759-amino acid protein
with a mass of 81.5 kDa (Supplemental Figure S1A). Using
the antisense strand of the cDNA as a probe, we performed
a Northern blot on RNA derived from cells either before or
during flagellar regeneration. An ?2.8-kb transcript is de-
tected that is up-regulated in response to deflagellation and
flagellar regrowth (data not shown), a hallmark of mRNAs
encoding flagellar proteins (Silflow et al., 1982).
IC97 Is a Non-WD Repeat IC That Has Orthologues in
The predicted protein was analyzed by BLAST and found to
have homology to several proteins, including a Ciona intes-
tinalis axonemal protein (BAB88834.1) and a murine protein,
Las1 (Zhang et al., 2003; Manenti et al., 2004), that is part of
the multigenic Pas1 locus associated with an inherited pre-
disposition to pulmonary carcinoma in mice. Alignment of
the Chlamydomonas and murine proteins shows the homol-
ogy spans the entire sequence (Figure 1). Other than a small
region in the N terminus that is predicted to form a coiled-
coil, the IC97 protein does not have any notable motifs
including WD-repeat domains that are found in other dy-
nein intermediate chains.
IC97 Antiserum Detects the IC97 Component of I1 Dynein
To determine whether the gene we identified by MS/MS
does indeed encode the IC97 subunit of I1 dynein, we made
a specific polyclonal immune sera to an epitope-tagged fu-
sion protein containing amino acids 443-693 of the predicted
IC97 protein (Supplemental Figure S1B). In Western blots of
isolated axonemes, the antibody detects a band having a Mr
of ?90,000 on SDS-PAGE (Figure 2A), well in agreement
with the predicted mass of 81.5 kDa (The Mrof 90,000 is
estimated from our SDS-PAGE gels. The mass of IC97 has
been previously estimated as 97 and 110 kDa; Porter et al.,
1992; Habermacher and Sale, 1997. This discrepancy is most
likely due to differences in the molecular weight standards
used). This band is specifically missing in I1-dynein–defi-
cient axonemes (ida1, ida3, and ida7), indicating that the
antiserum detects an I1-dynein component. To further verify
this, we purified I1 dynein by FPLC fractionation. The anti-
serum detects the same size band in purified I1-dynein
(dynein-f peak) fractions (Figure 2B). These results demon-
strate that our antibody specifically detects the IC97 compo-
nent of I1 dynein.
IC97 Interacts with ?- and ?-Tubulin Components
The IC97 protein sequence does not predict any notable
domains or motifs that would lend insight into the function
of IC97. Thus, to better understand the role of IC97 in I1
dynein, we took advantage of EDC chemical cross-linking to
identify IC97-binding proteins. EDC is a proven reagent for
identifying axonemal interactions, particularly for the axon-
emal dyneins (King et al., 1991; Yang and Sale, 1998;
Benashski and King, 2000; DiBella et al., 2004a; Hendrickson
et al., 2004). Western blots of EDC-treated axonemes were
probed with the IC97 antibody, which detected an EDC
cross-linked product, with an Mrof ?140,000, suggesting
I1-Dynein Assembly and Regulation
Vol. 20, July 1, 20093047
IC97 (Mrof ?90,000) is in direct contact with an axonemal
protein with an estimated mass of 50 kDa (Figure 3A).
To determine the identity of the IC97-interacting protein,
we used the IC97 antiserum to immunoprecipitate the IC97
cross-linked product in sufficient yield for identification
(Figure 3B). MS/MS analysis of the IC97 cross-linked prod-
uct identified peptides corresponding to both ?- and ?-tu-
bulin in addition to IC97. The identification of IC97 peptides
demonstrates that the band isolated for MS/MS is the IC97
cross-linked product. The Mrof the cross-linked product is
?140,000 on SDS-PAGE, suggesting that IC97 does not
cross-link to a tubulin dimer, but rather IC97 independently
cross-links to ?- and ?-tubulin. Hence, the cross-linked
product is a mixture of IC97-?-tubulin and IC97-?-tubulin
species. In support of this model, the ratio of IC97:?-tubulin:
?-tubulin peptides recovered is 1.5:1:1, indicating that both
?- and ?-tubulin are present in equivalent amounts com-
pared with a nearly twofold amount of IC97.
To further validate the results obtained by MS/MS, we
used several approaches. First, we immunoprecipitated the
IC97 cross-linked product (Figure 3C, IC97) from SDS-solu-
bilized EDC-treated axonemes containing an HA-tagged ?1-
tubulin (Kozminski et al., 1993). The IC97 cross-linked prod-
uct is detected using an antibody to the HA tag, indicating
that it contains the epitope-tagged tubulin (Figure 3C, HA).
Second, we bound purified I1 dynein to Taxol-stabilized
microtubules and then performed EDC cross-linking. The
IC97 cross-linked product is formed indicating that IC97
interacts with tubulin components in vitro (Figure 3D).
Third, we took advantage of a blot overlay assay that uses
biotinylated tubulin as a probe to identify tubulin-interact-
ing proteins (Yanagisawa and Kamiya, 2004). Blot overlays
of purified dynein fractions detect a tubulin-interacting
band at the expected size for IC97 in purified-I1 dynein
(dynein-f peak) fractions (asterisk in Figure 3E).
IC97 Interacts with IC138 and IC140 within I1 Dynein
To further characterize how IC97 assembles within I1-dy-
nein, we took advantage of unique I1-dynein mutants that
assemble partial I1-dynein complexes. Figure 4A shows
Western blots of isolated axonemes from three mutant
murine Las1. Alignment of the Chlamydomonas IC97
sequence and its murine orthologue, Las1, shows a
high degree of conservation throughout the two pro-
teins. 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).
Chlamydomonas IC97 is homologous to
M. Wirschell et al.
Molecular Biology of the Cell3048
strains: bop5-1–expressing and assembling a truncated IC138
but missing the LC7b and FAP120 subunits of the IC138
subcomplex (Hendrickson et al., 2004; Ikeda et al., 2008),
ida7-1::IC140 5A–expressing and assembling a truncated IC140
lacking the N terminus (see table 1 in Perrone et al., 1998), and
bop5-2—an IC138-null allele that assembles I1 dynein lacking
IC138, LC7b, and FAP120 (Ikeda et al., 2009; Bower et al.,
2009) ida7-1::IC140 5A, but not bop5-2 (Figure 4A). These
observations are consistent with other reports, indicating that
IC97 is a component of the IC138 subcomplex and dependent
upon IC138 for assembly into I1-dynein (Ikeda et al., 2008;
Bower et al., 2009). This interaction is not mediated by LC7b or
FAP120 because IC97 assembles in bop5-1, where LC7b and
FAP120 are missing (Hendrickson et al., 2004; Ikeda et al., 2008).
Thus, we conclude that IC97 is interacting with IC138 directly.
Furthermore, in the bop5-1 mutant, IC97 copurifies with
I1-dynein by FPLC fractionation, indicating that it is stably
associated with I1 dynein (Hendrickson et al., 2004; our
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 Mrof ?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 sub-
unit; bottom) confirming that the antibody specifically
recognizes the IC97 I1-dynein subunit (Porter et al., 1992;
King and Dutcher, 1997; Myster et al., 1997; Harrison et al.,
1998; Bowman et al., 1999; Pazour and Witman, 2000;
Perrone et al., 1998, 2000; DiBella et al., 2004b,c; Hendrick-
son et al., 2004).
Antibodies to C_850038 recognize the IC97
(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 immu-
noprecipitate the cross-linked product (arrow; uncross-
linked IC97 is marked by the arrowhead). The image is
a representative silver-stained gel of the immune com-
plexes showing that the IC97 antibody pulls down both
uncross-linked IC97 (arrowhead) and the EDC-gener-
ated cross-linked product (arrow). The band corre-
sponding 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 ?-tubulin.
(C) Western blots of the IC97-immune complexes de-
rived from EDC-treated axonemes containing HA-
tagged ?1-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 ?1-tubulin. (D) FPLC-purified I1 dy-
nein was bound to Taxol-stabilized microtubules (as-
sembled 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 (dia-
mond) 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 (?? ? e) are purified outer
dynein arm fractions; the right lanes (f) are purified I1-dynein fractions.
IC97 interacts with both ?- and ?-tubulin.
I1-Dynein Assembly and Regulation
Vol. 20, July 1, 20093049
unpublished data). In contrast, although IC97 assembles in
ida7-1::IC140 5A, it does not cofractionate with I1 dynein
(derived from the ida7-1::IC140 5A oda9 double mutant and
represented by IC138 in Figure 4B), indicating that IC97
requires the N terminus of IC140 for stable association with
the I1-dynein complex and suggesting that IC97 and IC140
also interact. Together, these results suggest that IC97 inter-
acts with both IC138 and IC140 within I1-dynein, further
defining interactions of I1-dynein subunits in the higher
order dynein motor complex.
IC97 Is Not Required for I1-Dynein Assembly or for
Anchoring in the Axoneme
The results from the bop5-2 mutant demonstrate that IC97 is
not required for I1-dynein assembly or anchoring in the
axoneme (Bower et al., 2009). To verify this, we tested whether
I1-dynein complexes that lack IC97 (derived by salt extraction
from strain ida7-1::IC140 5A oda9) can rebind I1-dynein–defi-
cient axonemes in an in vitro reconstitution assay. Supplemen-
tal Figure S2 shows that I1-dynein complexes can rebind to
I1-deficient axonemes in the absence of IC97, indicating that
IC97 is not required for I1-dynein anchoring to the axoneme.
Furthermore, analysis of a newly identified LC8 mutation
fla14-3 (identified as the defective gene in the motility mu-
tant pf5), demonstrates that I1 dynein (identified by IC140)
assembles without IC97 and FAP120 (Figure 4C). The fla14-3
mutant expresses a larger LC8 protein due to a read-through
mutation in the LC8 stop codon (Figure 4C; Yang, Yang,
Wirschell, and Davis, unpublished data). LC8 is a compo-
nent of several flagellar complexes, including the retrograde
intraflagellar transport (IFT) motor, the outer dynein arm, I1
dynein, and the radial spokes. This particular fla14 allele
affects assembly of the I1-dynein components listed above
and the radial spokes but does not affect assembly of the
outer dynein arm. There also may be an effect on the retro-
grade IFT motor as evidenced by assembly of half- to full-
length flagella. A detailed characterization of the assembly
defects in fla14-3 is described in Yang, Yang, Wirschell, and
Davis (unpublished data).
IC97 Is Required for Control of Microtubule Sliding
Because IC97 is not required for assembly of I1 dynein, we
predicted that IC97 may function with IC138 in the regula-
tion of I1-dynein activity. Based on analysis of bop5-1 axon-
emes, FAP120 and LC7b do not seem to function in the
regulatory pathway that controls I1 dynein (Hendrickson et
al., 2004; Ikeda et al., 2008). Therefore, we reasoned that we
could use the fla14-3 mutant to test the role of IC97 in
regulation of I1-dynein activity using the microtubule slid-
ing assay. We did not need to combine the fla14-3 mutation
with a mutation such as pf17 that disrupts radial spoke
assembly because fla14-3 itself is inherently defective in as-
sembly of the radial spokes (Huang et al., 1981; Yang, Yang,
Wirschell, and Davis, unpublished data). Defects in radial
spoke assembly lead to disruption of the signaling pathway
that regulates I1 dynein, resulting in hyperphosphorylated
IC138 and inhibition of microtubule sliding. Given the radial
spoke defect in fla14-3, we predict that IC138 is hyperphos-
phorylated in fla14-3 axonemes. As expected, IC138 is hy-
perphosphorylated, similar to the radial spoke mutant pf17,
indicating that there is a defect in the regulatory pathway in
fla14-3 axonemes, a result that is consistent with the radial
spoke defect present in this mutant (Figure 5A; compare
CIP-treated to untreated axonemes; also see Hendrickson et
To test whether IC97 plays a regulatory role, we used the
in vitro microtubule sliding assay to compare I1-dynein
function in axonemes that contain IC97 (wild-type and pf17)
with fla14-3 axonemes that lack IC97. Consistent with pre-
vious reports, relative to wild-type axonemes, microtubule
sliding is greatly reduced in pf17 axonemes (Figure 5B). Like
other radial spoke mutants, microtubule sliding velocities in
fla14-3 axonemes are greatly reduced (Figure 5B). However,
unlike other radial spoke mutants, the slow microtubule
sliding in fla14-3 axonemes is not rescued by kinase inhibi-
tors (Figure 5B). These results are consistent with the hy-
pothesis that IC97 plays a critical role, along with IC138, in
the regulation of I1-dynein activity.
The central pair/radial spoke signaling mechanism has
been shown to impinge upon the IC138 subunit of I1 dynein
and rescue of microtubule sliding velocities correlates with
dephosphorylation of IC138 (Habermacher and Sale, 1997).
To further understand the failure to rescue microtubule
sliding velocities with kinase inhibitors, we used an in vitro
phosphorylation assay to test whether there is a defect in
dephosphorylation of IC138 in fla14-3 axonemes. Isolated
or anchoring in the axoneme. (A) Western blots of par-
tial 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 cofrac-
tionate 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, unpub-
lished 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).
IC97 is not required for I1-dynein assembly
M. Wirschell et al.
Molecular Biology of the Cell 3050
axonemes from pf17 and fla14-3 were incubated with radio-
active ??-32P?ATP after treatment with the protein kinase
inhibitors CKI-7 or 5,6-dichloro-1-?-d-ribofuranosylben-
zimidazole (DRB) (shown to block IC138 phosphorylation as
well as rescue microtubule sliding in isolated axonemes;
Yang and Sale, 2000). Consistent with previous reports
(Yang and Sale, 2000), incorporation of radioactive phos-
phate is reduced when pf17 axonemes are treated with the
kinase inhibitors (Figure 5B, pf17, bottom). In fla14-3 axon-
emes, kinase inhibitor treatment also reduces radioactive
phosphate incorporation (Figure 5B, fla14-3, bottom). Al-
though this assay does not detect dephosphorylation at spe-
cific residues (see Discussion), the reduction of phosphate
incorporation indicates that IC138 is dephosphorylated and
that the kinases and phosphatases that regulate IC138 are
intact. Thus, although IC138 phosphorylation in axonemes
from fla14-3 is inhibited with CKI-7 or DRB, microtubule
sliding was not restored to wild-type velocity, further indi-
cating an essential role for IC97 in regulation of I1 dynein.
Here, we describe the IC97 subunit of the conserved I1
dynein motor. IC97 interacts directly with tubulin, and
along with IC138, plays a role in regulation of I1 dynein and
control of microtubule sliding.
IC97 Interacts with Tubulin Subunits in the Axoneme
Based on multiple lines of evidence, we determined that
IC97 interacts with both ?- and ?-tubulin subunits (Figures
3 and 6A and Supplemental Figure S2). Our data suggest
that IC97 is in direct contact with the A-tubule of the outer
doublet, near the I1-dynein docking site. This is consistent
with a close association of the I1-dynein IC/LC domain with
the wall of the A-tubule (Nicastro et al., 2006; Bui et al., 2008).
Given that IC97 is not required for I1-dynein assembly, an
IC97–tubulin interaction cannot explain the precise localiza-
tion of I1 dynein within the 96-nm repeat. The specific
localization of I1 dynein in the axoneme may still require
additional factors (King and Kamiya, 2009).
The precise function of the IC97–tubulin interaction is
unclear. One possibility is that it is part of a mechanism that
detects curvature of the axoneme for control of dynein ac-
tivity and axonemal bending. Experimental and theoretical
evidence indicate that mechanical feedback from bending of
the axoneme is involved in regulation of dynein activity
(Hayashibe et al., 1997; Brokaw, 2002; Lindemann, 2004;
Morita and Shingyoji, 2004; Brokaw, 2008; Hayashi and
Shingyoji, 2008). It is possible that these mechanically based
control mechanisms involve multiple interactions of the dy-
nein motors with the axoneme that include direct interac-
tions with tubulin. Of particular interest is whether these
tubulin interactions with dynein intermediate chains are
altered during axonemal bending.
IC97 Interactions with I1-Dynein Subunits
In Figure 6A, we propose a model for how IC97 interacts
with the axoneme and I1-dynein components. Shown in red
are interactions between IC97-tubulin, IC97-IC140, and the
IC97-IC138 subcomplex, including FAP120.
Based on the analysis of the bop5-2 mutant (Bower et al.,
2009), we conclude that IC97 assembles into I1 dynein in
an IC138-dependent manner, suggesting that these two
I1-dynein subunits interact. This interaction is not medi-
ated by the C terminus of IC138, LC7b, or FAP120 because
IC97 is present in bop5-1, where these proteins are missing
(Figure 4C) (Hendrickson et al., 2004; Ikeda et al., 2009;
Bower et al., 2009). This result suggests that IC97 and
IC138 interact directly.
The IC97-IC140 interaction is supported by analysis of the
ida7-1::IC140 5A strain (Perrone et al., 1998). Although an
intact I1 dynein assembles in this mutant, IC97 is specifically
lost upon extraction of I1 dynein from the axoneme (Figure
4B), indicating that IC97 requires the N terminus of IC140 for
a stable association with I1 dynein in vitro. No other I1-
dynein subunit seems to dissociate in the ida7-1::IC140 5A
mutant, suggesting that the IC97-IC140 interaction is also
The model also depicts a possible interaction between
IC97 and LC8. Analysis of the fla14-3 mutant indicates that
assembly of IC97 and FAP120 are specifically disrupted.
However, we predict that the loss of IC97/FAP120 from I1
dynein in fla14-3 is indirect (see below).
IC97 Is Required for I1-Dynein–mediated Control of
Microtubule Sliding In Vitro
I1 dynein is an essential part of a regulatory pathway that
controls microtubule sliding and regulates axonemal bend-
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 (hyperphosphory-
lated) in radial spoke mutants such as pf17 and in the fla14-3
mutant (compare the untreated vs. treated bands; Hendrick-
son et al., 2004). (B) Microtubule sliding velocities were mea-
sured in wild-type, pf17, and fla14-3 axonemes (bars indicate
relative to wild type—a result consistent with other radial
spokemutantaxonemes(pf17).Incontrastto 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 phos-
phorylation of pf17 or fla14-3 axonemes by using ??-32P?ATP
was performed in the presence or absence of CK1-specific
phosphate incorporation into IC138 is reduced, indicating
that IC138 is largely dephosphorylated.
IC97 is required for regulation of microtubule
I1-Dynein Assembly and Regulation
Vol. 20, July 1, 2009 3051
ing. Figure 6B is founded in part on analysis of microtubule
sliding in axonemes isolated from paralyzed flagellar mu-
tants defective in assembly of the radial spokes or central
pair structures (reviewed in Porter and Sale 2000; Smith and
Yang 2004; Wirschell et al., 2007).
Here, we provide evidence that assembly of IC97 is nec-
essary for regulation of I1 dynein and control of microtubule
sliding. fla14-3 axonemes assemble an intact I1-dynein that
appears to lack only the IC97 and FAP120 subunits (Figure
4C). This mutant allele of LC8 manifests assembly defects in
I1 as well as the radial spokes (Huang et al., 1981; Yang,
Yang, Wirschell, and Davis, unpublished data). The defect in
radial spoke assembly intrinsic to this mutant allele allowed
us to use the microtubule sliding assay to directly test the
effects of the loss of IC97 on the regulatory pathway that
controls I1 dynein. Similar to radial spoke mutants like pf17;
IC138 is hyperphosphorylated in fla14-3 (Figure 5A). Fur-
thermore, despite inhibition of IC138 phosphorylation,
fla14-3 axonemes do not increase microtubule sliding rates
upon treatment with kinase inhibitors, indicating that IC97
is required, along with IC138, for control of I1 dynein. Evi-
dently, dephosphorylation of IC138 is not sufficient for con-
trol of microtubule sliding; the assembly of IC97 also is
required (Figure 6B).
We cannot rule out the possibility that the microtubule
sliding defect in fla14-3 is a result of the mutation in LC8 and
a novel defect in radial spoke assembly that allows fla14-3 to
behave differently than other radial spoke mutants. How-
ever, we propose that the failure to rescue microtubule
sliding velocities in fla14-3 is due to the specific loss of IC97
for the following reasons. 1) Recent models propose that the
LC8 dimer functions in a structural capacity; establishing
and driving the formation of large macromolecular com-
plexes like the dynein motors (Williams et al., 2007; Barbar,
2008; King, 2008). For example, the IC of cytoplasmic dynein
is partially disordered and gains structure upon binding to
LC8 (Nyarko et al., 2004). Thus, loss of IC97/FAP120 from
fla14-3 I1-dynein may be the result of a defective I1-dynein
structure that is not competent to recruit these subunits. Such a
model would indicate that IC97/FAP120 and LC8 do not in-
teract directly. 2) More importantly, IC97 requires IC138 for
assembly into I1 dynein and together they are localized within
a subcomplex at the base of I1-dynein (Bower et al., 2009).
IC97 can assemble in the absence of LC7b and FAP120
and for control of I1-dynein activity. (A) The I1-dynein
interaction map showing the IC97 interactions deter-
mined 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 ?- and ?-tubulin; and IC97 may in-
teract 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 addi-
tion, 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 ki-
nase inhibitors) and subsequently activated. However
in the absence of IC97 (such as in fla14-3), I1 dynein
remains inactive even though IC138 dephosphorylation
Models for I1-dynein structural interactions
M. Wirschell et al.
Molecular Biology of the Cell 3052
(bop5-1; Hendrickson et al., 2004; Ikeda et al., 2008), thus it
is likely that IC97 is interacting directly with IC138. Be-
cause IC97 is intimately associated with IC138, we pro-
pose that the failure in regulation of microtubule sliding
in fla14-3 is a direct result of the loss of IC97.
The mechanism of how IC97 is contributing to control of
I1-dynein activity is unclear. IC138 is hyperphosphorylated
in fla14-3, indicating that the phosphoregulatory pathway is
disrupted. Furthermore, in vitro phosphorylation assays re-
veal that IC138 is dephosphorylated in response to kinase
inhibitors in fla14-3 axonemes indicating that IC97 functions
with IC138 in regulation of I1 dynein. Sakakibara et al. (2006)
have demonstrated that a conformational change occurs in
I1 dynein upon phosphorylation. We postulate that this
alteration in structure requires the assembly of both IC138
and IC97. Regardless of the mechanism, our results indicate
that IC97 is a critical regulatory protein that functions in
control of microtubule sliding and I1-dynein activity.
IC97 Is Related to Proteins Implicated in Pulmonary
IC97 has orthologues in many species that contain motile
cilia and flagella. Of interest is the observed homology be-
tween IC97 and Las1—a protein implicated as a lung tumor
susceptibility gene (Zhang et al., 2003). Las1 is highly ex-
pressed in lung and testis— tissues that contain motile cilia
and in the kidney—a tissue containing immotile, primary
cilia (Zhang et al., 2003). Moreover, in dividing cells, Las1 is
expressed in G2 and its degradation during mitosis is re-
quired for cell cycle progression (Liu et al., 2007). The finding
that Las1 is a microtubule-binding protein (Liu et al., 2007) is
consistent with the IC97–tubulin interaction described here,
suggesting that an interaction with tubulin may be a con-
served feature of IC97/Las1 function. The significance of the
homology between Chlamydomonas IC97 and the Las1 pro-
tein involved in lung tumorigenesis is unclear, but raises the
questions of whether Las1 localizes to motile cilia, as well as
primary cilia, and whether Las1 plays functional roles both
in cilia and outside of the axoneme.
We thank Jacque Hudak for generating the ida7-1::IC140 5A oda9 double
mutant strain; and Cheryl Jones for technical expertise in cloning the IC97
genomic sequences. We also thank Avanti Gokhale and Candice Elam for
helpful discussions. This study was supported by National Institutes of
Health grants GM-051173 (to W.S.S.), GM-55667 (M.E.P.), GM-30626 (to
G.B.W.), and GM-068101 (to P. Y.); National Institutes of Health National
Research Service Award postdoctoral fellowship GM-075446 (to M. W.); and
a grant from the Ministry of Education, Culture, Sports, Science and Tech-
nology (to R. K.).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990).
Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Barbar, E. (2008). Dynein light chain LC8 is a dimerization hub essential in
diverse protein networks. Biochemistry 47, 503–508.
Benashski, S. E., and King, S. M. (2000). Investigation of protein-protein
interactions within flagellar dynein using homobifunctional and zero-length
crosslinking reagents. Methods 22, 365–371.
Bower, R., VanderWaal, K., O’Toole, E., Fox, L., Perrone, C. A., Mueller, J.,
Wirschell, M., Kamiya, R., Sale, W. S., and Porter, M. E. (2009). IC138 defines
a sub-domain at the base of the I1 dynein that regulates microtubule sliding
and flagellar motility. Mol. Biol. Cell 20, 3055–3063.
Bowman, A. B., Patel-King, R. S., Benashski, S. E., McCaffery, J. M., Goldstein,
L.S.B., and King, S. M. (1999). Drosophila roadblock and Chlamydomonas LC 7,
a conserved family of dynein-associated proteins involved in axonal trans-
port, flagellar motility, and mitosis. J. Cell Biol. 146, 165–180.
Brokaw, C. J. (1994). Control of flagellar bending: a new agenda based on
dynein diversity. Cell Motil. Cytoskeleton 28, 199–204.
Brokaw, C. J. (2002). Computer simulation of flagellar movement VIII: coor-
dination of dynein by local curvature control can generate helical bending
waves. Cell Motil. Cytoskeleton 53, 103–124.
Brokaw, C. J. (2008). Thinking about flagellar oscillation. Cell Motil. Cytoskel-
eton Epub ahead of print.
Brokaw, C. J., and Kamiya, R. (1987). Bending patterns of Chlamydomonas
flagella: IV. Mutants with defects in inner and outer dynein arms indicate
differences in dynein arm function. Cell Motil. Cytoskeleton 8, 68–75.
Bui, K. H., Sakakibara, H., Movassagh, T., Oiwa, K., and Ishikawa, T. (2008).
Molecular architecture of inner dynein arms in situ in Chlamydomonas rein-
hardtii flagella. J. Cell Biol. 5, 923–932.
Burgess, S. A, Carter, D. A, Dover, S. D, and Woolley, D. M. (1991). The inner
dynein arm complex: compatible images from freeze-etch and thin section
methods of microscopy. J. Cell Sci. 100, 319–328.
DiBella, L. M., and King, S. M. (2001). Dynein motors of the Chlamydomonas
flagellum. Int. Rev. Cytol. 210, 227–268.
DiBella, L. M., Sakato, M., Patel-King, R. S., Pazour, G. J., and King, S. M.
(2004a). The LC7 light chains of Chlamydomonas flagellar dyneins interact with
components required for both motor assembly and regulation. Mol. Biol. Cell
DiBella, L. M., Smith, E. F., Patel-King, R. S., Wakabayashi, K., and King, S. M.
(2004b). A novel Tctex2-related light chain is required for stability of inner
dynein arm I1 and motor function in the Chlamydomonas flagellum. J. Biol.
Chem. 279, 21666–21676.
Dutcher, S. K., Gibbons, W., and Inwood, W. B. (1988). A genetic analysis of
suppressors of the PF10 mutation in Chlamydomonas reinhardtii. Genetics 120,
Freshour, J., Yokoyama, R., and Mitchell, D. R. (2006). Chlamydomonas flagellar
outer row dynein assembly protein Oda7 interacts with both outer row and I1
inner row dyneins. J. Biol. Chem. 282, 5404–5412.
Goodenough, U. W., and Heuser, J. E. (1985). Substructure of inner dynein
arms, radial spokes, and the central pair/projection complex of cilia and
flagella. J. Cell Biol. 100, 2008–2018.
Habermacher, G., and Sale, W. S. (1997). Regulation of flagellar dynein by
phosphorylation of a 138-kD inner arm dynein intermediate chain. J. Cell Biol.
Harris, E. H. (1989). The Chlamydomonas Sourcebook: A Comprehensive
Guide to Biology and Laboratory Use, San Diego, CA: Academic Press.
Harris, E. H. (2009). The Chlamydomonas Sourcebook: Introduction to Chlamy-
domonas and Its Laboratory Use. Oxford, NY: Academic Press.
Harrison, A., Olds-Clarke, P., and King, S. M. (1998). Identification of the t
complex-encoded cytoplasmic dynein light chain tctex1 in inner arm I1 sup-
ports the involvement of flagellar dyneins in meiotic drive. J. Cell Biol. 140,
Hayashi, S., and Shingyoji, C. (2008). Mechanism of flagellar oscillation-
bending-induced switching of dynein activity in elastase-treated axonemes of
sea urchin sperm. J. Cell Sci. 121, 2833–2843.
Hayashibe, K., Shingyoji, C., and Kamiya, R. (1997). Induction of temporary
beating in paralyzed flagella of Chlamydomonas mutants by application of
external force. Cell Motil. Cytoskeleton 37, 232–239.
Hendrickson, T. W., Perrone, C. A., Griffin, P., Wuichet, K., Mueller, J., Yang,
P., Porter, M. E., and Sale, W. S. (2004). IC138 is a WD-repeat dynein inter-
mediate chain required for light chain assembly and regulation of flagellar
bending. Mol. Biol. Cell 12, 5431–5442.
Howard, D. R., Habermacher, G., Glass, D. B., Smith, E. F., and Sale, W. S.
(1994). Regulation of Chlamydomonas flagellar dynein by an axonemal protein
kinase. J. Cell Biol. 127, 1683–1692.
Huang, B., Piperno, G., Ramanis, Z., and Luck, D. J. (1981). Radial spokes of
Chlamydomonas flagella: genetic analysis of assembly and function. J. Cell Biol.
Ikeda, K, Yamamoto, R., Wirschell, M., Yagi, T., Bower, R., Porter, M. E., Sale,
W. S., and Kamiya, R. (2008). A novel ankyrin-repeat protein interacts with
the regulatory proteins of inner arm dynein f (I1) of Chlamydomonas reinhardtii.
Cell Motil. Cytoskeleton Epub ahead of print.
Kagami, O., and Kamiya, R. (1992). Translocation and rotation of microtu-
bules caused by multiple species of Chlamydomonas inner-arm dynein. J. Cell
Sci. 103, 653–664.
Kagami, O., and Kamiya, R. (1995). Separation of dynein species by high-
pressure liquid chromatography. Methods Cell Biol. 47, 487–489.
I1-Dynein Assembly and Regulation
Vol. 20, July 1, 20093053
Kamiya, R. (2002). Functional diversity of axonemal dyneins as studied in Download full-text
Chlamydomonas mutants. Int. Rev. Cytol. 219, 115–155.
Kamiya, R., Kurimoto, E., and Muto, E. (1991). Two types of Chlamydomonas
flagellar mutants missing different components of inner-arm dynein. J. Cell
Biol. 112, 441–447.
King, S. J., and Dutcher, S. K. (1997). Phosphoregulation of an inner dynein
arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant
strains. J. Cell Biol. 136, 177–191.
King, S. M. (2008). Dynein-independent functions of DYNLL1/LC 8, redox
state sensing and transcriptional control. Sci. Signal 1, pe51.
King, S. M., and Kamiya, R. (2009). Axonemal Dyneins: Assembly, Structure,
and Force Generation. In: The Chlamydomonas Sourcebook: Cell Motility and
Behavior, vol. 3, ed. G. B. Witman, Oxford, NY: Academic Press, 131–208.
King, S. M., and Patel-King, R. S. (1995). The M. (r) ? 8,000 and 11,000 outer
arm dynein light chains from Chlamydomonas flagella have cytoplasmic ho-
mologues. J. Biol. Chem. 270, 11445–11452.
King, S. M., Wilkerson, C. G., and Witman, G. B. (1991). The Mr 78,000
intermediate chain of Chlamydomonas outer arm dynein interacts with alpha-
tubulin in situ. J. Biol. Chem. 266, 8401–8407.
Kotani, N., Sakakibara, H., Burgess, S. A., Kojima, H., and Oiwa, K. (2007).
Mechanical properties of inner-arm dynein-f (dynein I1) studied with in vitro
motility assays. Biophys. J. 93, 886–894.
Kozminski, K. G., Diener, D. R., and Rosenbaum, J. L. (1993). High level
expression of nonacetylatable alpha-tubulin in Chlamydomonas reinhardtii. Cell
Motil. Cytoskeleton 25, 158–170.
LeDizet, M., and Piperno, G. (1995). The light chain p28 associates with a
subset of inner dynein arm heavy chains in Chlamydomonas axonemes. Mol.
Biol. Cell 6, 697–711.
Lindemann, C. B. (2004). Testing the geometric clutch hypothesis. Biol. Cell
Liu, Y., Vikis, H. G., Yi, Y., Futamura, M., Wang, Y., and You, M. (2007).
Degradation of lung adenoma susceptibility 1, a major candidate mouse lung
tumor modifier, is required for cell cycle progression. Cancer Res. 67, 10207–
Manenti, G., Galbiati, F., Gianni-Barrera, R., Pettinicchio, A., Acevedo, A., and
Dragani, T. A. (2004). Haplotype sharing suggests that a genomic segment
containing six genes accounts for the pulmonary adenoma susceptibility 1
(Pas1) locus activity in mice. Oncogene 23, 4495–4504.
Mastronarde, D. N., O’Toole, E. T., McDonald, K. L., McIntosh, J. R., and
Porter, M. E. (1992). Arrangement of inner dynein arms in wild-type and
mutant flagella of Chlamydomonas. J. Cell Biol. 118, 1145–1162.
Matsuo, T., OK, Onai, K., Niwa, Y., Shimogawara, K., Ishiura, M. (2008). A
systematic forward genetic analysis identified components of the Chlamydo-
monas circadian system. Genes Dev. 22, 918–930.
McVittie, A. (1972). Flagellum mutants of Chlamydomonas reinhardii. J. Gen.
Microbiol. 71, 525–540.
Morita, Y., and Shingyoji, C. (2004). Effects of imposed bending on microtu-
bule sliding in sperm flagella. Curr. Biol. 14, 2113–2118.
Myster, S. H., Knott, J. A., O’Toole, E., and Porter, M. E. (1997). The Chlamy-
domonas Dhc1 gene encodes a dynein heavy chain subunit required for
assembly of the I1 inner arm complex. Mol. Biol. Cell 8, 607–620.
Myster, S. H., Knott, J. A., Wysocki, K. M., O’Toole, E., and Porter, M. E.
(1999). Domains in the 1alpha dynein heavy chain required for inner arm
assembly and flagellar motility in Chlamydomonas. J. Cell Biol. 146, 801–818.
Nicastro, D., Schwartz, C., Pierson, J., Gaudette, R., Porter, M. E., and McIntosh,
J. R. (2006). The molecular architecture of axonemes revealed by cryoelectron
tomography. Science 313, 944–948.
Nyarko, A., Hare, M., Hays, T. S., and Barbar, E. (2004). The intermediate
chain of cytoplasmic dynein is partially disordered and gains structure upon
binding to light-chain LC8. Biochemistry 43, 15595–15603.
Okagaki, T., and Kamiya, R. (1986). Microtubule sliding in mutant Chlamydo-
monas axonemes devoid of outer or inner dynein arms. J. Cell Biol. 103,
Okita, N., Isogai, N., Hirono, M., Kamiya, R., and Yoshimura, K. (2005).
Phototactic activity in Chlamydomonas‘non-phototactic’ mutants deficient in
Ca2?-dependent control of flagellar dominance or in inner-arm dynein. J. Cell
Sci. 118, 529–537.
Pazour, G. J., Agrin, N., Leszyk, J., and Witman, G. B. (2005). Proteomic
analysis of a eukaryotic cilium. J. Cell Biol. 170, 103–113.
Pazour, G. J., Wilkerson, C. G., and Witman, G. B. (1998). A dynein light chain
is essential for the retrograde particle movement of intraflagellar transport
(IFT). J. Cell Biol. 141, 979–992.
Pazour, G. J., and Witman, G. B. (2000). Forward and reverse genetic analysis
of microtubule motors in Chlamydomonas. Methods 22, 285–298.
Perrone, C. A., Myster, S. H., Bower, R., O’Toole, E. T., and Porter, M. E.
(2000). Insights into the structural organization of the I1 inner arm dynein
from a domain analysis of the 1beta dynein heavy chain. Mol. Biol. Cell 11,
Perrone, C. A., Yang, P., O’Toole, E., Sale, W. S., and Porter, M. E. (1998). The
Chlamydomonas IDA7 locus encodes a 140-kDa dynein intermediate chain
required to assemble the I1 inner arm complex. Mol. Biol. Cell 9, 3351–3365.
Piperno, G., Ramanis, Z., Smith, E. F., and Sale, W. S. (1990). Three distinct
inner dynein arms in Chlamydomonas flagella: molecular composition and
location in the axoneme. J. Cell Biol. 110, 379–389.
Porter, M. E., Power, J., and Dutcher, S. K. (1992). Extragenic suppressors of
paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that
alter the inner dynein arms. J. Cell Biol. 118, 1163–1176.
Porter, M. E., and Sale, W. S. (2000). The 9 ? 2 axoneme anchors multiple
inner arm dyneins and a network of kinases and phosphatases that control
motility. J. Cell Biol. 151, F37–F42.
Rozen, S., and Skaletsky, H. (2000). Primer3 on the WWW for general users
and for biologist programmers. Methods Mol. Biol. 132, 365–386.
Silflow, C. D., Lefebvre, P. A., McKeithan, T. W., Schloss, J. A., Keller, L. R.,
and Rosenbaum, J. L. (1982). Expression of flagellar protein genes during
flagellar regeneration in Chlamydomonas. Cold Spring Harb. Symp. Quant.
Biol. 46, 157–169.
Sakakibara, H., Burgess, S., and Sakai, Y. (2006). Conformational changes of
inner-arm dynein-f from Chlamydomonas coupled with phosphorylation of its
intermediated chain IC138. Mol. Biol. Cell 17 (Suppl), abstract 211/B156.
Smith, E. F., and Sale, W. S. (1991). Microtubule binding and translocation by
inner dynein arm subtype I1. Cell Motil. Cytoskeleton 18, 258–268.
Smith, E. F., and Yang, P. (2004). The radial spokes and central apparatus:
mechano-chemical transducers that regulate flagellar motility. Cell Motil.
Cytoskeleton 57, 8–17.
Williams, J. C., Roulhac, P. L., Roy, A. G., Vallee, R. B., Fitzgerald, M. C., and
Hendrickson, W. A. (2007). Structural and thermodynamic characterization of
a cytoplasmic dynein light chain-intermediate chain complex. Proc. Natl.
Acad. Sci. USA. 104, 10028–10033.
Wirschell, M., Hendrickson, T., and Sale, W. S. (2007). Keeping an eye on I 1, I1
dynein as a model for flagellar dynein assembly and regulation. Cell Motil.
Cytoskeleton 64, 569–579.
Wirschell, M., Zhao, F., Yang, C., Yang, P., Diener, D., Gaillard, A., Rosenbaum,
J. L., and Sale, W. S. (2008). Building a radial spoke: flagellar radial spoke protein
3 (RSP3) is a dimer. Cell Motil. Cytoskeleton 65, 238–248.
Witman, G. B. (1986). Isolation of Chlamydomonas flagella and flagellar axon-
emes. Methods Enzymol. 134, 280–290.
Yanagisawa, H.-A., and Kamiya, R. (2004). A Tektin homologue is decreased
in Chlamydomonas mutants lacking an axonemal inner-arm dynein. Mol. Biol.
Cell 15, 2105–2115.
Yang, P., Diener, D. R., Rosenbaum, J. L., and Sale, W. S. (2001). Localization
of calmodulin and dynein light chain LC8 in flagellar radial spokes. J. Cell
Biol. 153, 1315–1326.
Yang, P., and Sale, W. S. (1998). The Mr 140,000 intermediate chain of
Chlamydomonas flagellar inner arm dynein is a WD-repeat protein implicated
in dynein arm anchoring. Mol. Biol. Cell 9, 3335–3349.
Yang, P., and Sale, W. S. (2000). Casein kinase I is anchored on axonemal
doublet microtubules and regulates flagellar dynein phosphorylation and
activity. J. Biol. Chem. 275, 18905–18912.
Zhang, Z., Futamura, M., Vikis, H. G., Wang, M., Li, J., Wang, Y., Guan, K. L.,
and You, M. (2003). Positional cloning of the major quantitative trait locus
underlying lung tumor susceptibility in mice. Proc. Natl. Acad. Sci. USA 100,
M. Wirschell et al.
Molecular Biology of the Cell 3054