JCS ePress online publication date 22 January 2008
428
Research Article
Tetrahymena IFT122A is not essential for cilia
assembly but plays a role in returning IFT proteins
from the ciliary tip to the cell body
Che-Chia Tsao and Martin A. Gorovsky*
Department of Biology, University of Rochester, Rochester, NY 14627, USA
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 12 November 2007
J. Cell Sci. 121, 428-436 Published by The Company of Biologists 2008
doi:10.1242/jcs.015826
Summary
Intraflagellar transport (IFT) moves multiple protein particles
composed of two biochemically distinct complexes, IFT-A and
IFT-B, bi-directionally within cilia and is essential for cilia
assembly and maintenance. We identified an ORF from the
Tetrahymena macronuclear genome sequence, encoding
IFT122A, an ortholog of an IFT-A complex protein.
Tetrahymena IFT122A is induced during cilia regeneration, and
epitope-tagged Ift122Ap could be detected in isolated cilia.
IFT122A knockout cells still assembled cilia, albeit with lower
efficiency, and could regenerate amputated cilia. Ift172p and
Ift88p, two IFT-B complex proteins that localized mainly to
basal bodies and along the cilia in wild-type cells, became
Introduction
Cilia/flagella are present in most eukaryotic organisms, including
protozoans and humans, and share a highly organized and
conserved microtubule-based structure, the axoneme (Sleigh,
1974). Intraflagellar transport (IFT) is a bi-directional process that
moves particles along the axonemal microtubule tracks within the
cilia/flagella and plays an essential role in cilia assembly and length
maintenance (Kozminski et al., 1993; Marshall and Rosenbaum,
2001; Rosenbaum and Witman, 2002). Anterograde IFT, mediated
by kinesin-2 motor proteins, moves IFT particles and their cargos
from the cell body to the ciliary tip where the motor-cargo-IFT
complexes dissociate from the microtubules, interact with ciliary
tip proteins, change their configuration and probably exchange
their cargo (Iomini et al., 2001; Kozminski et al., 1995; Pedersen
et al., 2006; Pedersen et al., 2005). Retrograde IFT, mediated by
cytoplasmic dynein 1b, returns the modified complex from the
ciliary tip back to the cell body (Pazour et al., 1999; Porter et al.,
1999; Signor et al., 1999). When the turnaround step or retrograde
transport is impeded, ciliary proteins fail to return to the cell body
and accumulate at the distal region (Hou et al., 2004; Pazour et al.,
1998; Schafer et al., 2003).
IFT particles in the green alga Chlamydomonas reinhardtii
contain multiple proteins that can be separated into two complexes
using sucrose gradient centrifugation (Cole, 2003; Cole et al., 1998).
IFT complex A (IFT-A) contains five proteins including IFT140,
122A and 122B. IFT complex B (IFT-B) is composed of at least 11
proteins including IFT172, 88, 57 and 52. The physiological
significance of the two complexes is not fully understood, but recent
data suggest that they have distinct roles (Scholey, 2003). Numerous
loss-of-function mutant strains of IFT-B genes have been obtained
preferentially enriched at the ciliary tips in IFT122A knockout
cells. Our results indicate that Tetrahymena IFT122A is not
required for anterograde transport-dependent ciliary assembly
but plays a role in returning IFT proteins from the ciliary tip
to the cell body.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/121/4/428/DC1
Key words: Intraflagellar transport, Cilia/flagella, Ciliary assembly,
WD domain protein, Gene targeting
in Chlamydomonas, Tetrahymena, Caenorhabditis elegans,
Drosophila, zebrafish and mouse by forward genetic screening or
reverse genetic manipulation (Beales et al., 2007; Brazelton et al.,
2001; Brown et al., 2003; Deane et al., 2001; Follit et al., 2006; Han
et al., 2003; Haycraft et al., 2003; Hou et al., 2007; Huangfu and
Anderson, 2005; Pazour et al., 2000; Qin et al., 2001; Qin et al.,
2007; Sun et al., 2004). The common phenotype of these IFT-B
mutants is loss or abnormal shortening of cilia/flagella, arguing that
IFT-B proteins function in anterograde IFT and play an essential role
in cilia assembly (Scholey, 2003). Fewer mutant phenotypes of the
IFT-A genes have been described. For example, morpholino
knockdown of zebrafish IFT140 does not lead to the sensory
neuronal defect observed in IFT-B gene knockdown strains
(Tsujikawa and Malicki, 2004). The C. elegans mutations in CHE11/IFT140 and DAF-10/IFT122A cause the diameter of neuronal
cilia to become irregular and electron-dense material to accumulate
(Perkins et al., 1986). The Chlamydomonas retrograde IFT mutant
strains fla15, fla16 and fla17-1 all lack the same two IFT-A proteins
in the flagella (Iomini et al., 2001), but the actual mutated genes are
not yet identified. These observations suggest a correlation between
retrograde IFT and IFT-A complex proteins, but this relationship has
not yet been directly demonstrated.
The ciliated protozoan Tetrahymena thermophila possesses
hundreds of motile oral and somatic cilia with the canonical ciliary
structure found in most organisms (Gaertig, 2000). With the
recently available macronuclear genome sequence (Eisen et al.,
2006) and facile gene knockout and replacement methods (Hai et
al., 2000), Tetrahymena is an ideal model system to elucidate the
function of conserved ciliary genes. In this paper, we report
characterization of an IFT-A gene, IFT122A, in this organism. We
429
Tetrahymena IFT122A
identified an IFT122A ortholog from the Tetrahymena genome
database and generated IFT122A knockout strains. Phenotypic
studies revealed that, while IFT122A is not essential for cilia
assembly, it is required for the efficient return of IFT proteins from
the ciliary tip to the cell body. Our results strongly support the
hypothesis that IFT-A is involved in retrograde IFT.
A
IFT80
Hs Wdr56
Ce Che-2
Tt 225.m00042
Dm OSEG5
Tt IFT172
Ce Osm-1
Dm OSEG1
IFT122A
Hs SLB
Hs Wdr10
Tetrahymena IFT122A is induced during cilia regeneration
To determine whether the IFT122A gene was involved in ciliary
function, we first investigated whether its expression is induced
during regeneration after cilia are amputated, which is a property
of previously described Tetrahymena ciliary genes (Guttman and
Gorovsky, 1979). Northern blotting showed that in growing and
Journal of Cell Science
Dm OSEG2
957
788
Ce Daf-10
Results
Tetrahymena IFT122A is a conserved gene
Compared with studies on IFT-B proteins, little is known about the
physiological role of IFT-A proteins. To explore the functional
implication of an IFT-A protein in typical motile cilia, we used
reverse genetic techniques to study IFT122A in Tetrahymena.
IFT122A is the smallest IFT-A protein whose sequence was
available in the GenBank database, allowing us to search for
possible orthologs encoded in the recently sequenced Tetrahymena
macronuclear genome. Using the C. elegans DAF-10/IFT122A as
the query sequence, we found a predicted open reading frame
(ORF), 107.m00117, showing strong sequence homology (E-value
6.9e-100). Although the putative Tetrahymena IFT122A homolog
appears to be a single copy gene, the Tetrahymena genome contains
other genes with weak homology to it. Furthermore, several IFT
proteins, as well as OSEG proteins identified by a computational
genomic comparison and shown to be involved in the formation of
the neuronal cilium in Drosophila (Avidor-Reiss et al., 2004), share
similar domain features with IFT122A. To exclude the possibility
that the gene we identified is not the ortholog of IFT122A but one
of the other IFT or OSEG genes, we searched the Tetrahymena
genome database to find other similar sequences and performed a
phylogenetic analysis. The gene encoding 107.m00117 was the
only one that clearly grouped with the IFT122A orthologs from
other species on the un-rooted tree (Fig. 1A), arguing that it is the
Tetrahymena IFT122A and we have named it accordingly. Single
Tetrahymena genes from the other IFT/OSEG clades (Avidor-Reiss
et al., 2004; Cole, 2003) were also identified.
Further determination of Tetrahymena IFT122A coding
sequence by sequencing the cDNA and 5⬘ rapid amplification of
the cDNA end revealed that the actual first exon and intron were
missing in the predicted ORF and several splicing sites were also
wrongly predicted, causing in-frame insertions. Based on the
cDNA sequence, we deduced a revised ORF containing 1230
amino acid residues and encoding a 142 kDa protein with a pI of
~7.6. It shows 25% identity and 38% similarity to DAF-10, 32%
identity and 54% similarity to Chlamydomonas predicted protein
C510064, and 33% identity and 55% similarity to WDR10, the
closest human ortholog (Gross et al., 2001). As illustrated in Fig.
1B, a protein secondary structure analysis predicts that the Nterminal half of the protein forms ␤ strands while the rest of the
protein contains mainly ␣ helical structures and short loops.
Domain analysis identified six WD40 motifs in the ␤ strand-rich
N-terminal region (Fig. 1B, yellow boxes) and two highly
degenerate repeat units (Fig. 1B, green boxes; also see alignment
in Fig. 1C) in the C-terminal region. This two-domain organization
is similar to that of several other IFT proteins and OSEG genes.
1000 548948
1000 1000
1000
998
1000
1000
1000
768
735
956
Tt IFT122A
107.m00117
999
Hs Wdr35
Tt 230.m00035
Dm OSEG6
420
541
Hs Wdr19
Dm OSEG4
Ce 30425610
OSEG4
OSEG6
Tt 23.m00255
Tt 18.m00435
Ce IFTA-1
Dm OSEG3 Ce Che-11
Hs 14336756
IFT140
0.1
B
IFT172
Primary structure analysis:
WD40
TPR-like
Secondary structure prediction:
Beta-rich
C
Alpha-rich
Residue number:
719-752
963-996
QGNWKKGGELYIQAEVYKKAIEVYVTNNFSEGIV
SYLYYSLGKVSKQLEGYKTARICYEKLASYKIPT
G
Fig. 1. Sequence analysis of the Tetrahymena IFT122A gene. (A) An unrooted neighbor-joining tree of IFT122A and other IFT and OSEG proteins,
which share similar domain organization. Ce, Caenorhabditis elegans; Dm,
Drosophila melanogaster; Hs, Homo sapiens; Tt, Tetrahymena thermophila.
The number at the branches indicates the value of 1000 bootstraps.
(B) Primary and secondary structure analysis of Ift122Ap. Yellow boxes
indicate six WD motifs, and green boxes show two degenerate TRP-like repeat
units. Secondary structure prediction shows two distinct regions of Ift122Ap:
an N-terminal ␤-strand-rich region (yellow) and an ␣-helix-rich region
(green). (C) Sequence alignment of the degenerate repeat units on Ift122Ap.
Identical residues are shaded.
starved cells, IFT122A was expressed at a very low level. After 1
hour of cilia regeneration, however, the IFT122A RNA level was
highly elevated (Fig. 2A, upper panel). Thus, similar to tubulin
(Guttman and Gorovsky, 1979) and to other Tetrahymena IFT
genes such as IFT52 (Brown et al., 2003) and IFT172 (Tsao and
Gorovsky, 2008), the expression of IFT122A can be induced during
cilia regeneration, suggesting that IFT122A is a ciliary gene.
HA-tagged Ift122Ap localizes in isolated cilia
To determine if IFT122A indeed encodes a ciliary protein, we
constructed an IFT122A-HA strain in which Ift122Ap fused with
a hemagglutinin (HA) epitope at the C-terminus was expressed
from its endogenous locus. Unfortunately, we could not detect any
signal by immunofluorescence staining using anti-HA antibody
with various fixation methods. As an alternative approach to
examine whether Ift122Ap-HA was present in the cilia, we
extracted proteins from isolated cilia and cell body fractions and
analyzed them by immunoblotting using anti-HA antibody. We
observed a 140 kDa band from the cilia fractions prepared from
the IFT122A-HA cells, but no band was detected in the cell body
fraction (Fig. 2B, upper panel). As controls, the cilia and cell body
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Fig. 2. IFT122A is a ciliary gene. (A) Northern analysis of IFT122A
expression during cilia regeneration. CU428 wild-type cells were deciliated
and allowed to regenerate cilia. Total RNAs extracted from log phase cells and
from the cells collected at 0, 1 and 2 hours in the recovery buffer were probed
with isotope-labeled IFT122A cDNA. The upper panel shows that IFT122A
transcripts were highly induced during cilia regeneration in wild-type cells.
The lower panel shows the ethidium bromide staining as loading control.
(B) Detection of HA-tagged Ift122Ap in cilia by immunoblotting. Total
proteins from cilia and cell body fractions were isolated from HA-tagged
IFT122A, HA-tagged IFT172, and wild-type CU428 strains. In the upper
panel, the blot was probed with anti-HA monoclonal antibody. A 140 kDa
protein and 190 kDa protein were detected in the cilia fraction (Cil) from
IFT122A-HA and IFT172-HA cells, respectively. In the lower panel, the same
blot was re-probed with a control antibody against polyglycine. The
cytoplasmic protein Pgp1p, shown as multiple high-molecular mass bands,
was only present in the cell body fraction (CB).
fractions isolated from cells expressing Ift172p-HA and wild-type
CU428 cells were also probed with anti-HA antibodies. A 190 kDa
band was detected in the cilia fraction from IFT172-HA cells, and
no specific band was observed in the either fraction in CU428 cells
(Fig. 2B, upper panel). These results indicate that the 140 kDa band
we observed in the cilia fraction from the IFT122A-HA cells is
specific and co-fractionates with Ift172p, a protein previously
demonstrated by immunofluorescence to localize to cilia (Tsao and
Gorovsky, 2008) (also see Fig. 5). To assess the purity of the two
fractions, we re-probed the same blot with a control antibody
previously shown to detect Pgp1p, a cytoplasmic protein that
localizes to the endoplasmic reticulum (Xie et al., 2007). Ppg1p
appeared as multiple bands and could only be detected in the cell
body fraction but not in the cilia fraction (Fig. 2B, lower panel).
These results argue strongly that Ift122Ap is a ciliary protein. The
failure to detect a ciliary signal by fluorescence microscopy is
probably because the protein concentration is too low or the epitope
tag on Ift122Ap-HA is masked so that it is not accessible to the
antibody.
Ciliary assembly in the IFT122A knockout cells is affected but
not abolished
To explore the in vivo function of IFT122A, we created
Tetrahymena IFT122A germline knockout strains (Hai et al., 2000).
In these cells, the IFT122A coding sequence was disrupted with a
neo3 cassette in both the germline micronucleus and somatic
macronucleus. The genotype of the knockout cells was examined
by PCR using a locus-specific primer outside the targeting
Fig. 3. IFT122A knockout cells. (A) Strategy to generate knockout cells. The
wild-type IFT122A locus was homologously targeted by the disruption
construct, replacing most of the coding region with a neo3 cassette in the
knockout locus. Arrowheads indicate the primers that were used in the PCR
assay to determine the genotype. (B) PCR analysis of the genotype. Genomic
DNAs extracted from CU428 strains and from IFT122A knockout cells were
analyzed using a forward primer and two locus-specific primers. In the wildtype cells (WT), a 1.8 kb band was obtained, while in the IFT122A knockout
(KO), only the 2.4 kb corresponding to the knockout locus could be amplified.
(C) Ink uptake assay to test the function of oral cilia. IFT122A knockout cells
can ingest ink particles to form black food vacuoles. Scale bar: 10 ␮m.
(D) The proportion of cells that formed black food vacuoles at different time
points. Open squares, CU428 cell; filled (red) circles, IFT122A knockout cells.
(E-G). Immunostaining of IFT172 knockout (E), IFT122A knockout (F) and
CU428 (G) cells using anti-tubulin antibody. The arrowhead indicates the
anterior end of the cell. Scale bars, 10 ␮m.
construct and an allele-specific primer to either the wild-type
IFT122A allele or the knockout construct (Fig. 3A, primer sites
shown as arrow heads). In the wild-type cells, a predicted 1.8 kb
fragment corresponding to the undisrupted sequence was
amplified. In the IFT122A knockout cells, however, only a 2.4 kb
band that was specific to the knockout allele was amplified under
the same reaction conditions (Fig. 3B). These results confirmed
that the construct correctly targeted to the IFT122A locus and
replaced all copies of the endogenous wild-type allele in both
nuclei in the knockout cells.
The phenotypes of the IFT122A knockout cells clearly differed
from those previously described for either IFT52 (Brown et al.,
2003) or IFT172 knockout cells (Tsao and Gorovsky, 2008).
Disrupting one of these two IFT-B complex proteins in
Tetrahymena causes cells to quit swimming and become almost
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Tetrahymena IFT122A
431
Fig. 4. Distal tip localization of Ift88p-GFP in the IFT122A knockout cells. (A) Strategy to introduce IFT88-GFP constructs into the wild-type cells or IFT122A
knockout cells. (B) Ift88p-GFP fluorescence in the wild-type cells. (C) Enlarged image from the boxed region in B. Ift88p-GFP localized to the cilia in oral
apparatus (OA), along the somatic cilia (arrowheads) and to the basal body rows (arrows). (D) Ift88p-GFP fluorescence in the IFT122A knockout cells. (E) Ift88pGFP fluorescence merged with DIC images. (F,G), Enlarged images from the boxed region in E. In the IFT122A knockout background, Ift88p-GFP became
enriched at the distal tips of oral cilia (OA) and somatic cilia (arrowhead). Scale bars, 10 ␮m.
completely paralyzed. By contrast, the IFT122A knockout cells
could still swim. Close comparison with the wild-type cells
revealed that IFT122A knockout cells swam more slowly than the
wild-type cells (data not shown), suggesting that the somatic cilia
were affected. To test oral cilia function, we compared the ability
of the wild-type and IFT122A knockout cells to ingest ink particles,
which requires motile oral cilia to generate flow currents. Like the
wild-type cells, IFT122A knockout cells could ingest ink particles
and formed black food vacuoles (Fig. 3C) but did so more slowly
than the CU428 wild-type cells (Fig. 3D). This observation
suggests that the oral cilia can be assembled but function less
efficiently than wild-type oral cilia.
To compare the morphological differences between wild type,
IFT122A knockout and IFT172 knockout cells, we performed
immunostaining using anti-tubulin antibodies to visualize cilia.
While IFT172 knockout cells lacked or only had extremely short
cilia (Fig. 3E), IFT122A knockout cells still possessed long oral
and somatic cilia (Fig. 3F). The numbers of the cilia in IFT122
knockout cells, however, were fewer than those in the wild-type
cells (Fig. 3G). In addition, cilia length and distribution were less
homogeneous in IFT122A knockout cells than in wild-type cells,
with the density of cilia in the IFT122A knockout cells being higher
at the anterior (Fig. 3F, arrowhead) than in the rest of the cell. All
of these observations show that, while IFT122A clearly affects
cilia, unlike IFT52 and IFT172, it is not essential for cilia assembly
in Tetrahymena.
Deletion of IFT122A causes preferential localization of Ift88pGFP at the ciliary tips
Cilia assembly is dependent on anterograde IFT transport,
indicating that IFT122A is not required for this process. To
determine whether deletion of IFT122A affected retrograde
transport, we compared the distribution of another IFT protein in
cells with or without Ift122Ap. We created an IFT88-GFP targeting
construct carrying a cycloheximide-resistant selectable marker and
used it to transform both wild type and IFT122A knockout cells.
When the tagged strains were cultured with cycloheximide, a
number of the endogenous IFT88 genes in the polyploid
macronuclear genome were replaced by the introduced GFPtagged version, enabling the Ift88–GFP fusion protein to be
detected (Fig. 4A).
In wild-type cells (Fig. 4B,C), the Ift88p-GFP mainly localized
to basal bodies (Fig. 4C, arrow) and along the cilia (Fig. 4C,
arrowhead), especially in the oral cilia (Fig. 4B, OA). In the
IFT122A knockout cells (Fig. 4D-G), however, Ift88p-GFP was
rarely observed in basal bodies or along the cilia. Instead, strong
fluorescence was detected at the distal tip region of cilia (Fig. 4F,G,
arrowhead). The tip localization of Ift88p-GFP was not detected in
every cilium but was especially prominent at the tips of oral cilia.
(Fig. 4F, OA). These observations suggest that when IFT122A is
deleted, Ift88p (an IFT-B component) is retained at the distal tip of
the cilia, either because it is released from the complex and/or
because the entire complex inefficiently interacts with the
retrograde IFT machinery. Therefore, although IFT122A is not
essential for anterograde transport, it plays a role in returning Ift88p
from the tip to the base of the cilia.
Deletion of IFT122A causes a preferential accumulation of fulllength Ift172p-HA at the ciliary tips
In our strategy to introduce IFT88-GFP into IFT122A knockout
cells, both the native and GFP-fusion Ift88p were present in the
same cells, and we could not rule out the possibility that Ift88pGFP might behave differently from the untagged, endogenous
forms. To determine the effect of IFT122A on a complex B
protein but avoid this concern, we used an alternative strategy to
investigate the localization of another IFT protein, Ift172p, in the
IFT122A knockout cells (Fig. 5A). We generated IFT122A,
IFT172 double knockout cells by crossing the two single
knockout heterokaryon strains and made the cells homozygous
for both loci in both nuclei (Hai et al., 2000). The double
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Journal of Cell Science
should, therefore, localize like the untagged, wild-type
protein.
Using this knock-in strategy, we compared the
localization of Ift172p-HA by anti-HA antibody staining
in cells with intact IFT122A (Fig. 5C,D) or with IFT122A
deleted (Fig. 5E-H). Similar to what we observed with
Ift88p-GFP, Ift172p-HA accumulated in the ciliary tips on
some cilia when IFT122A was disrupted (Fig. 5E,F,
arrowhead). This is clearly distinct from Ift172p
localization in the cells with wild-type IFT122A, in which
Ift172p-HA associated mainly with basal bodies and
localized along the cilia, with more intense staining at the
proximal region (Fig. 5C,D). In some case, the staining of
the Ift172p-HA was observed in close proximity to the
ciliary tip (Fig. 5G,H, see Discussion). Our results show
that, when IFT122A is disrupted, more than one IFT
complex B protein accumulates at or close to the distal
region of the ciliary tip instead of localizing predominately
to the basal body and along the cilia.
IFT122A knockout cells can regenerate cilia after cilia
amputation and accumulate IFT proteins at the ciliary
tip
To analyze how IFT122A affects IFT, we deciliated
IFT122A knockout cells carrying HA-tagged IFT172. If
IFT122A is not essential for anterograde IFT, one
prediction is that, after cilia are amputated, cilia
regeneration should occur and the progress of cilia
regeneration should not be dramatically affected. As
predicted, when the deciliated IFT122A knockout cells
were transferred into regeneration buffer, the cells did
regenerate cilia (Fig. 6). Although the heterogeneity in
cilia length and numbers in IFT122A knockout cells
makes it difficult to compare with the regeneration
kinetics of the wild-type cells, we observed short nascent
Fig. 5. Distal tip localization of Ift172p-HA in the IFT122A knockout cells. (A) Strategy
to generate tagged strains with IFT172-HA knocked-in to the endogenous locus.
cilia in IFT122A knockout cells at 20 minutes (Fig. 6A(B) Growth curves of the CU428 wild-type cells and IFT knockout cells.
C, arrowhead), and the regeneration proceeded (Fig. 6D(C-H) Immunofluorescence staining of the cells with wild-type IFT122A (C,D) or with
F) until after 60 minutes (Fig. 6G-I). Similar to the wildIFT122A deleted (E-H). Cells were stained with anti-HA monoclonal antibody (red) and
type cells, newly formed long cilia could be observed in
anti-tubulin antiserum (green), and D, F and H are merged images of Ift172p-HA and
tubulin staining. Ift172p-HA became enriched at the distal region of some cilia (arrow).
the IFT122A knockout cells and the cells regained
Scale bars, 10 ␮m.
motility.
We also examined the localization of HA-tagged
Ift172p during cilia regeneration in the IFT122A
knockout cells lost normal motility and could not assemble
knockout cells. Ift172p-HA became localized in the nascent
normal length cilia. The morphology of the double knockout cells
cilia after 20 minutes of regeneration (Fig. 6B,C, arrow). Since
was indistinguishable from the IFT172 single knockout cells.
the nascent cilia are still short, it is difficult to discriminate any
Both IFT172 single knockout and IFT122A, IFT172 double
uneven distribution of Ift172p along the cilia if it occurred at
knockout strains had similar doubling times (~11–12 hours),
this time. However, by 40 minutes, when cells had longer cilia,
longer than the wild type or IFT122A single knockout cells (Fig.
we observed Ift172p accumulation in the distal regions of the
5B). Thus, IFT172 is epistatic to IFT122A as would be expected
cilia in IFT122A knockout cells (Fig. 6E,F, arrow). After 60
given that IFT172 encodes an IFT-B protein involved in
minutes of regeneration, the preferential distal localization of
anterograde transport, a step that precedes retrograde transport in
Ift172p in the IFT122A knockout cells became more prominent
which IFT122A has a predicted role. After the double knockout
and could be easily observed (Fig. 6H,I, arrow). This
strain was obtained, we then used a C-terminal HA-tagged
experiment confirms that the IFT122A knockout cells can
IFT172 construct to rescue the non-motile double knockout cells
assemble new cilia after cilia amputation and that the
and identified rescued strains based on their recovery of cilia and
preferential localization of IFT proteins at the tips occurs in the
motility. With this strategy, we ‘knocked in’ an HA-tagged
newly formed cilia. Collectively, these results argue that
IFT172 at its endogenous locus so that all the Ift172p produced
deletion of IFT122A does not abolish the anterograde IFT
in the rescued cells carried the epitope tag. Most importantly, the
process required for transporting building blocks into the cilia.
rescued cells swam and grew cilia like IFT122A single knockout
Instead, deletion of IFT122A affects the return of proteins from
cells, indicating that the HA-tagged Ift172p was functional and
the ciliary tip to the cell body.
Tetrahymena IFT122A
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IFT-A and IFT-B proteins have distinct functions
Although IFT proteins can be biochemically separated into
two complexes, IFT-A and IFT-B, they move coordinately
in the same particle in vivo (Ou et al., 2005; Qin et al.,
2001). This observation raises two questions: are IFT-A and
IFT-B functionally distinct complexes? What are their
respective roles in vivo?
Comparing the loss-of-function phenotypes of IFT-A and
IFT-B mutants in the same system addresses the first
question. Using Tetrahymena as the model, we and others
performed three knockout studies on IFT genes, including
one IFT-A gene, IFT122A (this study), and two IFT-B
genes, IFT52 (Brown et al., 2003) and IFT172 (C.-C. Tsao
and M. A. Gorovsky, submitted), enabling a direct
comparison of IFT-A and IFT-B gene functions in typical
motile cilia. We found that disruption of an IFT-A gene
shows a distinct phenotype from disruption of IFT-B genes
in Tetrahymena. Both IFT52 and IFT172 knockout cells
cannot assemble cilia or assemble only very short cilia, and
the knockout cells become virtually paralyzed (Brown et
al., 2003). The IFT122A knockout cells, however, still
assemble oral and somatic cilia during growth and
regenerate cilia after cilia amputation. The cells remain
motile although they swim more slowly. These results
clearly demonstrate that IFT-A and IFT-B proteins are
differentially involved in the assembly of motile cilia.
Other evidence suggests that IFT-A and IFT-B proteins
also play different roles in non-motile neuronal sensory
cilia. The cilia in C. elegans, IFT-B mutants osm-1/IFT172,
osm-5/IFT88, osm-6/IFT52 and che-13/IFT57 do not
extend beyond the transition zones and have shortened
axonemes, while the IFT-A mutants che-11/IFT140 and
daf-10/IFT122A have irregular sized axonemes and their
cilia are filled with material in the middle (Perkins et al.,
Fig. 6. Localization of Ift172p-HA during cilia regeneration in the IFT122A knockout
1986). In zebrafish, mutated IFT88 or morpholino
cells. IFT122A knockout cells with HA-tagged IFT172 were deciliated by pH shock
knockdown of IFT52 and IFT57, three IFT-B genes, cause
and allowed to regenerate cilia. Cells collected after 20 minutes (A-C), 40 minutes
(D-F) and 60 minutes (G-I) in the regeneration buffer were fixed and stained with antidefects in the maintenance of sensory cilia and lead to the
tubulin (green) and anti-HA antibody (red). The Ift172p-HA first appeared in nascent
degeneration of photoreceptors, olfactory sensory cells and
cilia and gradually became enriched at the tips of some cilia. Arrowhead, nascent cilia
auditory hair cells. When IFT140, an IFT-A gene, is
at 20 minutes. Arrow, the Ift172p-HA in the nascent cilia (C) and accumulation at the
knocked down, however, only the cilia in the anterior nasal
tips of cilia (F and I). Scale bar, 10 ␮m.
pit are significantly affected and the phenotype is weaker
and infrequent (Tsujikawa and Malicki, 2004). Together,
Discussion
these studies and our results argue that IFT-A and IFT-B proteins
IFT122A encodes a ciliary protein
have distinct functions in both canonical motile cilia and in nonSeveral lines of evidence argue that the gene we identified in the
motile sensory cilia derivatives.
Tetrahymena macronuclear genome is truly the IFT122A ortholog
An IFT-A protein is important for returning IFT proteins from
and encodes a ciliary protein. First, it encodes a protein that
the ciliary tip to the cell body
shares high sequence homology with those encoded by C. elegans
The different function of IFT-A or IFT-B proteins was initially
and human IFT122A orthologs and has a domain organization
inferred by comparative phenotypic studies among IFT mutants
characteristic of other IFT-A proteins (Cole, 2003; Jekely and
in different models. IFT-B proteins have been shown to be
Arendt, 2006). Phylogenetic analysis places this candidate
important for cilia formation in several organisms, indicating that
protein with IFT122A protein orthologs from other species and
they are essential for the anterograde process (for a review, see
distinguishes it from other IFT proteins with similar domain
Scholey, 2003). Chlamydomonas fla15, fla16 and fla17, three
organization. Second, IFT122A expression is induced during cilia
temperature-sensitive mutant strains in retrograde IFT, are
regeneration, a feature of ciliary genes (Guttman and Gorovsky,
deficient for the same two IFT-A proteins in the flagella at the
1979; Lefebvre and Rosenbaum, 1986). Third, the HA-tagged
restrictive temperature (Piperno et al., 1998). This correlation
protein encoded by this gene is detected in the isolated ciliary
between the absence of some IFT-A proteins and a defect in the
fraction. Fourth, disruption of this gene affects cilia and alters the
retrograde transport led Piperno et al. (Piperno et al., 1998) to
localization of two other IFT proteins. All these results argue
hypothesize that IFT-A is responsible for retrograde transport,
strongly that the putative IFT122A ortholog encodes a ciliary
although other interpretations were also possible. Here we deleted
protein that functions in IFT.
434
Journal of Cell Science 121 (4)
one of the IFT-A genes in Tetrahymena and demonstrated an
effect on retrograde transport of two other IFT proteins to provide
a direct verification of this hypothesis.
In Tetrahymena IFT122A knockout cells, Ift88p and Ift172p
became enriched at the distal region of the ciliary tip. The
accumulation of IFT proteins could be due to a defect in retrograde
transport, since the phenotype is similar to loss-of-function of a
heavy chain or of a light intermediate chain of the cytoplasmic
dynein retrograde motor in Chlamydomonas and C. elegans
(Pazour et al., 1999; Schafer et al., 2003; Signor et al., 1999). In
these mutants, dense materials accumulated and a bulge appeared
at the flagellar/ciliary tip. Accumulations of IFT proteins in cilia
were also reported in C. elegans IFT-A mutants che-11/IFT140 and
daf-10/IFT122A (Perkins et al., 1986). Another gene, ifta-1, which
phenocopied retrograde IFT defects when it was mutated, is a
strong candidate for an uncharacterized IFT-A gene, IFT122B
(Blacque et al., 2006). These results from Tetrahymena and C.
elegans collectively argue strongly that the IFT-A complex plays
an important role in retrograde IFT.
transition at the tip region. The very distal tip of Tetrahymena
cilia only contains the A-tubule of axonemal microtubule
doublets (Sale and Satir, 1976). This configuration is similar to
the outer segment of C. elegans sensory cilia, and Osm-3 motors
are essential to assemble this portion (Snow et al., 2004). In
IFT122A knockout cells, in some cases we observed the
accumulation of Ift172p in proximity to the ciliary tip, probably
at the transition to the region containing the A-tubule singlet. This
suggests a possible functional interaction between Ift122Ap,
Ift172p, Osm-3 motors and/or ciliary tip proteins at the distal
ciliary region where the cilia assembly and IFT particle
remodeling take place. The utilization of two anterograde motors
may contribute to neuronal cilia diversity in C. elegans (Evans et
al., 2006), and the IFT machinery was proposed to utilize a
combination of different ‘functional modules’ such as IFT-A/B,
BBS complex and motor proteins (Ou et al., 2007). Whether a
similar mechanism is also used in other species and the
physiological significance of using different complexes to
transport cilia/flagella proteins in the same direction remains to
be further studied.
Journal of Cell Science
Is IFT122A also involved in other IFT processes?
IFT122A knockout cells still assemble cilia, but in fewer numbers
and with less length homogeneity. One explanation is that
disruption of IFT122A causes accumulation of IFT-B proteins at
the ciliary tips, which eventually perturbs anterograde IFT particle
assembly. IFT122A could also have additional, non-essential roles
in anterograde transport and/or other IFT processes.
Recent studies using C. elegans as a model showed that IFT-A
and IFT-B are moved in the same particle in anterograde transport
(Ou et al., 2005). In the sensory cilium, anterograde IFT is
dependent on two kinesin-2 motors, heterotrimeric kinesin-II and
homodimeric kinesin Osm-3, whose functions are partially
redundant in some sensory neurons but are completely redundant
in others (Evans et al., 2006; Ou et al., 2005; Snow et al., 2004).
Interestingly, IFT-A is moved by kinesin-II and the IFT-B is carried
by Osm-3, respectively, and the concerted movement of the whole
particle also requires the non-kinesin proteins BBS-7/8 (Ou et al.,
2005). Tetrahymena kinesin-II and Osm-3 orthologs have been
cloned (Awan et al., 2004; Brown et al., 1999), and knockout of
the kinesin-II alone is sufficient to abolish cilia assembly (Brown
et al., 1999), indicating that the two types of kinesin-2 are not
functionally redundant in Tetrahymena. If this two-kinesin
mechanism is conserved in other species, such as Tetrahymena, one
prediction is that IFT-A also has a role in the anterograde transport
since it mediates the kinesin-II movement of the whole IFT
particle. Our knockout results imply that a complete IFT-A
complex is not essential for kinesin-II to move the IFT particles,
but the kinesin-II–IFT interaction could still be partially
compromised when IFT122A is deleted so that ciliary assembly is
not as efficient as in the wild-type cells.
The phenotype of Tetrahymena IFT122A knockout cells is
similar to Tetrahymena IFT172 mutants with a partial truncation
of the Ift172p C-terminal repeat domain, in which Ift88p and the
truncated Ift172p itself preferentially accumulated at the tips of
the cilia (C.-C.T. and M.A.G., submitted). Chlamydomonas
IFT172 has been implicated in anterograde/retrograde IFT
turnaround at the flagellar tip, probably through interaction with
the flagellar tip protein EB1 (Pedersen et al., 2006; Pedersen et
al., 2003; Pedersen et al., 2005). Given similarities between
IFT122A knockout and IFT172 partial truncation mutant cells, it
is possible that IFT122A also mediates the anterograde/retrograde
Materials and Methods
Tetrahymena strains, culture growth, and conjugation
Wild-type CU428 and B2086 strains of Tetrahymena thermophila were provided by
Dr Peter Bruns (Cornell University). Cells were grown in 1⫻ SPP medium
(Gorovsky et al., 1975) containing 1% peptone at 30°C, except that IFT172 knockout
and IFT122A, IFT172 double knockout cells were grown in MEPP medium (Orias
and Rasmussen, 1976). For starvation, mid-log-phase cells were washed and
resuspended in 10 mM Tris-Cl (pH 7.5) for 16–20 hours at 30°C. For conjugation,
equal numbers of the starved cells from two different mating types were mixed
together and incubated at 30°C without shaking.
Deciliation and cilia regeneration
Cells were grown to mid-log-phase and starved before deciliation. For cilia
regeneration analysis, the cells were deciliated by pH shock and immediately
incubated in Tris-Cl buffer (pH 7.5) containing 10 mM CaCl2 as previously described
(Calzone and Gorovsky, 1982).
Gene cloning and sequence analysis
The Tetrahymena IFT122A gene was identified by a BLAST search against the
Tetrahymena Genome Database (TGD, http://www.ciliate.org). A gene encoding
predicted protein, 107.m00117, was identified using the C. elegans DAF-10 sequence
(GI 133930820) as the query sequence. To verify the predicted coding sequence, total
RNA was extracted from the cells at 1 hour after deciliation using Trizol reagent
(Invitrogen). The cDNA was reverse transcribed by both the random hexamer and
d(T)17 primer using SuperScript II (Invitrogen) following manufacturer’s instructions
and amplified by PCR using specific primers based on the 107.m00117 sequence.
The 5⬘ end of the cDNA was determined by an RLM-RACE kit (Ambion) following
manufacturer’s instructions. The IFT122A ORF (Genbank EF601830) was deduced
from the cDNA sequence.
The consensus secondary structure was predicted by NPS@PBIL-IBCP
(http://npsa-pbil.ibcp.fr). The protein motif and repeat identification were analyzed
by SMART (Schultz et al., 1998) and REP v1.1 (Andrade et al., 2000), respectively.
To construct the phylogenetic tree, full-length protein sequences were aligned by
CLUSTALX (Thompson et al., 1997) using default parameters (see supplementary
material Tables S1 and S2 for the accession numbers of the sequences analyzed).
After the initial alignment, large gaps and un-aligned terminal sequences were
removed as described (Hall, 2001) to refine the alignment, and the distance was
calculated by the neighbor-joining method. The bootstrap values were calculated by
1000 re-samplings.
Germline gene knockout
The 5⬘ and 3⬘ flanking regions for homologous recombination were amplified from
CU428 genomic DNA. Overlapping PCR (Kuwayama et al., 2002) was used to join
the flanking regions and the neo3 cassette (Shang et al., 2002) to make the knockout
construct. The crude PCR products, mixed with EcoRI linearized pBluescript DNA
as carriers, were introduced into the conjugating CU428 and B2086 cells by biolistic
particle bombardment 3 hours after mixing as described (Cassidy-Hanley et al.,
1997). The germline transformants were selected, assorted and made homozygous
for the knockout allele in the micronucleus as described (Hai et al., 2000). After the
heterokaryon cells were mated, individual conjugating pairs were isolated between
6 and 10 hours post-mixing and transferred into drops of 1⫻ SPP medium on a Petri
Tetrahymena IFT122A
dish. These progeny were examined with an Olympus SZH-ILLD dissecting
microscope.
To generate IFT122A, IFT172 double knockout cells, the IFT122A and IFT172
single knockout homozygous heterokaryons (Tsao and Gorovsky, 2008) were
crossed, and the progeny cells were selected, assorted and made homozygous for the
micronuclear genome as previously described (Hai et al., 2000). To test the genotype
after ‘round 1’ genomic exclusion, the double knockout exconjugants were testcrossed with IFT122A and IFT172 single knockout cells. A total of 48 individual
conjugating pairs from each cross were isolated into drops of 1⫻ SPP medium 6
hours after mixing, and the phenotypes of the test-cross progeny cells were examined
using a dissecting microscope.
Genotyping by PCR
To analyze the genotype, genomic DNAs from CU428 and IFT122A knockout cells
were extracted as described (Liu et al., 2004) and used as templates for PCR analysis.
A common forward primer sitting outside the construct and two locus-specific
reverse primers (Fig. 3A, arrowhead) were added in the same PCR reaction (93°C,
30 seconds; 50°C, 30 seconds; 68°C, 3 minutes; 30 cycles).
Northern analysis
Total RNAs were extracted with Trizol reagent (Invitrogen) from mid-log-phase and
from deciliated CU428 cells incubated in cilia regeneration buffer for 0, 1 or 2 hours.
20 ␮g total RNA was resolved on 2.2 M formaldehyde-1% agarose gels and blotted
to MagnaGraph membranes (Osmonics). Hybridization was performed at 42°C
overnight in hybridization buffers containing 50% formamide as previously
described (Dou et al., 2005). The probe was labeled by random priming with ␣[32P]dATP using IFT122A cDNA as the template.
Journal of Cell Science
Ink particle uptake assay
Cells were washed and re-suspended in 10 mM Tris (pH 7.5). A drop of Higgins
India black ink was added to the cell suspension (~1 ␮l ink per ml of cells), and the
cells were kept at 30°C with gentle shaking for at least 30 minutes. Cells were fixed
with 10% formalin and examined using a bright field Olympus BH-2 microscope.
Epitope tagging
To make IFT122A-HA strains, the HA-tagged IFT122A construct was created by
overlapping PCR to introduce an HA-epitope at the C-terminus of Ift122Ap. The
PCR products mixed with the pBluescript II plasmid linearized by EcoRI were
introduced into starved IFT122A knockout cells by biolistic transformation (CassidyHanley et al., 1997). The cells were recovered in SPP medium for 4 hours, distributed
into 96-well microtiter plates, and maintained at room temperature for 2 days,
followed by incubation at 15°C for 3 days and returned to room temperature for
another 2 days. The transformed cells, which swim faster than the IFT122A knockout
cells, were identified by visual screening under an Olympus SZH-ILLD dissecting
microscope.
To introduce Ift172p-HA in the IFT122A knockout background, the HA-tagged
IFT172 constructs were introduced into the IFT172, IFT122A double knockout cells
to rescue the non-motile cells. The double knockout cells were grown to log phase
in the MEPP medium with vigorous shaking and washed with 10 mM Tris (pH 7.5)
before transformation. The IFT172-HA fragments released from the vector were shot
into the cells by biolistic bombardment as previously described (Cassidy-Hanley et
al., 1997). After bombardment, cells were incubated in SPP medium with gentle
shaking for 3 hours before aliquoting into microtiter plates. The rescued swimming
cells were examined under an Olympus SZH-ILLD dissecting microscope after 3
days of transformation.
To make IFT88-GFP strains, a targeting construct containing the IFT88 coding
region (TGD 252.m00027) fused with a GFP sequence at the C-terminus upstream
of the termination codon was created by overlapping PCR. For selecting the
transformant, a rpl29(K40M) gene conferring cycloheximide-resistance (Yao and
Yao, 1991) inserted into a MTT1 promoter-driven cassette with a BTU2 3⬘ terminator
(J. Bowen and M.A.G., personal communication) was included between the GFP and
the IFT88 3⬘ flanking region. The tagged construct was transformed into wild type
or IFT122A knockout cells. After biolistic bombardment, the cells were incubated
with 1⫻ SPP containing 1 ␮g ml–1 CdCl2 for 4 hours at 30°C followed by adding 5
␮g ml–1 cycloheximide into the medium at room temperature overnight. Before
aliquoting the cells into microtiter plates, additional cycloheximide was added to the
medium to make the final concentration 15 ␮g ml–1 . The cells were incubated at
30°C, and the cycloheximide-resistant transformants were observed under a
dissecting microscope after 3–5 days. The transformed cells were passaged every
2–3 days in 1⫻ SPP medium containing 0.5 ␮g ml–1 CdCl2 and 30 ␮g ml–1
cycloheximide for 2 weeks. To observe the Ift88p-GFP localization, the cells were
fixed with 0.05% formaldehyde in 10 mM Tris-Cl (pH. 7.5) at room temperature for
5 minutes, resuspended in phosphate-buffered saline (PBS) and examined using an
Olympus BH-2 fluorescence microscope.
Western blot analysis
IFT122-HA, IFT172-HA and CU428 cells were grown to 2⫻105 cells ml–1, washed
with 10 mM Tris buffer (pH 7.5) and deciliated with dibucaine-HCl as previously
435
described (Johnson, 1986). The cell body fraction was pelleted by low speed
centrifugation at 1500 g for 5 minutes and immediately dissolved in SDS-sample
buffer (5% beta-mercaptoethanol, 10% glycerol, 2% SDS, 60 mM Tris-HCl, pH 6.8).
The supernatant containing the cilia was centrifuged again at the same speed, and
the supernatant was collected by centrifugation at 15,000 g for 20 minutes. The cilia
pellet was washed, re-centrifuged at the same speed for 10 minutes and dissolved in
SDS sample buffer.
Proteins from ~5⫻105 cells were loaded onto 8% SDS-PAGE and transferred onto
an Immobilon-P membrane (Millipore) using a semidry electrophoretic transfer unit
(Bio-Rad). The membrane was blocked in 5% dry skimmed milk in PBS for 30
minutes at room temperature, incubated with anti-HA primary antibody (1:5000) at
4°C overnight and a rabbit anti-mouse IgG HRP-conjugant secondary antibody
(1:5000) at room temperature for 1 hour. The protein signal was detected using
Western Blot Chemiluminescence Reagent (NEN).
For re-probing the blot, the membrane was incubated with 62.5 mM Tris-HCl (pH
6.8), 2% SDS and 100 mM ␤-mercaptoethanol at 55°C for 4 hours and washed
several times with PBS. The membrane was blocked at room temperature for 1 hour,
incubated with rabbit anti-polyglycylation antiserum 2304 (1:5000) at 4°C overnight
and incubated with goat anti-rabbit HRP-conjugant (1:2500) at room temperature for
1 hour.
Immunofluorescent staining
Cells were doubly fixed with 2% paraformaldehyde-0.5% Triton-X100 in PHEM
buffer (Stuart and Cole, 2000) for 1 minute at room temperature and immediately
washed with 1% BSA in PBS and fixed with 45% ethanol-0.5% Triton-X100 on ice
overnight. The fixed cells were spread on poly-L-lysine coated cover glasses and
blocked with 10% normal goat serum and 3% bovine serum albumin in PBS with
0.1% Tween-20 at 37°C for 30 minutes. After blocking, the cells were incubated with
rabbit anti-tubulin antiserum (Van De Water, 3rd et al., 1982) (1:500) and mouse
anti-HA monoclonal antibody (1:500) at room temperature for 1 hour, The samples
were washed in PBS with 0.1% Tween-20 for 15 minutes, three times, followed by
incubation with goat anti-rabbit IgG FITC (1:500) and goat anti-mouse IgG+M Alexa
595 conjugant (1:500) at room temperature for 1 hour. The slides were washed in
PBS with 0.1% Tween-20 for 15 minutes, three times, and mounted with DABCO
and observed with a Leica TCS SP confocal microscope or Olympus BH-2
fluorescence microscope. The images were pseudo-colored and merged by Adobe
Photoshop 7.0.
This work was supported by National Institutes of Health grant GM26973. We thank Josephine Bowen for technical assistance and for
providing the Mrpl29B cycloheximide resistant cassette.
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