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The primary cilium originates from the mother centriole and participates in critical functions during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes. Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert syndromes. Here we generate a Cc2d2a(-/-) mouse that recapitulates features of Meckel syndrome including embryonic lethality and multiorgan defects. Cilia are absent in Cc2d2a(-/-) embryonic node and other somatic tissues; disruption of cilia-dependent Shh signalling appears to underlie exencephaly in mutant embryos. The Cc2d2a(-/-) mouse embryonic fibroblasts (MEFs) lack cilia, although mother centrioles and pericentriolar proteins are detected. Odf2, associated with subdistal appendages, is absent and ninein is reduced in mutant MEFs. In Cc2d2a(-/-) MEFs, subdistal appendages are lacking or abnormal by transmission electron microscopy. Consistent with this, CC2D2A localizes to subdistal appendages by immuno-EM in wild-type cells. We conclude that CC2D2A is essential for the assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for axoneme biogenesis.
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Received 7 Apr 2014 |Accepted 23 May 2014 |Published 20 Jun 2014
Ciliopathy-associated gene Cc2d2a promotes
assembly of subdistal appendages on the mother
centriole during cilia biogenesis
Shobi Veleri1, Souparnika H. Manjunath1, Robert N. Fariss2, Helen May-Simera1, Matthew Brooks1,
Trevor A. Foskett1, Chun Gao2, Teresa A. Longo1, Pinghu Liu3, Kunio Nagashima4, Rivka A. Rachel1,
Tiansen Li1, Lijin Dong3& Anand Swaroop1
The primary cilium originates from the mother centriole and participates in critical functions
during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes.
Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert
syndromes. Here we generate a Cc2d2a !/!mouse that recapitulates features of Meckel
syndrome including embryonic lethality and multiorgan defects. Cilia are absent in
Cc2d2a !/!embryonic node and other somatic tissues; disruption of cilia-dependent Shh
signalling appears to underlie exencephaly in mutant embryos. The Cc2d2a !/!mouse
embryonic fibroblasts (MEFs) lack cilia, although mother centrioles and pericentriolar pro-
teins are detected. Odf2, associated with subdistal appendages, is absent and ninein is
reduced in mutant MEFs. In Cc2d2a !/!MEFs, subdistal appendages are lacking or abnormal
by transmission electron microscopy. Consistent with this, CC2D2A localizes to subdistal
appendages by immuno-EM in wild-type cells. We conclude that CC2D2A is essential for the
assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the
mother centriole for axoneme biogenesis.
DOI: 10.1038/ncomms5207
1Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, NIH, Bethesda, Maryland 20892, USA. 2Biological Imaging Core, National
Eye Institute, NIH, Bethesda, Maryland 20892, USA. 3Genetic Engineering Core, National Eye Institute, NIH, Bethesda, Maryland 20892, USA. 4Electron
Microscope Laboratory, Advanced Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21701, USA. Correspondence
and requests for materials should be addressed to A.S. (email:
NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 | 1
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The primary cilium is a ubiquitous microtubule (MT)-based
organelle, projecting from the cell surface to perform
specialized sensory and signalling functions during
organogenesis and cellular/tissue homeostasis1–3. In quiescent
or post-mitotic cells, the migration of the centrosome towards the
plasma membrane sets the stage for biogenesis of the primary
cilium, which originates from the mother centriole (MC) acting
as the basal body4. The two centrioles in the centrosome are
ultrastructurally distinct; only the MC has distal and subdistal
appendages (SDA)5and is involved in membrane tethering/
docking, whereas the daughter centriole anchors cytoplasmic MT
arrays6. The formation of the ciliary axoneme is initiated at the
distal end of MC by the docking of a primary ciliary vesicle
followed by coordinated trafficking of hundreds of proteins
including signalling molecules and receptors via MT motors and
intraflagellar transport complexes7,8. The efforts to define critical
steps in cilia biogenesis have intensified lately because of the
association of ciliary function with human diseases.
Given the commonalities in cilia and centrosomes and their
nearly universal presence, it is not surprising that aberrant cilia
biogenesis and/or function can impact multiple tissues and cell
types and manifest as pleiotropic syndromic disorders, collec-
tively termed ciliopathies9–12. The ciliopathies encompass a
spectrum of clinically distinguishable phenotypes that include
retinal degeneration, cognitive impairment, neural tube defects,
hydrocephalus, polycystic kidney, polydactyly, situs inversus
and obesity. The clinical findings could include blindness (as in
Leber congenital amaurosis) and/or cystic kidneys (as in
nephronophthesis) as the primary manifestation. Alternately,
the phenotypes can encompass multiple system defects as in
Bardet–Biedl syndrome and Joubert syndrome (JBTS) or appear
as a rare lethal malformation in Meckel syndrome (MKS).
Strikingly, similar syndromic disorders can result from mutations
in many different genes, and mutations in one gene can lead
to distinct clinical manifestations. The observed phenotypic
diversity in ciliopathies might also reflect a cumulative
genetic load of variants/mutations and interactions among cilia-
associated genes12–14.
The search for homozygous genomic regions in 10 unrelated
MKS fetuses led to the identification of the ciliopathy gene,
CC2D2A15, which was independently discovered by
homozygosity mapping of consanguineous families with JBTS16.
Concurrently, a third group reported a homozygous splice site
mutation in a large Pakistani family with mental retardation and
retinitis pigmentosa17. Follow-up studies suggest that CC2D2A
mutations in JBTS patients are less deleterious than those causing
MKS18.CC2D2A encodes a coiled coil and C2 domain containing
protein that is required for cilia formation and predicted to be
involved in calcium-dependent membrane targeting15. The
CC2D2A protein also includes a catalytically inactive version of
the transglutaminase-like domain that may provide a peptide-
binding interface for MTs19. CC2D2A is localized to the basal
body and can physically interact with CEP290 (ref. 16), a cilia-
centrosomal protein associated with numerous ciliopathies20.
Interestingly, the multiprotein complex containing Tectonic1,
associated with regulation of Hedgehog signalling, includes
both CC2D2A and CEP290, and these proteins have been
localized to the transition zone between the basal body and ciliary
A fundamental requirement of CC2D2A in organogenesis
is implied from embryonic lethality in human MKS15. The
fibroblasts derived from a MKS embryo harbouring a CC2D2A
mutation are unable to extend ciliary axoneme even though the
basal body (that is, MC) is present, suggesting an essential role of
CC2D2A in cilia biogenesis15. Nevertheless, a nonsense cc2d2a
mutation identified in the sentinel zebrafish mutant did not
reveal defects in motile cilia number or morphology, although
some JBTS-like phenotypes (such as pronephric cysts) were
To elucidate the function of CC2D2A in cilia biogenesis and
produce a model of MKS, we generated a Cc2d2a-null allele in
mice. The loss of Cc2d2a (Cc2d2a !/!) results in embryonic
lethality with multiorgan defects related to cilia biogenesis. We
show that CC2D2A localizes to the SDA in the MC and its loss
prevents the assembly of SDA and anchoring of MTs. Our studies
further delineate the fundamental sequence of cilia biogenesis
from the MC and uncover ciliary ultrastructural defects
associated with the loss of CC2D2A function in MKS.
The loss of Cc2d2a in mouse leads to embryonic lethality.
Three protein-coding transcript variants are produced from the
Cc2d2a gene. To eliminate all transcripts, we replaced Cc2d2a
exons 6 to 8, shared by all variants, with a targeting vector con-
taining a b-gal reporter and a neomycin selection cassette
(Fig. 1a) through standard homologous recombination in ES cells.
Southern blotting of genomic DNA from ES clones using an exon
5–6 probe (Fig. 1a) showed 12.7 and 9.9 kb EcoRI fragments for
the wild-type and Cc2d2a !/!alleles, respectively (Fig. 1b,
Supplementary Fig. 1). We confirmed that the embryos homo-
zygous for the targeted allele (indicated as !/!or Cc2d2a !/!)
lacked the Cc2d2a transcript (Fig. 1c). Finally, we demonstrated
by immunoblot analysis that Cc2d2a !/!embryos lacked the
CC2D2A protein (Fig. 1d, Supplementary Fig. 2).
The analysis of F2 litters produced by crossing heterozygous
Cc2d2a þ/!mice identified live pups with only Cc2d2a þ/þand
Cc2d2a þ/!genotypes, suggesting lethality of Cc2d2a !/!
homozygous-null embryos. We then assessed at what age
Cc2d2a !/!embryos are lost. We were able to identify
Cc2d2a !/!embryos at close to predicted ratios (25%) on
embryonic day (E) 14 and E16. However, the ratio of Cc2d2a !/!
embryos declined sharply at E18 (to 4%), and null mutants rarely
survived beyond that age (Table 1). At E16–18, Cc2d2a !/!
embryos displayed pleiotropic phenotypes resembling MKS, with
many showing severe degeneration and resorption during early
embryogenesis (Fig. 1e). In addition to haemorrhage, open neural
tube, microphthalmia, and anophthalmia, Cc2d2a !/!embryos
often revealed situs inversus and dextrocardia, and on occasion,
lacked abdominal organs (Fig. 1f, boxed area). Polydactyly was
observed frequently in Cc2d2a !/!embryos (Fig. 1h, arrows).
Thus, Cc2d2a is broadly required for organogenesis in mice.
Among hundreds of Cc2d2a !/!mutants generated in our
laboratory, only one mouse survived for 27 days and showed
gross underdevelopment with marked lethargy, loss of hair on its
dorsal surface, hydrocephalus (Fig. 1g, compare arrows), and
retinal dystrophy (Supplementary Fig. 3).
Cc2d2a !/!embryos have defects in motile and sensory cilia.
The situs inversus phenotype suggested defects in the embryonic
node and establishment of left–right asymmetry. Scanning
electron microscopy of the E8 Cc2d2a !/!embryos revealed
flattening of the node with only a few cilia-like structures
(Fig. 2a–c). Immunostaining of the embryonic node using anti-
acetylated a-tubulin antibody validated these findings (Fig. 2d).
The analysis of other ciliated tissues also showed profound defects
in cilia biogenesis. The kinocilia in the cochlea were frequently
absent or abnormal (green signal in Fig. 2e, right) and
stereociliary bundles deformed (red signal in Fig. 2e, right) in
Cc2d2a !/!embryos. The cilia were also absent or abnormal in
Cc2d2a !/!embryonic liver (green signal in Fig. 2f, right).
Perinatal kidney tubules and tracheal epithelium revealed few
2NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 |
&2014 Macmillan Publishers Limited. All rights reserved.
cilia in Cc2d2a !/!embryos (green signal in Fig. 2g,h, right
panels). By comparison, kidney tubules in wild-type embryos
had prominent, primary cilia projecting into the lumen (Fig. 2g,
left), and tracheal epithelia presented tufts of motile cilia
(Fig. 2h, left).
Shh signalling is perturbed in Cc2d2a !/!embryos. As
Cc2d2a !/!embryos often exhibited exencephaly (see Fig. 1e),
we hypothesized that the open neural tube phenotype is due to
abnormal cilia biogenesis and perturbed Shh signalling22–24. Our
analysis of E 12 embryos revealed the presence of cilia in the
kb +/+
+/++/+ Cc2d2a–/–
Targeting construct
WT allele
ko allele
+/– +/+ +/– +/–+/–
E5 E6
E9 E10
mRNA level
–/– / +/+
E9 E10
Cc2d2a –/–
E5 E6 E7 E8 E9 E10 E11 E12
Figure 1 | The loss of Cc2d2a leads to embryonic lethality with pleiotropic defects in organogenesis. (a) The design for generating Cc2d2a knockout (ko)
allele in mouse. The wild-type Cc2d2a locus at mouse chromosome 5 (top), the targeting vector (middle) and the ko allele (bottom) are shown. Small lines
with diamond caps indicate the DNA fragment used as a probe for Southern blot analysis. Mice were genotyped using the forward and reverse primers
indicated by arrows. (b) Validation of Cc2d2a ko allele in ES cells by Southern blot analysis after digestion with EcoRI, as shown in a. Successful
recombination with the targeting vector would yield both wild-type (12.7kb) and targeted (9.9 kb) fragments. M, DNA ladder; þ, wild type allele; !,ko
allele. (c) Reverse transcriptase PCR analysis of Cc2d2a transcripts. The ratio of Hprt (control) or Cc2d2a transcripts in !/!versus þ/þtissues of
newborn (P0) mouse is shown. Cc2d2a transcripts were undetectable in the ko embryos. (d) Immunoblot analysis with anti-CC2D2A antibody on protein
extract from the wild-type and Cc2d2a ko embryos aged E14. A 187 kDa CC2D2A protein was detected in the wild type ( þ/þ), whereas in the !/!
embryos CC2D2A was undetectable. b-Actin was used as a loading control. (e) Gross phenotype of E16 Cc2d2a !/!embryos compared with wild type
(þ/þ) littermates. Mutant embryos display a range of developmental defects, including exencephaly or open rostral neural tube (arrow; middle), small
size (right) and microphthalmia (dotted oval). (f) Visceral phenotype of Cc2d2a !/!embryos. The !/!embryos display dextrocardia (arrow) and this
one lacks kidney, spleen on the left side, in addition to large part of intestine (dotted rectangle). (g) The phenotype of a single rare !/!survivor at P27,
along with its littermate control. The !/!(top) animal has a domed head representing profound hydrocephalus compared with its þ/þlittermate
(lower). See also Supplementary Fig. 3. (h) Hind limbs from wild-type and !/!embryos. Note the pre-axial polydactyly on the hind limb of !/!
embryo (arrow).
NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 | 3
&2014 Macmillan Publishers Limited. All rights reserved.
neural tube in the wild type (Fig. 3a, upper middle panel) but not
in the Cc2d2a !/!mutants (Fig. 3a, lower middle panel). We
noted the presence of basal bodies in the neural tube of both
genotypes (Fig. 3a, left panels).
We then investigated a potential defect in the patterning of
neural tube domain markers due to lack of cilia-mediated Shh
signalling. In wild-type embryos, Shh signal localizes to the floor
plate of neural tube and at the notochord (Fig. 3b). In contrast,
Cc2d2a !/!embryos lack Shh signal at the floor plate (Fig. 3b).
In contrast to the wild-type embryos showing Nkx2.2 and Nkx6.1
expression (Fig. 3b) at the ventral neural tube, Nkx2.2 signal was
found at the floor plate in the Cc2d2a !/!embryos and Nkx6.1
expressing domain shifted towards the ventral midline (Fig. 3b).
Furthermore, the Pax6 domain also expanded ventrally. These
data indicate the loss of floor plate cell fate and a reduction in V3
progenitors, which require high levels of Shh for induction. Olig2
signal, marking progenitors of the oligodendrocyte lineage that
also depend on Shh signalling for induction, was absent in the
Cc2d2a !/!embryos (Fig. 3b). We note that Pax7, HB9 and
Msx2 showed no discernable difference in Cc2d2a !/!embryos
compared with the wild type (Fig. 3b). Thus, the neural tube
patterning defect of Cc2d2a !/!embryos mostly affects ventral
cell fates, consistent with a perturbation of Shh signalling. The
Table 1 | Genotypes obtained from heterozygous ( þ/!)
Age Cc2d2a genotype (%) Total (N)
Predicted % 25 50 25
E14 29 54 17 41
E16 24 52 24 25
E18 32 64 4 25
P0 34 66 0.2 1,022
First column shows the age of the embryos/animals analysed, indicated as either embryonic (E)
day or as P0 for live-born pups. The last column shows total number of animals genotyped at
each age. Predicted percentages are italicized.
P0 trachea E18 cochleaE13.5 liver P0 kidney
Anti-Ac-tubulin/DAPI/phalloidinE8 embryonic node/anti-Ac-tubulin
+/+ Cc2d2a–/– +/+ Cc2d2a –/–
Figure 2 | Motile and sensory cilia biogenesis is defective in Cc2d2a !/!embryos. (ac) Scanning electron micrographs of E8 embryonic node in
increasing order of magnification. The wild-type node comprises ciliated cells, whereas the !/!embryo had only rare cilia (arrowheads in c).
(d) Immunostaining for cilia markers (anti-a-acetylated tubulin, green) on E8 embryonic node (within the dashed circled area). The wild-type embryo
(left panel) had many cilia compared with the !/!embryo (right panel) that had almost none. (e) E18 cochlea showing kinocilia (anti-a-acetylated
tubulin, green) and stereocilia (phalloidin, red) in wild-type ( þ/þ) and Cc2d2a!/!embryos. In the wild type, kinocilia were evenly spaced and aligned,
whereas those in the !/!were missing or misoriented (arrowhead). (f) E13.5 liver from wild-type ( þ/þ) and Cc2d2a !/!embryos showing cilia
staining (anti-a-acetylated tubulin, green). Cilia were underdeveloped in !/!liver (arrowheads). (g) Kidney tubule and (h) trachea from P0 wild-type
(þ/þ) and Cc2d2a !/!mice showing cilia staining (anti-a-acetylated tubulin, green). The wild-type kidney had many long luminal cilia that are not
detected in the !/!mice (arrowheads). Similarly, the !/!trachea had no cilia (arrowheads) compared with tufts of multiple motile cilia on each
epithelial cell in the wild type. The scale bars in all panels are 5 mm except in ‘a’, which is 50 mm. In dh, anti-a-acetylated tubulin is green and nuclear DAPI
counterstain is blue.
4NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 |
&2014 Macmillan Publishers Limited. All rights reserved.
Shh Olig2 Nkx2.2 Nkx6.1
Pax6 Pax7 HB9 Msx2
Arl13b DAPI+ merge Inset enlarged
Figure 3 | Cilia-mediated Shh signalling is perturbed in the developing neural tube of Cc2d2a!/!embryos. (a) Immunostaining for anti-gtubulin
(marker of basal bodies; red) and anti-Arl13b (highlighting cilia; green) in embryonic (E12) neural tube. In wild-type embryo ( þ/þ) neural tube
showed presence of both basal bodies (red) and cilia (green). In ko embryo ( !/!), the cilia are missing and the basal bodies are mislocalized.
The inset region is enlarged and shown at the right. (b) Immunostaining for indicated markers (red) in embryonic E12 neural tube. In the wild type ( þ/þ),
the Shh signal was clearly seen in the ventral floor plate and notochord whereas in the ko ( !/!) the Shh signal was absent in the floor plate.
Olig2 and Nkx2.2 were both evident in ventral domains near the floor plate in the wild type, but in the mutant Olig2 was absent and Nkx2.2 was seen
ectopically at the floor plate. Compared with the wild type, Nkx6.1 signal was shifted ventrally, and Pax6 domain also showed a slight expansion ventrally.
Pax7, HB9 and the roof plate maker Msx2 did not show any apparent difference in signal intensity and pattern in the wild-type and the ko embryos.
The nuclear staining is done with DAPI (blue). The images are taken in rostrocaudal axis approximately in the hind limb level. Scale bars, 50mm.
NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 | 5
&2014 Macmillan Publishers Limited. All rights reserved.
neural tube patterning defect in this mutant is similar to that of
the Mks1 mutant25.
Cc2d2a !/!fibroblasts have basal body but not ciliary
axoneme. To delineate the underlying defect in cilia biogenesis,
we isolated and cultured mouse embryonic fibroblasts (MEFs)
from Cc2d2a !/!mutants. Unlike MEFs from wild-type litter-
mates (Fig. 4a, upper panel), Cc2d2a !/!MEFs did not grow
cilia on serum starvation (Fig. 4a, lower panel). We, however,
noted that B10% of Cc2d2a !/!MEFs escaped this phenotype.
Both Cc2d2a !/!and wild-type MEFs showed similar MC
staining with g-tubulin, yet Arl13b labelling was compromised in
Cc2d2a !/!MEFs (Fig. 4a). In addition, MT staining with
acetylated a-tubulin showed presence of axoneme in the wild type
but not in Cc2d2a !/!MEFs; nonetheless, cytoplasmic MTs were
visible in Cc2d2a !/!MEFs (Fig. 4c,d). These data suggest that
ciliary axoneme biogenesis was not initiated from the MC in the
absence of Cc2d2a. The mouse Cc2d2a transgene could rescue the
axoneme assembly defect in Cc2d2a !/!MEFs (Fig. 4b; note that
the non-transfected cells in the same field do not have cilia),
demonstrating a direct role of CC2D2A in the genesis of cilia
from the MC.
The existence of a MC but lack of axoneme suggested
that Cc2d2a is needed in early ciliogenic processes. After
polarity-guided centriolar migration, the MC docks to the
membrane with distal appendages, whereas the anchoring of
MT arrays requires SDA6. Even though MT nucleation starts with
aster formation at both centrioles, only the MC is able to sustain a
stable MT array, a process requiring ninein26,27. Immunolabelling
with anti-ninein antibody revealed a significant reduction of
ninein signal at the MC in Cc2d2a !/!MEFs (Fig. 4d). It should
be noted that ninein is present predominantly at the distal end of
the MC, on SDA, while it is also detectable at the proximal ends
of both centrioles. Our results thus indicate a basic structural
defect in the MC.
% cells with cilia
Fluorescence intensity (a.u.)Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.) Fluorescence intensity (a.u.)
Arl13b γ-tub
AC-α-tub AC-α-tub
DAPI + merge
DAPI + merge
Odf2 DAPI + merge Trichoplein DAPI + merge
Ninein DAPI + merge
GFP Arl13b DAPI + merge
5748 65100
4555 77123
**** ****
+/+ –/–
+/+ –/– +/+ –/–
+/+ –/–
Figure 4 | Lack of cilia and defects in MC of Cc2d2a !/!MEFs can be rescued by Cc2d2a transgene. (a)Cc2d2a!/!MEFs have MC but no cilia.
Anti-Arl13b is used to highlight cilia. MC (basal body) is stained with g-tubulin antibody. (b)Cc2d2a transgene rescues cilia biogenesis in Cc2d2a!/!
MEFs. Anti-Arl13b staining is shown in red circle. GFP (green) represents the transfected cells. Non-transfected cells in the same field do not have any cilia.
Nuclei are stained with DAPI (blue). In c,d, cilia are marked by acetylated a- tubulin (green). (c) Pericentrin (a proximal end marker of MC) staining (in red)
is unaltered in Cc2d2a !/!MEFs. See also Supplementary Fig. 4. (d) Immunostaining of ninein that marks both SDA and the MC proximal end is
significantly reduced in Cc2d2a !/!MEFs. (e) Anti-Odf2 immunostaining, which is detected at SDA in wild type, is barely visible in Cc2d2a!/!MEFs.
(f) Trichoplein immunostaining is reduced in Cc2d2a !/!MEFs. Scale bars, 5 mm. Antibody is indicated above each panel. In merged images, blue
nuclear stain is DAPI. The number of cells used for quantification is indicated in all graphs. The graph in acompares percentage of cells with cilia (yaxis)
between wild-type ( þ/þ) and Cc2d2a !/!genotypes (xaxis). The graphs in remaining panels compare the level of antibody-generated fluorescence
(red signal in each panel) between the two genotypes, where xand yaxes are genotypes and corrected fluorescence intensity (arbitrary units, (a.u.)),
respectively. Bars indicate s.e.m. The sample size (n) is indicated on the bar diagram, and the experiment was repeated three times. P-value was
derived from unpaired two-tailed t-test. Statistical significance is marked with asterisks (* and **** indicate Pr0.05 and 0.0001, respectively).
6NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 |
&2014 Macmillan Publishers Limited. All rights reserved.
Distal end components are abnormal in MC of Cc2d2a !/!
MEFs. Immunolabelling of wild-type and Cc2d2a !/!MEFs
using rootletin and pericentrin antibodies revealed no significant
differences (Fig. 4c and Supplementary Fig. 4a), implying struc-
tural integrity of pre-proximal and proximal ends of the two
centrioles. Absence of the ciliary axoneme and reduced ninein
staining prompted us to examine Odf2, an established marker of
SDA28, which is needed for cilia biogenesis29. The anti-Odf2
signal at the MC was dramatically reduced in Cc2d2a !/!MEFs
compared with the wild type (Fig. 4e). We then performed
immunolabelling against trichoplein, which controls MT
anchoring at the MC by binding to Odf2 and ninein30.
Interestingly, trichoplein signal was also significantly reduced at
the MC in Cc2d2a !/!MEFs (Fig. 4f). Our results suggest that
recruitment of Odf2 and trichoplein to the SDA is compromised
when CC2D2A is not present, and that CC2D2A is needed for
SDA assembly.
CC2D2A is required to form SDA. The loss of cc2d2a in
zebrafish photoreceptors resulted in mislocalization of Rab8
(ref. 31), which interacts with Odf2 and is needed for cilia
biogenesis32. In Cc2d2a !/!MEFs, Rab8 staining is significantly
reduced compared with the wild type (Fig. 5a). Furthermore,
transfection of Rab8a-mCherry in wild-type MEFs showed sharp
localization at the base of the cilium, whereas the signal was
diffuse and cytoplasmic in Cc2d2a !/!MEFs (Fig. 5b). Notably,
immunoblotting demonstrated similar levels of Rab8a protein in
the wild-type and Cc2d2a !/!MEFs (Supplementary Fig. 5).
These results thus indicate defective vesicle docking or membrane
tethering at the MC in the absence of CC2D2A.
To further investigate cellular ultrastructure at and near the
MC, we took advantage of transmission electron microscopy
(TEM). In wild-type MEFs, normal ciliary structures were evident
with MTs anchored at SDA and oriented in a regular array
from the cytoplasm towards the basal body (Fig. 5c; left, arrow).
However, in Cc2d2a !/!MEFs, the MC lacked or had
abnormal SDA, had disorganized MTs and showed accumulation
of vesicle-like structures nearby (Fig. 5c, middle panel). Notably,
in a fraction of Cc2d2a !/!MEFs, the MC docked to ciliary
vesicles but apparently no transition zone developed (Fig. 5c,
right panel). This finding was not obtained in the wild-type
MEFs. We therefore conclude that CC2D2A is needed for the
assembly of SDA at MC, for stable MT anchoring and docking
of transport vesicles, and for further elaboration of the
transition zone.
MT regrowth assays using a- and g-tubulin33 were then
performed to evaluate whether MT anchoring at the MC requires
CC2D2A. In wild-type and Cc2d2a !/!MEFs, MT regrowth did
not initiate immediately after removing nocodazole (Fig. 6,
0 min). At 5 min, the MT started reappearing at the MC in the
wild type, but not seen in Cc2d2a !/!MEFs. At 30 min of
Fluorescence intensity (a.u.)
mCherry Arl13b
N=27, SDA present = 23 N=24, SDA absent or abnormal = 18
40 32
Figure 5 | Accumulation of transport vesicles and lack of or abnormal SDA in Cc2d2a!/!MEFs. (a) Immunostaining of Rab8a (red) that marks
transport vesicles in wild-type and Cc2d2a !/!MEFs. Rab8a localization is diffuse in cytoplasm in Cc2d2a !/!MEFs compared with the wild type. The
xand yaxes are genotypes and corrected fluorescence intensity (arbitrary units, (a.u.)), respectively. Bars indicate s.e.m. Signal intensity quantification
shows a highly significant difference (P-value was derived from unpaired two tailed t-test; **** indicates P-value r0.0001). The sample size (n) is indicated
on the bar diagram, and the experiment was repeated three times. (b) Immunostaining for anti-Arl13b (green) and Rab8a-mCherry (red) transfected
in wild-type and Cc2d2a !/!MEFs. Rab8a-mCherry signal is sharp at the base of the cilium (arrowhead) in wild-type cells but diffuse in !/!MEFs.
Nuclei are stained with DAPI (blue). (c) Transmission electron micrographs of cilia from wild-type and Cc2d2a !/!MEFs. In wild type, the MC has
well-defined SDA (arrow head), to which MTs (arrow) are anchored for trafficking of vesicles (V). In Cc2d2a !/!MEFs, the SDA is not visible at a
comparable location (middle panel, arrowhead) or an abnormal SDA with compromised transition zone (right panel, arrowhead) and the MTs (arrow)
are not anchored. The vesicles accumulate in the cytoplasm around the MC. The total number (N) of MEFs analysed for the presence of SDA or abnormal
SDA is indicated. MC and daughter centriole (DC) are mother and daughter centriole, respectively. Scale bars in aand bare 5 mm and in cis 500 nm.
NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 | 7
&2014 Macmillan Publishers Limited. All rights reserved.
recovery from nocodazole MT aster anchoring at the MC
was visible in wild-type cells (Fig. 6, 30 min). At 30 min,
although MTs were visible in Cc2d2a !/!MEFs, these did not
form an aster and anchor at the basal body. These data
demonstrate a critical role of CC2D2A in MT anchoring at the
basal body.
CC2D2A localizes to SDA. Immunolabelling in ciliated IMCD3
cells revealed the localization of CC2D2A protein to the MC.
The CC2D2A staining partially overlapped with RPGR, a cilia
transition zone marker34,andanti-GT335(Fig.7a,b),which
labels polyglutamylated tubulin and marks both the basal
body and the proximal axoneme35,36. Interestingly, CC2D2A
immunostaining appeared similar to that of centriolin35 and Odf2
(ref. 28). To further refine the localization of CC2D2A, we took
advantage of immuno-EM. Immunogold labelling for CC2D2A
was clearly visible on the SDA both in IMCD3 cells and in
monkey retina (Fig. 7c,d). Our data are concordant with
preceding MEF studies and strengthen the proposition of a role
for CC2D2A in SDA assembly.
No cell cycle defect in Cc2d2a !/!MEFs. We detected similar
anti-GT335 immunostaining in wild-type and Cc2d2a !/!MEFs
(Supplementary Fig. 4b, lower panels), consistent with the
hypothesis that CC2D2A includes a catalytically inactive trans-
glutaminase-like domain19. To test whether CC2D2A might be
required for sensing Ca2þlevel changes during cell cycle37, we
analysed the cell cycle profile in MEFs. No significant difference
was identified between the wild-type and Cc2d2a !/!MEFs
(Supplementary Fig. 6).
Embryonic lethality observed in MKS represents the most severe
of all phenotypes in ciliopathies. CC2D2A is one of the
10 centrosome-cilia genes associated with MKS. Mutations in
CC2D2A also cause JBTS, another relatively severe disease with a
plethora of clinical findings. Previous studies implicate a
fundamental role of CC2D2A in cilia biogenesis that when
interrupted would lead to MKS or JBTS. However, the precise
disease mechanism and CC2D2A function have been elusive.
Here, we demonstrate that CC2D2A is required for the assembly
of SDA that are critical for anchoring of MTs, vesicle docking
and initiation of ciliary axoneme. Loss of CC2D2A, as in
the Cc2d2a !/!embryos reported here, appears to prevent
cilia-mediated functions of motile or sensory cilia, thereby leading
to defects in organogenesis in MKS. We suggest that the less
severe JBTS ciliopathy phenotype likely results from residual
function of certain mutant CC2D2A alleles or from partial
compensation by a modifier gene14,38,39.
The Cc2d2a !/!mouse model recapitulates the phenotype
reported in human MKS fetuses that inherit a nonsense mutation
in CC2D2A15. The lethality observed in cc2d2a-null mutants of
zebrafish40 reflects the evolutionary conservation of Cc2d2a
function in cilia biogenesis. Interestingly, although a Cc2d2a
gene-trap mouse exhibits embryonic lethality, the MEFs derived
from this mutant could extend a ciliary axoneme21, suggesting
that the gene-trap allele may not be a complete null. Different
mouse mutants therefore offer an opportunity to better elucidate
underlying cilia biogenesis defects and pathologies in distinct
syndromes caused by mutations in CC2D2A.
We note that the situs inversus phenotype in Cc2d2a !/!
embryos can be explained by the lack of nodal cilia, as in IFT-
defective Kif3b mice41. However, the cilia defects are remarkably
different in Cc2d2a !/!mutants compared with the IFT
mutants. Generally, in IFT mutants, cilia are formed with
different degrees of abnormality depending on the mutation:
shorter, rounder or of normal length but with bulges near the
tip42. By contrast, the cilia are not formed in the Cc2d2a mutant,
suggesting that CC2D2A is required at an early step of
ciliogenesis. IFTs are utilized as transport modules at a later
stage to build and maintain the cilia.
The Shh pathway is initiated in primary cilia for both neural
tube and limb bud patterning22,23. Notably, Shh signalling is
perturbed in the developing Cc2d2a !/!neural tube (see Fig. 3b).
Ventral progenitor domains marked by Shh and Olig2 are absent
in Cc2d2a !/!embryos. Nkx2.2 is shifted and confined to the
floor plate, and both Nkx6.1 and Pax6 show a slight shift towards
the ventral midline. These data indicate lost floor plate and
diminished V3 progenitor domain, both of which require high
levels of Shh signalling for induction. On the other hand, dorsal
and lateral fates are largely unaffected in Cc2d2a !/!embryos.
The neural tube patterning defect is consistent with reduced Shh
signalling and is similar to those reported for Mks1 mutant25 and
some other ciliogenesis or trafficking mutants23,43. Polydactyly is
a common finding in MKS and JBTS44, and is caused by aberrant
Shh signalling45 via its effector Gli proteins localized on the
ciliary membrane22,46,47. We conclude that both the neural tube
0 min 5 min 30 min
Figure 6 | CC2D2A is required for MT anchoring at the basal body. The MT array (a-tubulin, green) formation at the basal body (g-tubulin, red) is
not visible at 0 min in the wild-type ( þ/þ) and the ko cells ( !/!). At 5 min, the MT array is apparent at the basal body in the wild-type cells but
not in the ko cells. At 30 min, a profuse array of MT forming an aster is observed at the basal body in the wild type but not in the ko cells even
though cytoplasmic MTs are detected. The area in the inset is shown at the higher magnification on the right side of each image. Scale bar, 5 mm.
8NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 |
&2014 Macmillan Publishers Limited. All rights reserved.
defect and polydactyly in Cc2d2a !/!embryos result from a
disruption in cilia-based Shh signalling.
The two centrioles in the centrosome constitute the primary
centre for cytoplasmic MT nucleation, anchoring and elongation
in animal cells. Distinct ultrastructural characteristics, specifically
the presence of distal appendages and SDA, distinguish the MC
from daughter centriole and provide cellular architecture for
building the ciliary axoneme using molecular motors and
intraflagellar transport6,48,49. Initiation of cilia biogenesis
requires the assembly of MT bundles, aster formation and their
stabilization at the MC; the absence of CC2D2A will compromise
this process. While aberrant immunostaining of ninein, Odf2 and
trichoplein indicate a SDA defect, TEM of Cc2d2a !/!MEFs
clearly shows the absence or abnormality of SDA. Thus, SDA
assembly at the MC requires CC2D2A along with ninein, Odf2
and trichoplein. Odf2 appears to be a core component since SDA
are missing in the Odf2 !/!model28. Ninein and E-tubulin
are also essential because their depletion halts MT aster
formation50,51. In addition, centriolin and Cep170 are
associated with SDA35,52, although whether they play a direct
role is less clear. Additional investigations are required to
determine whether distinct proteins are assembled directly at
the MC, or if a preassembled complex is attached to the MC to
generate SDA. The catalytically inactive transglutaminase-like
domain in CC2D2A is a good candidate for providing the MT
interaction surface for SDA assembly (see Fig. 8 for a schematic).
A transition zone membrane complex required to build cilia
contains CC2D2A, in addition to other MKS–JBTS-associated
Ciliary vesicle extension
Ac-α−tub RPGR DAPI + mergeCC2D2A
CC2D2A DAPI + mergeGT335
Figure 7 | CC2D2A localizes to SDA where ciliary transition zone begins. (a) Immunostaining for anti-GT335 (green) and anti-CC2D2A (red) in IMCD3
cells that were induced to generate cilia. Anti-GT335 signal marks the MC and the proximal part of the axoneme, whereas anti-CC2D2A staining is
restricted to the MC (third panel, circled). Inset shows magnified view of the circled cilium. (b) Immunostaining for anti-a-acetylated tubulin (magenta),
anti-RPGR (green) and anti-CC2D2A (red) in IMCD3 cells. RPGR is a cilia transition zone marker. The CC2D2A immunostaining partially overlaps with anti-
RPGR signal (circled, enlarged in inset) at the base of axoneme marked by anti-a-acetylated tubulin. Nuclei are stained with DAPI (blue) in aand b.
(c) Immuno-EM of MCs labelled with anti-CC2D2A from ciliated IMCD3 cells. Gold particles (10 nm) are localized to SDA on MC in both micrographs.
The upper micrograph shows a ciliary vesicle of an extending cilium axoneme (arrow). (d) Immuno-EM of a monkey retina, using CC2D2A antibody in c.
The gold particles align on the SDA. MC is supported by a rootlet. Scale bars in aand bare 5 mm, and cand dare 500 nm.
NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 | 9
&2014 Macmillan Publishers Limited. All rights reserved.
proteins such as CEP290, Tectonic1 and B9D1 (ref. 21). It
appears that the SDA region is at the base of the ciliary transition
zone where triplet MTs of the MC terminate and doublet MTs of
the axoneme begin4,53,54. The overlap in RPGR and CC2D2A
immunostaining, reported here (see Fig. 7b), is consistent with
this model (see Fig. 8) and with their interaction with CEP290
(refs 16,55); however, these proteins might exist in distinct
protein complexes in SDA or transition zone12,14,56. A substantial
fraction of mutant MEFs revealed the docking of the MC with the
ciliary vesicle but no transition zone was apparent, suggesting a
requirement of CC2D2A in transition zone formation (Fig. 5c,
right panel). We hypothesize that CC2D2A interacts with Odf2,
which in turn facilitates the delivery of Rab8a vesicles to tether
the membrane at the SDA32. Accumulation of vesicles observed
in Cc2d2a !/!MEFs (see Fig. 5) and mislocalization of Rab8 in
zebrafish-mutant photoreceptors31 further support the role of
CC2D2A and other SDA proteins in membrane loading and/or
tethering during cilia biogenesis.
In summary, we demonstrate an essential role of CC2D2A in
the formation or stabilization of subdistal appendages to initiate
the process of cilia biogenesis from the basal body. Defects in
nodal and primary cilia, and consequently Shh signalling, can
explain severe pleiotropic phenotypes. Our studies thus provide
molecular insights into the MKS disease caused by CC2D2A
Generation of Cc2d2a !/!mice and MEFs for phenotyping.All animal
experiments were performed after obtaining approval of the animal care and use
committee of the National Eye Institute (ASP-NEI 650).
The mouse Cc2d2a gene on chromosome 5 spans almost 80 kb and includes
40 exons (gene ID: 231214) with three protein-coding splice variants. Following
standard homologous recombination41,57, exons 6–8 were replaced with a
gene-targeting cassette containing homology arms and a b-gal reporter. Southern
blotting and PCR validated the correct integration of the targeting cassette.
Quantitative reverse transcriptase PCR analysis of different tissues was performed
using a TaqMan assay (Applied Biosystems; Mm01211431_m1). The method for
RNA-seq analysis of MEFs has been reported58. The procedures for
electroretinography, histology and immunohistochemistry are reported
elsewhere59. Analysis of the neural tube for cilia and Shh signalling-associated
proteins was largely performed as detailed43.
MEFs were prepared from E12.5–E13.5 mouse embryos, as instructed
(Millipore Tech. Publications). For immunostaining, 70,000 MEFs were plated in
each well of a chamber slide (ibidi, WI) with 300 ml of medium. After overnight
growth, cilia biogenesis was induced with serum-free medium. The cells were
grown to confluence for 48 h, washed with PBS, fixed with 4% paraformaldehyde at
room temperature for 15 min and subjected to standard immunostaining protocol.
MEFs were imaged on LSM 700 (Zeiss, Germany) confocal microscope, and the
images were edited with Adobe Photoshop CS5 and assembled on Adobe
Illustrator CS5. The fluorescence intensity in the images was analysed with
ImageJ64 (NIH) as performed earlier60. Briefly, fluorescence signal (pixel area) in
an image was selected with a tool (circle) and integrated density (mean grey value)
of the area was measured. Similarly, from the same field, an area with no
fluorescence signal (next to a cell) was measured for background intensity.
The corrected fluorescence was calculated by the formula, corrected
fluorescence ¼integrated density !(area of pixel with signal $mean fluorescence
body (MC)
Pericentriolar material (PCM)
Ninein Subdistal
Distal appendage
pocket Vesicle
Plasma membrane
Figure 8 | A schematic of the base of primary cilium showing the MC (basal body) and the proposed function of CC2D2A at SDA. The MC
includes triplet MTs (purple cylinders) that continue as the doublet MTs of the axoneme (pink cylinders). The plus and minus ends of MTs are marked.
The proteins localized to the cilium and MC are indicated. Nine extensions of each MT bundle at the border of MC and transition zone represent the
SDA (blue–green–yellow pyramids) and distal appendages (blue rods). The proteins localized to SDA are shown in an order from centre to periphery, based
on the current experimental evidence. The SDA anchors MT arrays and helps in the docking of transport vesicles at the base of cilium. The distal
appendages help in tethering the MC to the plasma membrane, separating the cilium compartment from the cytoplasm. The green circle at the far
end of the distal appendage, on the right side, represents a Rab8a-tagged vesicle. Rab8a may help tether plasma membrane to distal appendages
with support from Odf2 and CC2D2A. Inset shows the top view of MC at the level of SDA, which are shown anchored to MT. The electron micrographs on
the right show a cross-sectional view at the level indicated: doublet axoneme (top), at the level of appendages (middle) and triplet MC (basal body)
10 NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 |
&2014 Macmillan Publishers Limited. All rights reserved.
of background readings). Statistical analysis of difference in fluorescence intensity
was performed by t-test (two-tailed, type 2) in Excel (Microsoft).
Antibodies.The following primary antibodies were used: anti-CC2D2A (Rb,1:300,
custom made), anti-acetylated a-tubulin (Ms, 1:500; Sigma, T6793), anti-a-tubulin
(Ms, 1:2,000; Sigma, T6199 (DM1A)), anti-g-tubulin (Ms, 1:500; Sigma, T6557),
anti-GT335 (Ms, 1:500; Adipogen, AG-20B-0020), anti-Arl13b (Rb, 1:500; Pro-
teintech, 17711-1-AP;), anti-trichoplein (Rb, 1:100; Masaki Inagaki), anti-human
cenexin (ODF2 Rb, 1:50; Kyung Lee), anti-ninein-L77 and –L79 (Rb, 1:2,500;
Michel Bornens), anti-Rab8a (Rb, 1:250; Proteintech, 55296-1-AP), anti-rootletin6
( Rb, 1:500; Tiansen Li) and anti-RPGR-S3 (Chk, 1:300; Tiansen Li), anti-
pericentrin (Rb, 1:500; Abcam, ab448), Anti-Olig2 (Rb, 1:200; R&D, AF2418). The
following monoclonal antibodies were used (Ms, 1:200, DSHB): Shh, Nkx2.2,
Nkx6.1 (clone F55A10), HB9, Msx2, Islet-1 (clone 40.2.D6) Pax6 and Pax7. The
secondary antibodies for immune-fluorescence analysis were coupled to Alexa
Fluor 488 or 568 dyes (Molecular Probes).
MT regrowth assay.MEFs were plated at a high confluence onto the chamber
slide (70–80%) and the following day, the cells were treated with 5 mM nocodazole
(Sigma) in complete media (DMEM) and incubated for 1.5 h at 37 !C. Subse-
quently, the media was aspirated and the cells were washed once with PBS and
replaced with complete media. After treatment, the cells were fixed at time points
of interest with 100% methanol 3 min on ice and then processed for immuno-
fluorescence staining, with anti-g-tubulin and anti-a-tubulin, sequentially.
The images were captured on LSM 700 confocal microscope (Zeiss, Germany).
Scanning electron microscopy.Embryos (E8) were fixed in paraformaldehyde
(4%) and glutaraldehyde (2%) in cacodylate buffer (0.1 M) for 2 h at room tem-
perature and then washed in cacodylate buffer thrice (10 min each). The embryos
were treated for 1 h in osmium tetroxide (1%), washed thrice (10 min each) in
cacodylate buffer and dehydrated in graded ethanol (35%, 50%, 70%, 95%, two
times for 10 min each, and then three times with 100%). After treating with tet-
ramethylsilane solution (three times for 10 min each), the embryos were mounted
on a SEM stub and sputter coated with gold/palladium using an EMITECH K575
high-resolution coater. Imaging was performed on S-3000N scanning electron
microscope (Hitachi, Japan).
TEM and immuno-EM.The serum-starved MEFs were fixed and processed for
TEM as described61. Sections of cells in Epoxy blocks (80 nm) were prepared with
Leica UCT Ultramicrotome, mounted on 200 mesh copper grids and coated with
carbon. The sections were imaged using H-7600 transmission electron microscope
Post-embedding immuno-EM.The procedure of post-embedding immuno-EM
was previously described62. Briefly, cultured IMCD3 cells (CRL-2123, ATCC) were
fixed in phosphate-buffered saline containing formaldehyde (4% v/v) and
glutaraldehyde (0.05% v/v) for 2 h, then dehydrated in a series of cold ethanol
(35%, 50%, 70%, 95% and 100%). The cells were infiltrated in 1:1 and 1:2 mixtures
of 100% ethanol and LR White resin for 1 h each, then in LR White resin overnight.
The cells were embedded in LR White resin and cured in a 55 !C oven for 24 h.
Thin sections (90–100 nm) were then mounted on 300-meshed nickel grids that
were first blocked by a commercial blocking buffer for goat antibodies and then
incubated in a serial dilution of primary antibody, followed by immunogold-
conjugated secondary antibody (10 nm particles). The grids were washed in Tris
buffer (pH 7.4) containing BSA (0.1% w/v), NaCl (250 mM) and Tween-20 (0.01%
v/v). The grids were stained in uranyl acetate and lead citrate and examined by
electron microscope.
Post-embedding immunolabelling of monkey photoreceptors.Monkey eyes
(Macaca mulatta) were obtained from the Washington National Primate Research
Centre at the University of Washington. Eyes were fixed in 2% paraformaldehyde
and 0.5% glutaraldehyde in phosphate buffer for 1 h, then en bloc stained in uranyl
acetate, dehydrated and infiltrated in LR White as previously described63. Thin
sections were collected on nickel grids, blocked in normal goat serum and
incubated in anti-CC2D2A antibody (1:100) for 2 h, washed and incubated in goat
anti-rabbit 10 nm gold secondary for 1 h. Sections were stained with uranyl acetate
and lead citrate and examined on a JEOL 1010 transmission electron microscope.
Negative controls (no primary) were run in parallel.
Flow cytometry.DNA content was assayed by flow cytometry using propidium
iodide. Samples were acquired using a FACSCalibur (BD Bioscience, CA) and the
data analysed with FlowJo V9.5 (TreeStar, OR) using Watson Pragmatic Modeling.
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We are grateful to Michel Bornens and James Sillibourne for anti-Ninein, Masaki Inagaki
and Akhito Inoko for anti-Trichoplein, Kyung Lee for anti-hOdf2 antibodies, James
Goldenring for Rab8a-mCherry plasmid, and Andrew Kodani and Jeremy Reiter for
microtubule regrowth assay protocol. We thank Megan Kopera and Seid Ali with mouse
matings, Rafael Villasmil with flow sorting, Adam Harned with EM, and Ethan Tyler for
an illustration. We thank Jacob Nellissery, Sharda Yadav, and Haiyan Guo for technical
assistance. The following monoclonal antibodies—Shh, Nkx2.2, HB9, Msx2, Islet-1
(clone 40.2.D6) (developed by T.M. Jessell and S. Brenner-Morton) ; Nkx6.1 (clone
F55A10) (developed by O.D. Madsen); Pax6, and Pax7 (developed by A. Kawakami)—
were obtained from the Developmental Studies Hybridoma Bank created under the
auspices of the NICHD and maintained by The University of Iowa, Department of
Biology, Iowa City, IA 52242. Our studies are supported by the Intramural Research
Program of the National Eye Institute and by a contract to SAIC from National Cancer
Author contributions
S.V., T.L. and A.S. conceived the study, designed the experiments, analysed the data and
wrote the manuscript. R.A.R. performed some of the data analysis and was involved in
manuscript revision. S.V., S.H.M., H.M.-S, M.B., T.A.F. and T.A.L. performed the
experiments. P.L., S.V. and L.D. generated the knockout mice. S.V., C.G., K.N. and R.N.F.
performed confocal or electron microscopy.
Additional information
Supplementary Information accompanies this paper at
Competing financial interests: The authors declare no competing financial interests.
Reprints and permission information is available online at
How to cite this article: Veleri, S. et al. Ciliopathy-associated gene Cc2d2a promotes
assembly of subdistal appendages on the mother centriole during cilia biogenesis.
Nat. Commun. 5:4207 doi: 10.1038/ncomms5207 (2014).
12 NATURE COMMUNICATIONS | 5:4207 | DOI: 10.1038/ncomms5207 |
&2014 Macmillan Publishers Limited. All rights reserved.
... [8][9][10] Although a number of related coding genes have been reported and included in RetNet (, [11][12][13][14][15][16][17][18][19][20] the noncoding genes that influence the macula and cones have rarely been considered. miR-183C (composed of miR-182, miR-183, and miR-96) is abundantly expressed in human and animal retinas. ...
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Purpose: Abundant retinal microRNA-183 cluster (miR-183C) has been reported to be a key player in photoreceptor development and functionality in mice. However, whether there is a protagonist in this cluster remains unclear. Here, we used a mutant mouse model to study the role of miR-96, a member of miR-183C, in photoreceptor development and functionality. Methods: The mature miR-96 sequence was removed using the CRISPR/Cas9 genome-editing system. Electroretinogram (ERG) and optical coherence tomography (OCT) investigated the changes in structure and function in mouse retinas. Immunostaining determined the localization and morphology of the retinal cells. RNA sequencing was conducted to observe retinal transcription alterations. Results: The miR-96 mutant mice exhibited cone developmental delay, as occurs in miR-183/96 double knockout mice. Immunostaining of cone-specific marker genes revealed cone nucleus mislocalization and exiguous Opn1mw/Opn1sw in the mutant (MT) mouse outer segments at postnatal day 10. Interestingly, this phenomenon could be relieved in the adult stages. Transcriptome analysis revealed activation of microtubule-, actin filament-, and cilia-related pathways, further supporting the findings. Based on ERG and OCT results at different ages, the MT mice displayed developmental delay not only in cones but also in rods. In addition, a group of miR-96 potential direct and indirect target genes was summarized for interpretation and further studies of miR-96-related retinal developmental defects. Conclusions: Depletion of miR-96 delayed but did not arrest photoreceptor development in mice. This miRNA is indispensable for mouse photoreceptor maturation, especially for cones.
... The most common features of ciliopathy include cystic liver and/or kidney, blindness, neural tube defects, brain anomalies, mental disability, skeletal abnormalities, obesity, and infertility, among others (Oud et al., 2017). Genomic and bioinformatics research has revealed that some primary cilia genes are linked to psychiatric disorders, such as the genes CC2D2A and Disc1, which are involved in ciliogenesis (Shen et al., 2008;Marley and von Zastrow, 2010;Veleri et al., 2014), and their defects can lead to psychiatric disorders, including Joubert syndrome (Bachmann-Gagescu et al., 2012), mental retardation (Noor et al., 2008;Shi et al., 2012), Meckel syndrome (Tallila et al., 2008), and Bardet Biedl syndrome (BBS) (Haq et al., 2019). In addition, several signaling pathways and crucial factors highly associated with schizophrenia, such as Wnt signaling, the fibroblast growth factor signaling system, neuronal migration, and the dopamine hypothesis, are dependent on the complete functionality of the cilium, although the specific mechanism is not yet well understood (Marley and von Zastrow, 2010;Muraki and Tanigaki, 2015;Narla et al., 2017;Hoseth et al., 2018). ...
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The gene Unc51-like kinase 4 (ULK4) belongs to the Unc-51-like serine/threonine kinase family and is assumed to encode a pseudokinase with unclear function. Recently, emerging evidence has suggested that ULK4 may be etiologically involved in a spectrum of neuropsychiatric disorders including schizophrenia, but the underlying mechanism remains unaddressed. Here, we summarize the key findings of the structure and function of the ULK4 protein to provide comprehensive insights to better understand ULK4-related neurodevelopmental and neuropsychiatric disorders and to aid in the development of a ULK4-based therapeutic strategy.
... However, it remains unknown whether this recruitment is mediated by direct interactions or through indirect mediators. The base of the conical SDA is formed by CC2D2A, ODF2 and CEP128, which form the inner SDA layer (Ishikawa et al., 2005;Kashihara et al., 2019;Tateishi et al., 2013;Veleri et al., 2014). Subsequently, CCDC120, CEP110 (also known as CNTRL), CCDC68 and NDEL1 interact with ODF2 and CEP128, establishing an intermediate layer (Chong et al., 2020;Huang et al., 2017;Kashihara et al., 2019;Tateishi et al., 2013). ...
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Centrosomes are the main microtubule-organizing centres, playing essential roles in the organization of the cytoskeleton during interphase, and in the mitotic spindle, which controls chromosome segregation, during cell division. Centrosomes also act as the basal body of cilia, regulating cilium length and affecting extracellular signal reception as well as the integration of intracellular signalling pathways. Centrosomes are self-replicative and duplicate once every cell cycle to generate two centrosomes. The core support structure of the centrosome consists of two molecularly distinct centrioles. The mother (mature) centriole exhibits accessory appendages and is surrounded by both pericentriolar material and centriolar satellites, structures that the daughter (immature) centriole lacks. In this Review, we discuss what is currently known about centrosome duplication, its dialogue with the cell cycle and the sequential acquisition of specific components during centriole maturation. We also describe our current understanding of the mature centriolar structures that are required to build a cilium. Altogether, the built-in centrosome asymmetries that stem from the two centrosomes inheriting molecularly different centrioles sets the foundation for cell division being an intrinsically asymmetric process.
... The transition zone membrane complex, which is required for cilia formation, contains CC2D2A together with Meckel-Gruber syndrome (MKS)-Joubert syndrome (JBTS)related proteins, such as centrosomal protein 290 (CEP290), tectonic family member 1 (TCTN1), and B9 domain containing 1 (B9D1) [42]. CC2D2A plays an essential role in the formation or stabilization of sub-basal appendages and the initiation of the process of ciliogenesis from the basal body; it is also essential for primary ciliogenesis [43]. Although few studies have been conducted on these proteins in chondrocytes, it has been reported that the expression of B9D1 is significantly reduced in osteoarthritis chondrocytes [44]. ...
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Introduction In cartilage regenerative medicine, transplanted chondrocytes contain a mixture of populations, that complicates the regeneration of uniform cartilage tissue. Our group previously reported that chondrocytes with higher chondrogenic ability could be enriched by selection of rapidly growing cells. In this study, the detailed properties of rapidly growing chondrocytes were examined and compared to slowly growing cells. Methods Human auricular chondrocytes were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) and analyzed using flow cytometry, focusing on division rates as indicated by fluorescence intensity and cell morphology according to the forward scatter and side scatter. Rapid and slow growing cell groups were harvested on days 2 and 4 after CFSE labeling, and their ability to produce cartilage matrix in vitro was examined. To compare the chondrogenic ability in vivo, the cells were seeded on poly-l-lactic acid scaffolds and transplanted into nude mice. Gene expression differences between the rapid and slow cell groups were investigated by microarray analysis. Results On day 2 after CFSE labeling, the rapidly growing cell group showed the highest proliferation rate. The results of pellet culture showed that the rapid cell group produced more glycosaminoglycans per cell than the slow cell group. The amount of glycosaminoglycan production was highest in the rapid cell group on day 2 after CFSE labeling, indicating high chondrogenic ability. Furthermore, microarray, gene ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed upregulation of genes that promote cell division such as origin recognition complex subunit 1 and downregulation of genes that inhibit cell division such as cyclin dependent kinase inhibitor 1A. Besides cell cycle-related genes, chondrocyte-related genes such as serpin family B member 2, clusterin, bone morphogenetic protein 2, and matrix metalloproteinase 3 were downregulated, while fibroblast growth factor 5 which is involved in stem cell maintenance, and coiled-coil and C2 domain containing 2A, which is required for cilia formation, were upregulated. Conclusion The results showed that the rapid cell group proliferated well and had more undifferentiated properties, suggesting a higher stemness. The present findings provide a basis for the use of the rapid cell group in cartilage regeneration.
... Gene Ontology (GO) analysis revealed that genes containing RetMICs are enriched for functions important for PR homeostasis such as cilium assembly and vesicle-mediated intracellular transport ( Fig. 1F and Table S2). We identified RetMIC-containing genes that localize to the cilia basal body and transition zone (POC1B, ARL6, CC2D2A [23][24][25], where they control ciliogenesis and vesicle anchoring to the OS microtubule axoneme. Others regulate OS disks morphogenesis (PROM1 26 ), PR homeostasis (IMPDH1 27 ), are involved in anterograde and retrograde transport (IFT88, DYNC2H1, KIF1B 28,29 ) or are part of the centrosomal complex (RPGRIP1L 30 ) (Fig. 1G). ...
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Retinal photoreceptors differ in their transcriptomic profiles from other neuronal subtypes, likely as a reflection of their unique cellular morphology and function in the detection of light thorough the ciliary outer segment. We discovered a new layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and in vesicle-mediated transport. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors and its deletion in zebrafish results in widespread downregulation of microexon inclusion, severe photoreceptor alterations and blindness. These results shed light into photoreceptor's transcriptomic specialization and functionality, uncovering new cell type-specific roles for Srrm3 and microexons with implication for retinal diseases.
... There was no electron dense deposit CC2D2A is expressed in brain, prostate, pancreas, kidney, lung, liver, and retina. It is a centrosome-cilia protein essential for the assembly of subdistal appendages, anchoring cytoplasmic microtubules and priming the mother centriole for axoneme biogenesis [7]. In zebrafish, the role of CC2D2A is suggested to be ciliary trafficking [8]. ...
Mutations in the ciliary gene TTC21B, NPHP4, and CRB2 cause familial focal and segmental glomerulosclerosis (FSGS). We report a girl with a mutation of the ciliary gene CC2D2A presenting with FSGS and nephronophthisis. The patient had mental retardation, postaxial polydactyly, and ataxic breathing, and was diagnosed as having compound heterozygous CC2D2A missense mutations at age 5. Retrospectively, azotemia at 1 year and proteinuria at 5 years were recorded but not investigated. At age 6, she was referred to the pediatric nephrology service because of hypertension, pretibial pitting edema, heavy proteinuria, and hematuria. eGFR was 66 ml/min/1.73 m2, total protein 5.3 g/dl, albumin 2.4 g/dl, and cholesterol 317 mg/dl. Ultrasonography showed normal-sized kidneys with a cyst in the right. Losartan was started. On renal biopsy, 8 out of 24 glomeruli were globally sclerosed, and three showed segmental sclerosis and/or hyalinosis with no immune deposits. Mild tubular dilatation, tubular atrophy, and interstitial fibrosis were observed. On electron microscopy, glomeruli showed focal foot process effacement with no electron dense deposits. Since losartan did not exert an obvious effect, treatment with prednisolone was tried. Urine protein decreased from 6.6 to 3.7 g/gCr. Prednisolone was discontinued after 10 days, however, because she developed duodenal ulcer perforation that necessitated omentoplasty. Subsequently, she was treated with losartan only. Her renal function deteriorated and peritoneal dialysis was initiated 8 months later. FSGS in this patient could be primary glomerular associated with CC2D2A mutation, rather than the consequences of tubulointerstitial fibrosis.
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The Ciliary Adhesion (CA) complex forms in close association with the basal bodies of cilia during the early stages of ciliogenesis and is responsible for mediating complex interactions with the actin networks of multiciliated cells (MCCs). However, its precise localization with respect to basal body accessory structures and the interactions that lead to its establishment in MCCs are not well understood. Here, we studied the distribution of the CA proteins using super-resolution imaging and possible interactions with the microtubule network. The results of this study reveal that the apical CA complex forms at the distal end of the basal foot and depends on microtubules. Our data also raise the possibility that CAs may have additional roles in the regulation of the organization of the microtubule network of MCCs.
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Meiosis is essential for evolution and genetic diversity in almost all sexual eukaryotic organisms. The mechanisms of meiotic recombination, such as synapsis, have been extensively investigated. However, it is still unclear whether signals from the cytoplasm or even from outside of the cell can regulate the meiosis process. Cilia are microtubule-based structures that protrude from cell surface and function as signaling hubs to sense extracellular signals. Here, we reported an unexpected and critical role of cilia during meiotic recombination. During gametogenesis of zebrafish, cilia were specifically present in the prophase stages of both primary spermatocytes and primary oocytes. By developing a germ cell-specific CRISPR/Cas9 system, we demonstrated that germ cell-specific depletion of ciliary genes resulted in compromised double-strand break repair, reduced crossover formation, and increased germ cell apoptosis. Our study reveals a previously undiscovered role for cilia during meiosis and suggests that extracellular signals may regulate meiotic recombination via this particular organelle.
Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3 , a paralog of the neural microexon regulator Srrm4 . Despite the fact that both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.
Vertebrates have an elaborate and functionally segmented body. It evolves from a single cell by systematic cell proliferation but attains a complex body structure with exquisite precision. This development requires two cellular events: cell cycle and ciliogenesis. For these events, the dynamic molecular signaling is converged at the centriole. The cell cycle helps in cell proliferation and growth of the body and is a highly regulated and integrated process. Its errors cause malignancies and developmental disorders. The cells newly proliferated are organized during organogenesis. For a cellular organization, dedicated signaling hubs are developed in the cells, and most often cilia are utilized. The cilium is generated from one of the centrioles involved in cell proliferation. The developmental signaling pathways hosted in cilia are essential for the elaboration of the body plan. The cilium's compartmental seclusion is ideal for noise-free molecular signaling and is essential for the precision of the body layout. The dysfunctional centrioles and primary cilia distort the development of body layout that manifest as serious developmental disorders. Thus, centriole has a dual role in the growth and cellular organization. It organizes dynamically expressed molecules of cell cycle and ciliogenesis and plays a balancing act to generate new cells and organize them during development. A putative master molecule may regulate and coordinate the dynamic gene expression at the centrioles. The convergence of many critical signaling components at the centriole reiterates the idea that centriole is a major molecular workstation involved in elaborating the structural design and complexity in vertebrates. This article is protected by copyright. All rights reserved.
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Intraflagellar transport (IFT) proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left–right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh) signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli) transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R) are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction.
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We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence micros-copy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts), or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G 1 /S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments , during G 1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.
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Comment on: Essential role of Cenexin1, but not Odf2, in ciliogenesis. [Cell Cycle. 2013]
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Formation of cilia, microtubule-based structures that function in propulsion and sensation, requires Kif3a, a subunit of Kinesin II essential for intraflagellar transport (IFT). We have found that, Kif3a is also required to organize centrioles. In the absence of Kif3a, the subdistal appendages of centrioles are disorganized and lack p150(Glued) and Ninein. Consequently, microtubule anchoring, centriole cohesion and basal foot formation are abrogated by loss of Kif3a. Kif3a localizes to the mother centriole and interacts with the Dynactin subunit p150(Glued). Depletion of p150(Glued) phenocopies the effects of loss of Kif3a, indicating that Kif3a recruitment of p150(Glued) is critical for subdistal appendage formation. The transport functions of Kif3a are dispensable for subdistal appendage organization as mutant forms of Kif3a lacking motor activity or the motor domain can restore p150(Glued) localization. Comparison to cells lacking Ift88 reveals that the centriolar functions of Kif3a are independent of IFT. Thus, in addition to its ciliogenic roles, Kif3a recruits p150(Glued) to the subdistal appendages of mother centrioles, critical for centrosomes to function as microtubule-organizing centres.
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Ciliopathies encompass a broad array of clinical findings associated with genetic defects in biogenesis and/or function of the primary cilium, a ubiquitous organelle involved in the transduction of diverse biological signals. Degeneration or dysfunction of retinal photoreceptors is frequently observed in diverse ciliopathies. The sensory cilium in a photoreceptor elaborates into unique outer segment discs that provide extensive surface area for maximal photon capture and efficient visual transduction. The daily renewal of approximately 10% of outer segments requires a precise control of ciliary transport. Here, we review the ciliopathies with associated retinal degeneration, describe the distinctive structure of the photoreceptor cilium, and discuss mouse models that allow investigations into molecular mechanisms of cilia biogenesis and defects. We have specifically focused on two ciliary proteins -- CEP290 and RPGR -- that underlie photoreceptor degeneration and syndromic ciliopathies. Mouse models of CEP290 and RPGR disease, and of their multiple interacting partners, have helped unravel new functional insights into cell type-specific phenotypic defects in distinct ciliary proteins. Elucidation of multifaceted ciliary functions and associated protein complexes will require concerted efforts to assimilate diverse datasets from in vivo and in vitro studies. We therefore discuss a possible framework for investigating genetic networks associated with photoreceptor cilia biogenesis and pathology.
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Primary cilia are microtubule-based solitary sensing structures on the cell surface that play crucial roles in cell signaling and development. Abnormal ciliary function leads to various human genetic disorders, collectively known as ciliopathies. Outer dense fiber protein 2 (Odf2) was initially isolated as a major component of sperm-tail fibers. Subsequent studies have demonstrated the existence of many splicing variants of Odf2, including Cenexin1 (Odf2 isoform 9), which bears an unusual C-terminal extension. Strikingly, Odf2 localizes along the axoneme of primary cilia, whereas Cenexin1 localizes to basal bodies in cultured mammalian cells. Whether Odf2 and Cenexin1 contribute to primary cilia assembly by carrying out either concerted or distinct functions is unknown. By taking advantage of odf2 (-/-) cells lacking endogenous Odf2 and Cenexin1, but exogenously expressing one or both of these proteins, we showed that Cenexin1, but not Odf2, was necessary and sufficient to induce ciliogenesis. Furthermore, the Cenexin1-dependent primary cilia assembly pathway appeared to function independently of Odf2. Consistently, Cenexin1, but not Odf2, interacted with GTP-loaded Rab8a, localized to the distal/subdistal appendages of basal bodies, and facilitated the recruitment of Chibby, a centriolar component that is important for proper ciliogenesis. Taken together, our results suggest that Cenexin1 plays a critical role in ciliogenesis through its C-terminal extension that confers a unique ability to mediate primary cilia assembly. The presence of multiple splicing variants hints that the function of Odf2 is diversified in such a way that each variant has a distinct role in the complex cellular and developmental processes.
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In addition to their role in motility, eukaryotic cilia serve as a distinct compartment for signal transduction and regulatory sequestration of biomolecules. Recent genetic and biochemical studies have revealed an extraordinary diversity of protein complexes involved in the biogenesis of cilia during each cell cycle. Mutations in components of these complexes are at the heart of human ciliopathies such as Nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS) and Joubert syndrome (JBTS). Despite intense studies, proteins in some of these complexes, such as the NPHP1-4-8 and the MKS, remain poorly understood. Using a combination of computational analyses we studied these complexes to identify novel domains in them which might throw new light on their functions and evolutionary origins. First, we identified both catalytically active and inactive versions of transglutaminase-like (TGL) peptidase domains in key ciliary/centrosomal proteins CC2D2A/MKS6, CC2D2B, CEP76 and CCDC135. These ciliary TGL domains appear to have originated from prokaryotic TGL domains that act as peptidases, either in a prokaryotic protein degradation system with the MoxR AAA+ ATPase, the precursor of eukaryotic dyneins and midasins, or in a peptide-ligase system with an ATP-grasp enzyme comparable to tubulin-modifying TTL proteins. We suggest that active ciliary TGL proteins are part of a cilia-specific peptidase system that might remove tubulin modifications or cleave cilia- localized proteins, while the inactive versions are likely to bind peptides and mediate key interactions during ciliogenesis. Second, we observe a vast radiation of C2 domains, which are key membrane-localization modules, in multiple ciliary proteins, including those from the NPHP1-4-8 and the MKS complexes, such as CC2D2A/MKS6, RPGRIP1, RPGRIP1L, NPHP1, NPHP4, C2CD3, AHI1/Jouberin and CEP76, most of which can be traced back to the last common eukaryotic ancestor. Identification of these TGL and C2 domains aid in the proper reconstruction of the Y-shaped linkers, which are key structures in the transitional zone of cilia, by allowing precise prediction of the multiple membrane-contacting and protein-protein interaction sites in these structures. These findings help decipher key events in the evolutionary separation of the ciliary and nuclear compartments in course of the emergence of the eukaryotic cell.
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Two intraflagellar transport (IFT) complexes, IFT-A and IFT-B, build and maintain primary cilia and are required for activity of the Sonic hedgehog (Shh) pathway. A weak allele of the IFT-A gene, Ift144, caused subtle defects in cilia structure and ectopic activation of the Shh pathway. In contrast, strong loss of IFT-A, caused by either absence of Ift144 or mutations in two IFT-A genes, blocked normal ciliogenesis and decreased Shh signaling. In strong IFT-A mutants, the Shh pathway proteins Gli2, Sufu, and Kif7 localized correctly to cilia tips, suggesting that these pathway components were trafficked by IFT-B. In contrast, the membrane proteins Arl13b, ACIII, and Smo failed to localize to primary cilia in the absence of IFT-A. We propose that the increased Shh activity seen in partial loss-of-function IFT-A mutants may be a result of decreased ciliary ACIII and that the loss of Shh activity in the absence of IFT-A is a result of severe disruptions of cilia structure and membrane protein trafficking.
Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.