When cilia go bad: cilia defects and ciliopathies

Transcription

REVIEWS
Mechanisms of Disease
When cilia go bad: cilia defects
and ciliopathies
Manfred Fliegauf*, Thomas Benzing‡ and Heymut Omran*
Abstract | Defects in the function of cellular organelles such as peroxisomes, lysosomes and
mitochondria are well-known causes of human diseases. Recently, another organelle has also
been added to this list. Cilia — tiny hair-like organelles attached to the cell surface — are
located on almost all polarized cell types of the human body and have been adapted as
versatile tools for various cellular functions, explaining why cilia-related disorders can affect
many organ systems. Several molecular mechanisms involved in cilia-related disorders have
been identified that affect the structure and function of distinct cilia types.
Intraflagellar transport
(IFT). A cilia-specific and
flagella-specific transport
system that relies on at least
16 different proteins that
assemble into transport rafts
and move ciliary components
across the compartment
border and along the
peripheral axonemal
microtubules to the ciliary tip
and back to the cell body.
IFT was first described in
bi-flagellate green algae
(Chlamydomonas reinhardtii).
Ciliogenesis
The processes of cilia assembly
and growth that follow and/or
accompany cell polarization.
*Department of Paediatrics
and Adolescent Medicine,
University Hospital Freiburg,
79106 Freiburg, Germany.
‡
Department of Medicine,
University of Cologne,
50937 Köln, Germany.
Correspondence to H.O.
e-mail: heymut.omran@
uniklinik-freiburg.de
doi:10.1038/nrm2278
Cilia (and flagella) are microtubule-based hair-like
organelles that extend from the surface of almost all cell
types of the human body (FIG. 1, see also the Primary
Cilia Resource web page). Although these highly conserved structures are found across a broad range of
species, a nearly ubiquitous appearance is observed
only in vertebrates. Cilia can be structurally divided
into subcompartments that include a basal body, trans
ition zone, axoneme, ciliary membrane and the ciliary
tip (BOX 1). Most cell types assemble only one cilium (a
monocilium or primary cilium), whereas some cells
build cilia bundles that consist of 200–300 individual
organelles (multiple cilia).
In contrast to other cell organelles, cilia are only
assembled when cells exit the cell cycle from mitosis
into a stationary or quiescent and/or differentiated state;
and vice versa, entry into the cell cycle is preceded by
ciliary resorption1. Cilia and flagella are highly complex structures that comprise >650 proteins (see the
Ciliomics, Cilia Proteome and Chlamydomonas Flagellar
Proteome web pages). The formation of cilia comprises
targeting of specific proteins to the basal body area
where pre-assembly of axonemal substructures (such
as outer dynein arms) occurs2. The transport of proteins
and multiprotein precursors across the ciliary compartment border and along the length of the axonemes to
their functional assembly site is dependent on intra
flagellar transport (IFT)3. Proteins are loaded onto the
IFT particles at the ciliary base within the cytoplasm
and transferred across the ciliary compartment border
in a process known as compartmentalized ciliogenesis4.
Mutations in genes encoding proteins that participate
in IFT cause ciliogenesis defects of both motile and
immotile cilia5–8. By contrast, cytosolic ciliogenesis is
independent of IFT.
880 | november 2007 | volume 8
Cilia can either be motile or immotile. The 9+2
axonemes of most motile cilia are assembled by nine
peripheral doublet microtubules surrounding two central
single microtubules (central pair complex) and contain
associated structures including inner and outer dynein
arms, radial spokes and nexin links (BOX 1). The 9+0
axonemes that are found in most non-motile cilia lack
the two central microtubules and are devoid of dynein
arms. The 9+0 axonemes of the motile nodal cilia lack
the central microtubules but have dynein arms. Although
there are many different classes of cilia with a diversity of
variations, all cilia types share the basic structural units
composed of the outer microtubule doublets and the
ciliary membrane. For example, cilia with a 9+4 axoneme
on the notochordal plate of the rabbit embryo have been
identified, and it has been proposed that the axonemal
structures may vary widely within the vertebrates9.
Four cilia types have been identified in humans and
all have been associated with human disease: motile 9+2
cilia (such as respiratory cilia, ependymal cilia); motile
9+0 cilia (nodal cilia); non-motile 9+2 cilia (kinocilium
of hair cells10); and non-motile 9+0 cilia (renal monocilia,
photoreceptor-connecting cilia). Recent advances have
indicated that all cilia (motile or non-motile) throughout
the diverse groups of organisms from protists to humans
might carry out sensory functions; thus, we prefer to avoid
the term ‘sensory cilia’, which is often used interchangeably
with ‘non-motile monocilia’ or ‘primary cilia’. In addition,
although they have not yet been identified, there might be
other motile monocilia types besides nodal cilia.
Although the basic structure of the different types
of cilia is obviously similar, they exert various tissuespecific functions during development, tissue morpho
genesis and homeostasis11–13, as discussed in more detail
below. Because cilia are located on almost all polarized
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© 2007 Nature Publishing Group
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Airways
Respiratory cilia
Brain
Ependymal cilia
Embryo
Nodal cilia
Female reproductive
system
Fallopian tube cilia
Motile 9+2
Motile 9+0
Non-motile 9+2
Inner ear
Kinocilium (red)
Stereocilia (green)
Non-motile 9+0
Kidney
Renal cilia
Male reproductive
system
Sperm flagella
Bile duct
Cholangiocyte cilia
Pancreas
Pancreatic duct cilia
Bone/cartilage
Osteocyte/
chondrocyte cilia
Eye
Photoreceptor
connecting cilia
Figure 1 | Ciliary dysfunction in human diseases. A monociliated cell is shown in the centre. Motile monocilia (9+0
axoneme, middle left panels) are found at the embryonic node and generate the nodal flow that is essential for
Nature Reviews | Molecular Cell Biology
determination of left–right body asymmetry. Multiple motile cilia (9+2 axonemal structure, top panels) that transport
extracellular fluid along the epithelial surface are located on respiratory epithelial cells, brain ependymal cells and
epithelial cells lining the fallopian tubes (panel reproduced with permission from REF. 59  (2005) Elsevier). The sperm
flagellum (top right panel; co-stained with antibodies against the dynein heavy chain DNAH5, red) represents a
specialized, elongated motile cilium (9+2) that confers motility. Non-motile monocilia (9+0, bottom panels) extend from
the surface of most quiescent cells of the body and sense environmental signals such as fluid flow and/or fluid composition.
Well-known examples are the monocilia of the tubular epithelia of the kidney, and the epithelia of the bile duct
(panel reproduced with permission from REF. 75  (2006) American Physiological Society) and pancreatic ducts (panel
reproduced with permission from REF. 129  (2006) Elsevier). The chondrocyte and osteocyte monocilia probably function
to sense the amount of strain in bones. The connecting cilia of photoreceptor cells are specialized non-motile cilia (9+0)
that connect the inner and outer segments. Non-motile 9+2 cilia (middle right panel) are found in the inner ear (kinocilium,
red, arrowhead; stereocilia, green) (panel reproduced with permission from REF. 10  (2005) John Wiley & Sons, Inc.).
Besides the four cilia types shown, there might be a high variability of the axonemal structures within vertebrates. In all
panels, axonemes were stained (red or green) by indirect immunofluorescence using an antibody against the cilia-specific
acetylated a‑tubulin isoform. Nuclei were stained using Hoechst or 4′,6-diamidino‑2-phenylindole (DAPI).
Notochordal plate
An epithelial primordial
structure of the notochord
(a cylindrical rod of cells).
The sheet of notochordal cells
is laterally in contact with the
roof of the primitive gut and
dorsally in contact with the
midline cells of the neural
plate. The notochordal plate
folds off from the roof of the
primitive gut to form the
notochord.
cell types of the human body, cilia-related disorders
— ciliopathies — can affect many organ systems.
Ciliopathies can either involve single organs or can occur
as multisystemic disorders with phenotypically variable
and overlapping disease manifestations14. Here, we focus
on the mechanisms by which ciliary dysfunction causes
human disorders. Notably, most of our knowledge about
cilia biology is based on genetic studies of model systems
such as Chlamydomonas reinhardtii and mutant mouse
models that have also enabled the development of novel
therapeutic options for human ciliopathies (BOX 2).
nature reviews | molecular cell biology
Functions of cilia
The existence of different cilia types indicates that this
organelle is likely to have numerous functions.
Motile functions of cilia. The ciliary axoneme comprises nine peripheral doublets, which have attached
dynein arms. Within these large multiprotein complexes, axonemal dynein heavy chains exert ciliary
movement by ATP-dependent conformational changes
and transient binding to neighbouring doublets. The
beating of each individual cilium is generated by
volume 8 | november 2007 | 881
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Box 1 | Ciliary subcompartments
The ciliary tip harbours the microtubule plus (+) ends (from which axonemes grow) and
the switch between the anterograde (kinesin) and retrograde (dynein) intraflagellar
transport (IFT) motors. It contains signalling molecules and can undergo morphological
changes in response to signalling processes.
The axoneme (see figure part 9) is the structural core of a cilium (without a membrane
or soluble material). Its peripheral microtubule doublets, comprising an A‑ and a
B‑tubule, are continuous with the microtubules at the transition zone. The doublets
are connected by nexin links and are held in place by radial spokes that extend into
the axonemal centre. In motile cilia, inner and outer dynein arms are attached to the
A‑tubules and mediate ciliary bending by reversibly binding to the neighbouring
B‑tubule. The central microtubule pair is surrounded by a sheath. The radial
spoke–central pair complex is involved in beat regulation.
The transition zone (parts 5–8) converts the triplet microtubular structure of the basal
body into the axonemal doublet structure. Proximal transition fibres (parts 4 and 5)
connect each microtubule doublet (without dynein arms) to the membrane and mark
the compartment border at which IFT proteins accumulate. The distal part contains
stellate fibre arrays (parts 6 and 8) and an amorphous disk structure (part 7) and gives
rise to the central microtubules in 9+2 axonemes121,122. The transition zone might contain
a gate that controls access to the ciliary compartment.
The basal body (parts 1–4) of each cilium is a specialized centriole (9 × 3 microtubular
structure; the tubules of one triplet are depicted as A, B and C in part 3) embedded in
pericentriolar material (dark orange) with a proximal amorphous disc (part 1), a
cartwheel structure (part 2), a middle piece that lacks appendages (part 3) and
transition fibres at the distal end (part 4). In most quiescent cells, the centrioles move to
the apical plasma membrane and the mother centriole functions as the microtubuleorganizing centre to nucleate the axonemal microtubules. The daughter centriole
remains perpendicular to the mother centriole. In multiciliated cells, centriolar
replication is required first.
The ciliary membrane is continuous with the plasma membrane but contains specific
signalling molecules that are essential for the function of cilia as antennae. IFT rafts move
between the ciliary membrane and the peripheral microtubules and carry membrane
anchors. Figure modified with permission from REF. 122  (2003) Blackwell Publishing.
Ciliary tip
B A
9
9+2 axoneme
8
4
7
Transition zone
3
C
B
A
6
2
1
Basal body
5
coordinated activation and inactivation of the dynein
motor proteins within the inner and outer dynein arms
along the length of the axoneme15. Ciliary motility is
required to move extracellular fluid: the motile 9+0
monocilia at the embryonic node generate an extraembryonic fluid flow (nodal flow) that is required to
determine embryonic left–right asymmetry16. Motility
of the multiple 9+2 cilia of respiratory epithelial cells
is responsible for mucociliary clearance. Analogously,
the multiple 9+2 ependymal cilia mediate ependymal
flow17. Furthermore, flagellar motility is required for
sperm cells to propel through the female reproductive
system.
Non-motile functions. Functions of cilia that are not
related to motility are thought to involve sensing of
environmental cues. Because cilia protrude from the
cell surface, they might act as antennae that receive signals from the periphery. The remote information may
be converted into signalling cascades that are initiated
within the ciliary compartment and then transduced
to the cell body. Consistently, the ciliary membrane
(which is continuous with the plasma membrane)
contains various cilia-specific receptors, ion channels
and signalling molecules. For example, flow-induced
passive cilia bending is required for mechanosensation
of extracellular fluid flow (for instance, tubular fluid,
urine)18. Studies in Caenorhabditis elegans have shown
that transient receptor potential vanilloid channels in
the sensory cilia membranes are transported bidirec
tionally by the IFT system. Therefore, IFT is not only
necessary for transport of axonemal components, but is
also important for the sensory activity of cilia19. Recent
observations indicate that chemosensation, as well as
signalling through receptor-dependent pathways such as
the sonic hedgehog (SHH), platelet-derived growth factor receptor (PDGFR) pathways or non-canonical Wnt
(also known as planar cell polarity (PCP)) pathways,
is also mediated through cilia20–22.
Cilia in development
Cilia in left–right asymmetry. A link between motile
cilia dysfunction and defects in establishing left–right
body asymmetry is apparent from the observation
that half of individuals with primary ciliary dyskinesia
(PCD) exhibit situs inversus totalis (also referred to
as Kartagener’s syndrome), which is consistent with
randomization of left–right asymmetry 11. A similar phenotype was observed in mice with recessive
mutations in Lrd, the orthologue of DNAH11, a
human axonemal dynein β‑chain gene23. During early
embryonic development (~7.5 days postcoitum), the
rotational movement of nodal cilia at the ventral pole
of the murine embryo creates a leftward fluid flow
(nodal flow) that is thought to induce breaking of the
body symmetry. Indeed, an artificially generated nodal
flow independent from ciliary motility was found to
be sufficient to determine laterality24. Similar laterality breaking mechanisms have also been proposed for
zebrafish (involving Kupffer’s vesicle), birds (Hensen’s
node), and amphibians (Spemann’s organizer)25.
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Box 2 | Therapeutic options
On the basis of the knowledge of the distinct mechanisms involved in many ciliarelated diseases, novel therapeutic options are currently being evaluated to attenuate
disease progression. In cystic kidney disease, several molecular mechanisms have been
identified that can be targeted by pharmacological therapy.
Dysregulated cell cycle. Pharmacological interventions to slow down the cell-cycle
progression involved in cystic kidney disease have been evaluated123. The cyclindependent kinase (CDK) inhibitor roscovitine (CYC202; a potent inhibitor of CDK2–
cyclin E) retarded cystogenesis and improved renal function with a long-lasting effect
in two mouse models of slowly progressing polycystic kidney disease. Roscovitine
treatment inhibited the formation of cysts from distinct nephron segments by blocking
the cell cycle, by transcriptional regulation and by reduction of apoptosis. This CDKselective inhibitor has minimal off-target kinase activities, and might therefore be a
promising candidate for clinical trials of human cystic kidney disease.
Altered downstream signalling. The expression of several mitogen-activated protein
kinases (MAPKs) is dysregulated in the cyst epithelium of pcy (polycystic kidney
disease) mice that carry a missense mutation in Nphp3 (encoding nephrocystin‑3),
the orthologous gene that is responsible for adolescent nephronophthisis124.
This dysregulation is probably a downstream consequence of disturbed renal monocilia
function125. Inhibition of extracellular signal-regulated kinase (ERK)–MAPK signalling
attenuated the progression of renal disease in pcy mice, implying that selective
targeting of downstream signalling events might be beneficial. Other potential
downstream targets for inhibition in polycystic kidney disease include the
vasopressin‑2 receptor, epidermal growth-factor receptor and Src126.
Increased mammalian target of rapamycin (mTOR) activity. Hamartin and tuberin,
proteins that are implicated in the formation of renal cysts in tuberous sclerosis, have
been found at the ciliary base. They form a heteromeric complex that inhibits mTOR, a
kinase that controls cell growth and proliferation. Inhibition of mTOR activity retards
cyst formation in rats with polycystic kidney disease127,128. Studies using mTOR inhibitors
in patients with cystic kidney disease and tuberous sclerosis are currently underway.
Anterograde
The transport direction from
the ciliary base to the tip.
Retrograde
The transport direction from
the ciliary tip back to the cell
body.
Mucociliary clearance
The process by which the
continuous coordinated
beating of respiratory cilia
moves the thin mucus layer
that covers the airway epithelia
towards the pharynx to defend
against inhaled pathogens
trapped in the mucus.
Ependymal flow
The laminar flow of
cerebrospinal fluid through the
brain ventricles and the
cerebral aqueduct generated
by the coordinated beating of
ependymal cilia.
Nodal flow hypotheses. Two models have been proposed
for how the nodal flow might contribute to left–right
asymmetry (FIG. 2). The ‘two cilia’ model26 predicts that
nodal flow generated by motile cilia is sensed by nonmotile, mechanosensory cilia at the periphery of the
node. This model is mainly based on the observation
that expression of polycystin‑2, a protein thought to be
involved in mechanosensation, is restricted to cilia at the
periphery of the node, whereas cilia in the centre of
the node express LRD but lack polycystin‑2. The second
model expands on the morphogen gradient model, and
predicts that nodal flow results in a leftward gradient
of a hypothetical morphogen. Tanaka et al. identified
nodal vesicular parcels filled with sonic hedgehog and
retinoic acid molecules, which bud off the nodal surface
to be transported leftwards by nodal flow where they
are smashed to release their contents27. Both models
reported an asymmetric Ca2+ release that is probably
involved in the subsequent events of left–right determin
ation, which, in turn, is based on asymmetric expression
of signalling molecules such as nodal and lefty, and
transcription factors such as PITX2 (paired-like homeo
domain-2). Nakamura et al. recently suggested that a
mechanism known as self-enhancement and lateralinhibition system (SELI) is necessary to generate robust
asymmetry28.
Current nodal flow hypotheses cannot sufficiently
explain the complex laterality defects that are observed
in humans and mice with inborn ciliary motility
defects (FIG. 2). Although most patients with PCD who
have DNAH5 or DNAI1 mutations or Dnahc5-mutant
mice exhibit situs solitus or situs inversus totalis, a small
proportion show partial laterality defects such as situs
inversus abdominalis and situs inversus thoracalis29–32. These
observations indicate that the reversal of left–right
body asymmetry can independently occur along the
anterior–posterior axis (corresponding to the upper
and lower part of the human body) and is controlled by
nodal cilia function. The site of the future diaphragm
appears to be the border of this determination. It can be
postulated that functional differences of the anterior and
posterior part of the node, distinct signalling molecules
for determining upper–lower body asymmetry and/or
temporal diversity of left–right determination (that is,
first the lower and then the upper part) might explain
partial laterality defects.
Disorders of development and growth
Numerous cilia-related diseases have been described that
are associated with developmental defects affecting the
central nervous system, the skeleton or other organ systems. Several signalling pathways have been implicated
in ciliary function.
Hedgehog signalling. Loss of activity of the Hedgehog
(Hh) signalling pathway33 can cause various birth defects,
including holoprosencephaly, polydactyly, craniofacial defects
and skeletal malformations34. These abnormalities resemble the developmental defects observed in IFT mutant
mice (Tg737∆2-3bGal and Tg737orpk mutations; Tg737 is
also known as Ift88 (intraflagellar transport-88 homologue) or polaris)6,35. An ENU (N-ethyl‑N-nitrosourea)
screen for embryonic patterning defects identified two
mouse mutants with phenotypes that are reminiscent of
defects of the Hh signalling pathway20. In both strains,
mutations in the genes encoding the IFT proteins
IFT172 and IFT88 were identified. Further analyses
showed that the IFT machinery is essential for Hh signalling downstream of the Hh receptor patched-1 and
upstream of direct transcriptional targets of Hh (FIG. 3a).
Analyses in the developing limb buds of IFT-mutant
mice also suggested altered Hh signalling downstream
of patched-1 (Refs 36–37).
In mammals, the main targets of Hh signalling are
the glioma (GLI) transcription factors GLI1, GLI2 and
GLI3. Haycraft et al. demonstrated that GLI2 and fulllength (the activator form) GLI3 functions are disrupted
in the Tg737 mutant cells, but that GLI1 and GLI3R (the
repressor form) can induce or repress the Hh pathway,
respectively, regardless of IFT function. Localization of
all GLI proteins as well as suppressor of fused (SUFU)
to the distal tip of cilia in primary limb-bud cell cultures
confirmed a prominent role for Hh signalling in the
cilium37. Proteolysis of GLI3 into the GLI3R repressor
form probably involves SUFU function at the ciliary tip
(FIG. 3a). Hh signalling therefore appears to require the IFT
machinery to shuttle GLI proteins and SUFU to the
ciliary tip. In the absence of Hh binding to patched-1,
smoothened (SMO) is not released from patched-1 and so
GLI3 is constantly proteolytically cleaved into the repressor
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a Laterality defects
Situs solitus
Situs inversus totalis
Left isomerism (polysplenia)
Situs inversus abdominalis
Right isomerism (asplenia)
Lung
Lung
Heart
Liver
Spleen
Stomach
Situs inversus thoracalis
ul
lu
b
Extra-embryonic fluid
Right
NVP
Nodal flow
Left
Intracellular
Ca2+ release
Epithelial cell
c
Extra-embryonic fluid
Right
Nodal flow
Left
Intracellular
Ca2+ release
Sensory cilia
Motile cilia
Sensory cilia
Figure 2 | Human laterality disorders and current models for establishing left–
Nature
Reviews
| Molecular
Cell Biology
right asymmetry. a | Schematic illustration of normal
left–right
body
asymmetry
(situs solitus) and five laterality defects that affect the lungs, heart, liver, stomach and
spleen. By their vigorous circular movements, motile monocilia at the embryonic node
generate a leftward flow of extra-embryonic fluid (nodal flow). b | The nodal vesicular
parcel (NVP) model predicts that vesicles filled with morphogens (such as sonic hedgehog
and retinoic acid) are secreted from the right side of the embryonic node and transported
to the left side by nodal flow, where they are smashed open by force27. The released
contents probably bind to specific transmembrane receptors in the axonemal membrane
of cilia on the left side. The consequent initiation of left-sided intracellular Ca2+ release
induces downstream signalling events that break bilaterality. In this model, the flow of
extra-embryonic fluid is not detected by cilia-based mechanosensation. c | In the two-cilia
model, non-sensing motile cilia in the centre of the node create a leftward nodal flow that
is mechanically sensed through passive bending of non-motile sensory cilia at the
periphery of the node26. Bending of the cilia on the left side leads to a left-sided release of
Ca2+ that initiates the establishment of body asymmetry.
GLI3R. GLI3R is then transported by cytoplasmic
dynein motors to the cell body and subsequently targeted for nuclear entry. Hh binding to patched-1 induces
the release of SMO, which can suppress GLI3 processing. The active GLI3A form is then transported to the
nucleus, where it can activate target genes (FIG. 3a). This
concept is compatible with the finding that mutations
in Shh, anterograde and retrograde IFT motors (such as
Kif3a (kinesin family member-3A) and Dync2h1 (dynein
cytoplasmic-2 heavy chain-1), respectively) as well as
IFT proteins and GLI3 result in similar developmental
defects20,38–41.
Wnt signalling. Mice with defective IFT proteins and
mutations in the Bardet–Biedl syndrome (BBS) genes
Bbs1, Bbs4 or Bbs6 (also known as Mkks, which encodes
McKusick–Kaufman syndrome protein) exhibit pheno
types resembling those observed in mutants of the
non-canonical Wnt pathway (also known as the PCP
pathway). These include open eyelids, neural tube defects
and disrupted cochlear stereociliary bundles42. The evolutionarily conserved PCP pathway (FIG. 3b) contributes
to the development of polarity in the plane of a cell layer,
controlling cellular processes such as cell migration or
mitotic spindle orientation43.
Genetic interaction of BBS genes and the PCP gene
Ltap (also called Vangl2) in double heterozygous mouse
mutants as well as in zebrafish (vangl2), and the observation that the PCP protein VANGL2 colocalizes with
BBS proteins to the basal body and ciliary axoneme,
confirmed the hypothesis that cilia are involved in PCP
signalling22. Further evidence that ciliary dysfunction
contributes to neural tube defects is provided by the demonstration that mutations in MKS1 and MKS3, which
encode the ciliary proteins MKS1 and meckelin44–45,
respectively, are associated with the multisystemic
Meckel–Gruber syndrome (MKS type 1–3). This is an
autosomal recessive lethal malformation disorder that,
among other characteristics, is regularly associated with
proximal neural tube defects (encephaloceles). Closure
of the neural tube during embryogenesis requires the
orientation of polarized epithelial cells in a single plane
perpendicular to the apical–basal axis as well as convergent extension, which leads to the narrowing and
lengthening of tissues during development. Therefore,
disruption of the PCP pathway owing to defective ciliamediated signalling (FIG. 3b) might explain the neural
tube defects. Recent studies have also shown that the
vertebrate PCP proteins inturned (Int) and fuzzy (Fuz)
are essential for ciliogenesis and that, as a consequence,
mutant Xenopus laevis embryos also have Hh signalling
defects46.
Signalling involving receptors attached to cilia. Besides
skeletal patterning defects, Tg737- and polycystin-1
(Pkd1)-mutant mice show stunted growth after birth,
which implies defects in appositional and endochondral development. An essential role for the primary cilia
of osteoblasts and osteocytes in bone development is
further supported by the observation that Pkd1-mutant
mice show severe skeletal defects, including abnormal
REVIEWS
formation of the axial skeleton, long bones and spines,
disrupted structure and organization of the vertebrae,
spina bifida occulta and osteochondro-dysplasia47,48. Thus,
it has been hypothesized that cilia from chondrocytes,
osteoblasts, odontoblasts, fibroblasts and myocytes
might sense mechanical strain or chemosensory signals
to aid interaction of the cell with its surrounding
extracellular matrix. Receptors for extracellular matrix
proteins, such as α2, α and β1 integrins, localize to the
ciliary membrane of chondrocytes, an observation that
supports this concept49.
In NIH3T3 fibroblasts and cultures of mouse
embryonic fibroblasts, the primary cilium is important
in growth control21. In growth-arrested fibroblasts,
the PDGFRα localizes to the primary cilium. Liganddependent activation of PDGFRα within the ciliary
Primary ciliary dyskinesia
(PCD). A genetically and
phenotypically heterogeneous
group of disorders
characterized by defective
ciliary motility.
Nephronophthisis
An autosomal recessive cystic
kidney disease characterized
by normal or reduced
kidney size, cysts at the
corticomedullary border and
predominant tubulointerstitial
fibrosis. Phthisis is a Greek
word meaning shrinking or
wasting.
a
SUFU
Anterograde
IFT
GLI
Retrograde
IFT
Transition zone
Basal body
PTCH1
Hh
Ciliary membrane
Plasma membrane
PTCH1
SUFU
GLI
Axoneme
GLIR
GLIA
SMO
SMO
Gene X
Nucleus
b
Canonical Wnt
signalling
Gene X Nucleus
Non-canonical Wnt (PCP) signalling
Ca2+
Flow
WNT
Frizzled
Frizzled
β-cat
DSH
Ca2+
Inversin
Inversin
DSH
APC/C
DSH
Nucleus
Axin GSK
APC
Nucleus
Gene X
Inversin
c
PDGF
PDGFRα
Flow
MEK/ERK
Nucleus
Gene X
Nucleus
Gene X
membrane is followed by activation of AKT and the
MEK1/2 (mitogen activated protein kinase (MAPK)–
extracellular signal-regulated kinase (ERK) kinase)–
ERK1/2 pathways, with MEK1/2 being phosphorylated
within the cilium and at the basal body (FIG. 3c). Thus,
the MEK1/2–ERK1/2 pathway is an example of a distinct signalling pathway that can be activated within the
subcellular ciliary compartment. For other signalling
pathways (for example, the RAS pathway), see also the
Cilia Proteome database.
Hydrocephalus formation. In the brain ventricles,
the synchronized beating of the ependymal 9+2 cilia
generates a laminar flow of cerebrospinal fluid above
the ependymal cell surface and through the cerebral
aqueduct, which is termed ependymal flow17. A link
Figure 3 | Models of cilia-generated signalling
mechanisms. a | Hedgehog (Hh) signalling in cilia involves
the intraflagellar transport machinery, which moves
components of the Hh signalling pathway to their
functional sites. The transcription factors glioma (GLI) and
suppressor of fused (SUFU) are transported to the ciliary
tip. GLI is processed to create a transcriptional repressor,
which is transported back to the cell body (left panel).
On Hh ligand binding to its receptor patched-1 (PTCH1),
smoothened (SMO) is released and transported to the
ciliary tip, where it turns off GLI processing by interacting
with SUFU. The activator form of the GLI transcription
factor is transported to the cell body and enters the
nucleus where it induces the expression of genes, such as
those involved in renal patterning (PAX2, SALL1), cell-cycle
regulation (CCND1 (cyclin D1), N‑MYC) and GLI family
members themselves (GLI1, GLI2) and PTCH1 (selfinduction). b | Cilia-mediated signalling acts as a switch
between canonical and non-canonical Wnt signalling
pathways. In the absence of fluid flow, canonical Wnt
signalling predominates. WNT binds to the receptor
frizzled, dishevelled (DSH) is recruited to frizzled, and
glycogen synthase kinase‑3β (GSK3β) is inactivated.
β‑catenin (β-cat) translocates to the nucleus where it acts
as a transcriptional co-activator with members of the
lymphoid enhancer binding factor (LEF)–T-cell-specific
transcription factor (TCF) family and induces transcription
of WNT target genes such as cMYC, AXIN2 or L1CAM (left
panel). On mechanosensation of fluid flow, intracellular
Ca2+ release causes increased inversin expression. Inversin
targets cytoplasmic DSH for anaphase-promoting
complex/cyclosome (APC/C)-dependent ubiquitylation
and degradation, making it unavailable for canonical Wnt
signalling. In non-canonical Wnt signalling (planar cell
polarity (PCP) pathway), β‑catenin undergoes degradation
by a complex of axin, adenomatous polyposis coli (APC)
and GSK3β. Inversin does not affect DSH recruitment to
the plasma membrane, where DSH is available for noncanonical Wnt signalling. c | The chemosensation-/
receptor-based signalling model. Receptors such as
platelet-derived growth factor receptor-α (PDGFRα) are
located within the axonemal membrane (left panel).
Ligands in the tubular flow bind to their receptors,
inducing cellular responses through downstream signalling
pathways such as the MEK/ERK cascade. IFT, intraflagellar
transport; MEK, mitogen activated protein kinase (MAPK)–
extracellular signal-regulated kinase (ERK) kinase. Part a
modified with permission from REF. 130  (2006) Elsevier.
REVIEWS
Subarachnoid space
Tel
Mes
Rho
Lv
III
Spinal cord
IV
Brain cortex
Lv
III
Aq
IV
P0.5
Lv
E10
E11.5
III
Aq
IV
E14.5
Adult
Situs inversus totalis
The complete mirror-image
arrangement of all thoracic and
abdominal organs.
Situs solitus
The normal position of the
viscera (stomach and spleen on
the left side, liver on the right
side). The three-lobed lung is
positioned on the right, with
the two-lobed lung on the left,
and the left and right cardiac
atria are positioned normally.
Situs inversus abdominalis
The isolated inversion of
abdominal organs, but a
normal composition of thoracic
organs.
Situs inversus thoracalis
The isolated inversion of
thoracic organs, but a normal
composition of abdominal
organs.
Holoprosencephaly
A developmental disorder of
the brain due to a failure of
the embryonic forebrain (the
prosencephalon) to form
bilateral hemispheres of the
cephalon. This causes defects
in brain structure and function
and also affects the
development of the face.
Polydactyly
Supernumerary fingers or toes.
The presence of six fingers or
six toes on one or both hands
or feet is usually called
hexadactyly.
Craniofacial defects
The developmental
abnormalities that affect the
head or skull and structures of
the face.
Figure 4 | Hydrocephalus in mice as a result of a lack of ependymal flow. Schematic illustration of the ventricular
system during mouse brain development (at embryonic day (E)10, E11.5, E14.5, postnatal
dayReviews
(P)0.5 and
in the adult).
Nature
| Molecular
Cell Biology
At E10, the brain has developed from the anterior end of the neural tube into three primary vesicles: telencephalic
(Tel; yellow), mesencephalic (Mes; blue) and rhombencephalic (Rho; orange). Later (at E14.5), the two lateral (Lv I and II),
third (III) and fourth (IV) ventricles develop. Cerebrospinal fluid, which is predominantly produced in the lateral ventricles
(Lv), is transported through the ventricular system and enters the subarachnoid space through foramina at the fourth
ventricle (not shown), where it is finally re-absorbed. During late embryonic brain development, the cerebral aqueduct
(Aq) connecting the third and fourth ventricle is formed and becomes the narrowest part of the cerebrospinal fluid
system (P0.5). In Dnahc5-mutant mice, the lack of ependymal flow owing to immotile ependymal cilia causes closure of
the aqueduct and subsequent formation of triventricular hydrocephalus during early postnatal brain development.
Figure reproduced with permission from REF. 17  (2004) Oxford University Press.
between ependymal cilia dysfunction and hydrocephalus
(enlargement of the brain ventricles) became apparent
by analysis of different animal models. Mice lacking the
axonemal dynein heavy chain Dnahc5, the axonemal
protein SPAG6 or proteins involved in ciliogenesis,
such as IFT88 or FOXJ1 (forkhead box J1; also known
as HFH‑4), develop hydrocephalus7,17,50,51. Moreover,
the spontaneous hydrocephalic WIC-Hyd rat mutant
exhibits impaired ependymal ciliary motility52. Hydin
(also known as Hy3)-mutant mice develop hydrocephalus and have reduced ependymal cilia53. In C. reinhardtii,
hydin is required for flagellar motility and localizes to a
specific projection on a single microtubule of the central apparatus54. Mutant flagella are arrested in one of
two switch points in the beat cycle: the beginning of the
effective stroke or the beginning of the recovery stroke.
Therefore, hydrocephalus caused by hydin mutations
probably involves defects in the central pair apparatus
that result in impaired ciliary motility and ciliary degeneration and makes the human orthologue an interesting
candidate for causing hydrocephalus in humans.
Two cilia-related disease mechanisms have been
implicated in hydrocephalus formation. The first was
described in Dnahc5-mutant mice, which have dysmotile cilia31. The consequent lack of ependymal flow
causes a secondary closure of the aqueduct and subsequent formation of triventricular hydrocephalus during
early postnatal brain development (FIG. 4). In humans,
ependymal ciliary dysmotility is not sufficient to cause
hydrocephalus but increases the risk of aqueduct closure;
hence, there is a higher incidence of this rare disorder in
patients with PCD.
The second mechanism for hydrocephalus formation
was described for Tg737orpk mice, which exhibit ciliogenesis defects of both motile and immotile cilia owing to a
mutation of the IFT protein IFT88 (REF. 55). However, in
these mice, hydrocephalus formation starts at postnatal
day 1, and loss of both the coordinated ciliary beat on
ventricular ependymal cells and consequent ependymal flow does not seem to be the initiating factor for
hydrocephalus formation. Rather, Tg737orpk mice have
aberrantly formed cilia of the choroid plexus, in which
polycystin‑1 is mislocalized to the bulb-like structure at
the tip, rather than to basal bodies and the distal ciliary
axoneme. Therefore, it has been speculated that cilia
in Tg737orpk mice have altered mechanosensory functions that might lead to altered ion transport activity in
the choroid plexus epithelium and, subsequently, to a
marked increase in cerebrospinal fluid production.
Dysfunction of the reproductive system
Sperm immotility (Supplementary information S1,S2,S3
(movies)) significantly contributes to male infertility.
The composition of sperm tails and respiratory cilia
is similar but not identical, which might explain why
sperm flagella dyskinesia is often, but not necessarily,
associated with PCD and vice versa. The understanding of these differences is aided by the demonstration
of cell-type-specific spatial localization of axonemal
proteins such as outer dynein arm components along
the length of the ciliary and flagellar axonemes. In addition, it can be speculated that the assembly processes
of human cilia and sperm flagella are not completely
identical. For instance, in flies, sperm tail generation
REVIEWS
Bardet–Biedl syndrome
(BBS). A clinically pleiotropic
disorder that has a primarily
autosomal recessive
inheritance pattern (twelve loci,
BBS1–BBS12, have been
identified so far) and a
multitude of symptoms
including rod–cone dystrophy,
retinitis pigmentosa, obesity,
polydactyly, renal
abnormalities (such as cystic
kidneys), learning disabilities or
mental retardation, male
hypogonadism and congenital
heart defects. BBS proteins
localize either to the ciliary
base or the axoneme and are
involved in subcellular targeting
of ciliary proteins. Seven of the
known BBS proteins assemble
into a core complex called the
BBSome.
Spina bifida
A developmental abnormality
that results from an incomplete
closure of the embryonic
neural tube and an
incompletely formed spinal
cord that protrudes through an
open gap in the unfused spines
of the vertebrae (spina bifida
aperta). In the milder form,
spina bifida occulta, the spinal
cord does not protrude
because only a small part of
one vertebra is missing and
there is no opening to the skin.
Osteochondro-dysplasia
An abnormal growth of
cartilage and bone (individual
bones or group of bones).
Growth defects of the long
bones and/or spine usually
cause shortened limbs or a
disproportionately shortened
body.
Metachronal wave
A wave-like movement that is
propagated along the epithelial
surface, created when cilia on
one segment of the epithelium
move after another.
Cholangiocytes
The epithelial cells of the bile
ducts.
involves cytosolic ciliogenesis, in which the axoneme
is assembled inside the cytoplasm of the spermatid,
independent of IFT4,56. However, observations in Tg737mutant mice indicate that spermiogenesis in vertebrates
requires IFT (G. Pazour, personal communication).
In the female reproductive system, the fallopian tube
epithelium undergoes hormonally mediated cyclical
morphological changes that affect the ciliated cells
and the ciliary beat frequency57,58. The mechanism by
which the ovarian duct epithelium responds to various
hormonal and neuronal stimuli is incompletely understood, although the cilia might function in sensory perception and signal transduction from the extracellular
environment59–61.
The propulsion of gametes and embryos is accomplished by complex interaction between muscle contractions, ciliary activity and the flow of tubal secretions62.
It is therefore still unclear whether ciliary motility has
a prominent role in transport of the fertilized ovum. In
female patients with PCD, fertility has been reported
to be slightly reduced and, thus, the activity of the
oviduct cilia is thought to be partially involved in egg
transport63.
Airway disease
Respiratory cilia characteristically beat with a forward
effective stroke and a backward recovery stroke in the
same plane along the cell surface. Moreover, beating of
the cilia bundles located on each single cell, as well as the
cilia of the entire cell layer, follow a highly regulated synchronized beating mode (Supplementary information
S4,S5 (movies)). This coordination generates a contin
uous series of metachronal waves that are essential for
mucus transport and airway clearance. In patients with
PCD, impaired mucociliary clearance causes chronic airway diseases of the upper (nasopharynx, sinus, middle
ear) and lower (bronchi, bronchioles) airways64. Airway
cilia from such patients often show characteristic
ultrastructural defects of the axoneme, such as absence
of some or all inner and/or outer dynein arms, radial
spoke defects or microtubule malposition, most of
which are associated with characteristic aberrant beating patterns65 (Supplementary information S6,S7,S8,S9
(movies)).
Outer dynein arm multiprotein complexes. Identification
of genes involved in PCD66 has aided understanding of
ciliary beat generation and regulation. Mutations in
DNAH5 and DNAI1, which encode axonemal motor
components responsible for ciliary movement generation, cause various defects of the distinct outer dynein
arm (ODA) multiprotein complexes along the respiratory ciliary axoneme. Human respiratory cilia contain
at least two distinct ODA types: type 1 is located within
the proximal ciliary axoneme and contains DNAH5 but
not DNAH9, and type 2 contains both DNAH5 and
DNAH9 and localizes to the distal ciliary axoneme67.
Interestingly, DNAH5 mutations regularly affect ODA
assembly of both known human ODA types, whereas
DNAI1 mutations mainly affect the assembly of the
distally located axonemal ODA complexes. Consistent
with these findings, immotile cilia often arise from
DNAH5-mutant axonemes, whereas DNAI1 mutant cilia
often retain some residual flickery movement capacity
(Supplementary information S6,S7 (movies)). Similar to
mutations of C. reinhardtii orthologues, mutations in both
human genes probably result in abnormal pre-assembly
of ODA multiprotein complexes. Consequently, dynein
heavy chain DNAH5 accumulates at the ciliary base,
which is consistent with the current hypothesis that preassembly of axonemal multiprotein complex precursors
occurs at the basal bodies2,67–69.
Rarely, PCD is caused by mutations in the genes
OFD1 (mutated in orofacialdigital syndrome; OFD) and
RPGR (retinitis pigmentosa guanosine triphosphatase
(GTPase) regulator), which do not primarily affect
axonemal motor proteins70,71. Both proteins are localized
at the ciliary base. In respiratory cells they are involved
in ciliary beat regulation and mutations result in altered
beating patterns (Supplementary information S9
(movie)). In addition, these proteins are also important
in the sensory function of 9+0 cilia (see below).
Cystic disorders of the kidney, liver and pancreas
Evidence for a common mechanism involving cilia.
IFT mutant Tg737orpk mice develop progressive kidney
and pancreatic cysts as well as abnormalities involving
the hepatic bile ducts5,7,72,73, which suggests that ciliary
dysfunction affects the morphogenesis and integrity of
kidney, liver and pancreas. All three organs contain similar functional units, tubular systems that transport urine,
bile fluid or pancreatic secretions; monocilia extend into
the lumen of each. In humans, biliary dysgenesis can
occur as an isolated disorder or in combination with
polycystic kidney disease.
The frequent association of renal and hepatic cysts
in autosomal dominant polycystic kidney disease
(ADPKD) and autosomal recessive polycystic kidney
disease (ARPKD), or nephronophthisis (NPHP) and
liver fibrosis (which occurs in Boichis disease) also suggests that a common mechanism might explain each of
these conditions. A rodent model of human ARPKD
develops cystic kidney and liver disease along with
bile-duct defects and shows malformed monocilia of
cholangiocytes74,75.
Most proteins associated with various forms of
human cystic kidney disorders (sometimes referred to
as cystoproteins) indeed localize to distinct ciliary subcompartments, supporting the hypothesis of cilia-related
disease mechanisms76. Proteins involved in BBS, NPHP,
OFD type 1, ARPKD and ADPKD localize either to the
ciliary base or the ciliary axoneme.
Polycystin, Ca 2+ and mechanosensation. The first
insights into the functional role of monocilia in tubular cells came from studies in cultured renal collecting
duct epithelial cells, which demonstrated a persistent
intracellular Ca2+ increase in response to flow-mediated
ciliary bending18,77. Subsequent analyses showed that
this Ca2+ influx required polycystin‑1 and polycystin‑2,
the proteins mutated in ADPKD78. Because depletion
of extracellular Ca2+ (as well as inhibition of ryanodine
REVIEWS
PC1–PC2 complex
(Ca2+ channel)
Ca2+
Axoneme
Flow
Transition zone
Basal body
Ca2+
Ryanodine/Ca2+sensitive Ca2+ pools
Nucleus
Ca2+
Nucleus
Ca2+ release
Subcellular
activity
Figure 5 | The mechanosensation-based cilia signalling model. The polycystin‑1–
polycystin‑2 complex (PC1–PC2), which is sensitive to shear stress, is localized within the
ciliary membrane (left panel). Fluid-induced ciliary bending activates this Ca2+channel.
The Ca2+ influx (right panel) causes Ca2+ release from ryanodine-sensitive intracellular
stores and subsequent downstream responses such as activating protein-1 (AP1)dependent gene transcription by the Ca2+-dependent kinase PKCa. Mutations in PC1 or
PC2 might disable cilia-mediated mechanosensation, which is normally required for
tissue morphogenesis, and thus can cause polycystic kidney disease.
receptors) abolished the physiological flow response in
wild-type cells, the authors proposed a mechanosensory model in which cilia sense fluid movement (FIG. 5).
Polycystin‑1, located within the cilium, was proposed
to function as a flow sensor and to transmit the signal
from the extracellular fluid environment to the interacting Ca2+-channel polycystin‑2. Polycystin‑2 then
mediates sufficient Ca2+ influx to activate intracellular
ryanodine receptors resulting in intracellular Ca2+
release; the increase in intracellular Ca2+ levels probably
regulates numerous molecular activities inside the cell
that contribute to tissue development and homeostasis
in response to tubular flow. Several other reports have
confirmed that ciliated cells in culture can react to fluid
flow79,80. It was also shown that cholangiocyte monocilia
can detect and transmit signals from the luminal bile
duct flow to the epithelial cells, which is reminiscent of
the function of renal monocilia80. However, so far only the
studies by Praetorius and Spring77 have demonstrated a
ciliary mechanosensory capacity by direct mechanical
stimulation. Alternatively, considering the mechanisms
involved in the nodal vesicular parcel model27, it is also
feasible that molecules within the fluid flow might activ
ate chemosensory or receptor-based signalling events
(FIG. 3c) to induce the subsequent intracellular Ca2+release response. Mechanical or chemical fluid-flow
sensing might provide morphogenic cues that regulate
tubule diameters. Defects due to mutant polycystin proteins might generate false signals that indicate a ‘lack of
flow’ and might cause a compensatory growth of the
tubular cells and subsequent cyst formation.
Mechanosensation and Wnt signalling. Recent findings
suggest that signalling from the mechanosensory cilia
of tubule cells might involve the PCP pathway (FIG. 3b).
Similar cystic kidney phenotypes in transgenic mice
overexpressing an activated form of β‑catenin and in
inv/inv mice (a mouse model for infantile NPHP)
implied a role for inversin in Wnt signalling79,81. An
inhibitory role for inversin in canonical Wnt signalling upstream of the β‑catenin degradation complex
was demonstrated in renal epithelial cells and X. laevis
embryos79. Inversin interacts with the protein dishevelled and targets it for degradation by the anaphasepromoting complex/cyclosome (APC/C). Consistent
with a role in non-canonical Wnt signalling, inversin
is required for convergent extension movements in
X. laevis gastrulation and elongation of X. laevis animal
caps. Furthermore, pronephric cysts in zebrafish caused
by inversin knockdown can be rescued by the canonical
Wnt signalling inhibitor diversin 82. Recently, it was
demonstrated that physiological flow conditions lead
to upregulated inversin expression and a reduction of
β‑catenin levels in kidney cells79. Together, these findings indicate that inversin might function as a switch
between the β‑catenin-dependent canonical and the
non-canonical PCP pathway. It is therefore tempting
to speculate that the start of urine flow during renal
development terminates canonical Wnt signalling to
facilitate the β‑catenin-independent PCP pathway83.
PCP and BBS proteins. Another indication of perturbed
PCP signalling in cystic kidney disease came from the
analysis of targeted mutations of BBS genes, which are
also associated with renal cysts. On the basis of the
observation that patients and mice carrying BBS gene
mutations display phenotypic defects similar to those of
classical defects in PCP, a putative PCP function of BBS
genes was explored22. A well-known example of PCP
in mammals is the uniform orientation of the hair-cell
stereociliary bundles within the cochlea. This orientation is perturbed in Bbs1–/–, Bbs4–/– and Mkks–/– mice,
which supports a role for BBS proteins in the establishment of PCP. In addition, genetic interaction of both
Bbs1 and Mkks in mice and bbs1 and bbs4 in zebrafish
with the classical PCP gene Vangl2 was demonstrated22.
Silencing of Bbs4 results in defective targeting or
anchoring of pericentriolar proteins and disorganization of microtubules. Because BBS4 interacts with pericentriolar material-1 (PCM1) and p150glued (a subunit
of dynactin that links dynactin with dynein), BBS4 is
speculated to function as an adaptor that facilitates
the loading of cargo onto dynein–dynactin molecular
motors, thereby recruiting cargo within the cytosol to
the centriolar satellites84. At the basal body, the cargo
is then prepared for IFT-dependent transport along
the ciliary axoneme. Thus, the correct function of BBS
proteins at the basal body might be a prerequisite for the
correct localization and function of PCP proteins such
as VANGL2 in the cilium22.
Recently, seven of the known BBS proteins (BBS1, -2,
-4, -5, -7, -8 and -9) have been shown to assemble into a
~450 kDa core complex (BBSome) that is proposed to
regulate RAB8 (a small GTPase)-dependent vesicular
trafficking of membrane proteins from the Golgi into the
ciliary membrane85. The BBSome localizes to centriolar
satellites in the cytoplasm and to the ciliary membrane.
Through the interaction of BBS1 with RABIN8, which
localizes at the basal body, the BBSome is recruited to the
REVIEWS
a
BM
RPE cell
Outer
segment
Connecting
cilium
basal body from the neighbouring centriolar satellites
and activates RAB8, which in turn promotes docking and
fusion of Golgi vesicles containing newly synthesized
ciliary membrane proteins. Disruption of these processes
may therefore be the cause of BBS.
b
Outer segment
PCP, cilia and tubule elongation. The PCP pathway has
a key role during tubular elongation, a process in which
epithelial cells divide along the longitudinal axis of renal
tubules, pancreatic ducts and bile ducts. Establishment
and maintenance of the tubular geometry requires
precise spatial information and correct positioning of
the spindle axis, which is controlled by the PCP pathway.
The monocilium appears to provide the spatial cues (by
sensing tubular fluid flow, for example) to position the
centrosome and the mitotic spindle before the next cell
division86. Defective cilia function or PCP signalling
might cause the tubular epithelial cells to lose spatial
orientation and apical–basolateral polarity, to proliferate
uncontrollably and to form cysts.
Inner segment
ONL
Inner
segment
Nucleus
Synaptic
terminal
OPL
Nucleus
INL
Outer segment
d
Kinesin-II
Cytoplasmic
dynein-2/-1b
Connecting cilium
c
Transition zone
IFT particle
Axoneme
Basal body
Rootlet
RPGR
Nephrocystin
Inner segment
Usherin
BBS proteins
Mitochondrion
Golgi apparatus
Figure 6 | Structure and function of the photoreceptor-connecting cilium. The
connecting 9+0 cilia of photoreceptors represent specialized cilia that are the sole
transport corridor between the outer and inner photoreceptor segments. These cilia are
essential in photoreceptor physiology and, therefore, their dysfunction contributes to
retinal degeneration. a | Schematic illustration of a photoreceptor cell and its
substructures. b | Immunofluorescence staining of retinal sections using specific markers
for ciliary subcompartments. Antibodies against acetylated α‑tubulin mark the axoneme
(green, left panel). The transition zone was visualized using antibodies against
nephrocystin (red, left and right panels), and the basal bodies were stained using
antibodies against the pericentriolar marker γ‑tubulin (green, right panel). c | Electron
microscopy image of a retina showing the localization of the photoreceptor-connecting
cilium between the outer and inner segments. d | Schematic illustration of the
physiological function of a connecting cilium. Biosynthesis products from the inner
segment and turnover products from the outer segment are shuttled through the
connecting cilium by the IFT machinery. The localization of several proteins implicated in
retinal diseases is indicated: RPGR and nephrocystin are found in the transition zone,
usherin is located in the ciliary membrane and BBS proteins are found in basal bodies.
BBS, Bardet–Biedl syndrome; BM, Bruch’s membrane; IFT, intraflagellar transport; INL,
inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal
pigment epithelium; RPGR, retinitis pigmentosa guanosine triphosphatase (GTPase)
regulator. Part a modified with permission from REF. 100  (2006) Elsevier.
Defects in vision, smell and hearing
Disturbed IFT function in retinal degeneration. In retinal
rods and cones, 9+0 cilia connect the inner segment of
photoreceptors (which contains the nucleus) with the
outer segment, which contains the membrane stacks that
contain the photo pigment (FIG. 6). All components that are
necessary for assembly, maintenance and continuous
turnover of the outer segment are synthesized in the cell
body and are moved through the connecting cilium by
IFT. In mice, targeted mutations that disrupt IFT cause
severe retinal degeneration87,88.
A link between human retinal degenerative diseases
and dysfunction of the connecting cilium became
evident from the observation that mutations in RPGR
(which localizes to the connecting cilium) account for
20% of retinitis pigmentosa cases 89. Because RPGR
interacts with IFT88 as well as with several microtubule
motor proteins90, it probably has a role in IFT. In addition, both the RPGR-interacting protein RPGRIP1 and
RP1 (retinitis pigmentosa-1), which are involved in the
pathogenesis of retinitis pigmentosa, also localize to
connecting cilia.
Approximately 20% of all cases of NPHP are associated with retinal disorders, which might originate
from defective IFT within the connecting cilium.
Direct interaction of various NPHP proteins with the
RPGR–RPGRIP1 complex has been demonstrated91–93,
so it can be speculated that the NPHP proteins assemble
with RPGR–RPGRIP1 into a large multimeric protein
complex within the connecting cilium.
Retinal degeneration (rod–cone dystrophy) is also a
main feature of BBS94. All BBS proteins that have been
analysed localize to the ciliary axonemes or to the ciliary
base95. BBS1, BBS2 and BBS5 contribute either to cilia
formation or function, whereas BBS4, BBS7 and BBS8
have been specifically shown to be involved in IFT pro
cesses. Retinitis pigmentosa is also present in Alstrom
syndrome (ALMS), which shares many features with
BBS. The responsible gene ALMS1 might also be involved
in IFT96,97.
REVIEWS
Usher syndrome (USH), is a heterogeneous disease,
which, in rare cases, can be associated with bronchiectasis,
chronic sinusitis and reduced nasal mucociliary clearance,
which is indicative of ciliary dyskinesia98. Dysfunction of
the vestibular system can be associated with progressive
retinitis pigmentosa. Most USH proteins localize to the
photoreceptor-connecting cilia or the periciliary membrane99,100, where they are involved in IFT, specifically
rhodopsin transport101.
Reduced sense of smell in BBS. Binding of odorant mole
cules to specialized receptors on the membrane of olfactory sensory cilia initially induces Ca2+ signals, which are
then converted to action potentials via sensory neurons.
A link between olfactory cilia dysfunction and hypoosmia
and anosmia has become apparent by the observation
that patients with BBS are frequently unable to smell102.
Mice that lack BBS1 or BBS4 have a severely reduced
olfactory ciliated border, defects in the highly specialized
ciliated dendritic knobs and trapping of olfactory ciliary
proteins in dendrites and cell bodies, which links
ciliary function with the sense of smell102,103.
Loss of hearing and balance in Usher syndrome. In vertebrates, both hearing and balance require sensory hair cells
of the inner ear that carry multiple actin-based stereocilia
and a single tubulin-based kinocilium. Auditory signals are
probably transduced via mechanically gated ion channels
that convert vibrations into electrical signals by depolarization of the hair cell104. Patients with Usher syndrome
display sensorineural hearing loss and lack vestibular
function; the relevant USH proteins possibly function in
sterocilia as well as in photoreceptor cilia100,105.
Bronchiectasis
A bag-like or cylindrical
widening of parts of the
bronchial tree, which is usually
caused by localized injury of
bronchial tissue due to
bacterial infections. Affected
bronchi are irreversibly
damaged.
Chronic sinusitis
A permanent or recurrent
inflammation of the paranasal
sinuses often caused by
infections.
Hypoosmia
A decreased ability to smell
odours.
Anosmia
The absence of the ability to
smell odours.
Oncogenesis
Cilia and cell division. Cilia assembly–disassembly seems
to be closely linked to cell division. Direct evidence
comes from the observation that the IFT protein IFT27
— a Rab-like small G protein —is required for the normal
completion of cell division in C. reinhardtii, in addition
to its role in flagellar assembly106. Partial knockdown
results in cytokinesis defects and elongation of the cell
cycle, whereas a more complete knockdown is lethal. In
addition, IFT88 localizes to the centrosome throughout
the cell cycle and prevents the G1–S transition upon
overexpression; it also promotes cell-cycle progression
when depleted by RNAi in HeLa cells107. Conversely, the
centrosomal Aurora A kinase, which promotes mitotic
entry in mammalian cells, also induces the rapid disassembly and resorption of cilia through activation of
histone deacetylase-6 (HDAC6)-dependent tubulin
deacetylation108. Most vertebrate cells assemble a primary
cilium in the G0–G1 phase of the cell cycle. The centriole
from a previously dividing cell can function as the basal
body for cilia assembly in a quiescent cell, and centrioles
released by cilia disassembly in G1 phase or before or
during S phase might function as microtubule-organizing centres that are essential for spindle formation109.
Therefore, primary cilia are dynamically assembled and
resorbed throughout the cell cycle. Dysregulation of
these processes might result in oncogenesis, for example,
as a consequence of centrosomal amplification and
subsequent genomic instability that is observed in many
cancers. The NEK (NIMA (never in mitosis gene A)related kinase) cell-cycle proteins also link the cell cycle
to the cilia–centrosome complex110–112. Support for the
involvement of NEK proteins in ciliary function came
from the observation that mutations in Nek1 and Nek8
cause cystic kidney disease in mice and zebrafish113,114.
Cystoproteins involved in cell-cycle regulation. Poly
cystin‑2 regulates cell proliferation and differentiation
by directly interacting with and influencing the nuclear
translocation of ID2 (inhibitor of DNA binding-2),
a member of the helix–loop–helix protein family 115.
Overexpressing polycystin‑2 blocks cell-cycle progression through upregulation of the cyclin-dependent
kinase (CDK) inhibitor p21. Overexpression of poly
cystin‑1, which is required for the ID2–polycystin‑2
interaction 115, induces p21 expression and directly
activates signalling through Janus kinase (JAK)–signal
transducer and activator of transcription (STAT) to
regulate the cell cycle116.
In response to fluid flow, the C‑terminal tail of the
integral plasma membrane protein polycystin‑1 is
cleaved off by proteolysis, enters the nucleus and directly
initiates signalling processes (such as Wnt- and the activating protein-1 (AP1)-mediated pathways) that are
modulated by polycystin‑2. Because the transcription
factor AP1 is involved in various processes, including
proliferation, transformation and apoptosis, mechanosensation in renal tubule epithelia might be directly
linked to cell-cycle regulation117.
These polycystin studies indicate that cilia-related
proteins could be good tumour-suppressor candidates.
Biallelic inactivation of the VHL (von Hippel Lindau)
tumour-suppressor gene is associated with most spor
adic renal clear cell carcinomas; here, tumorigenic transformation is preceded by the formation of renal cysts,
which, in turn, are commonly caused by renal monocilia dysfunction. The VHL protein localizes to the
axonemes of renal monocilia and controls ciliogenesis
in kidney cells118–120. In addition, VHL appears to associate with microtubules and coordinates directional
microtubule growth, a prerequisite for ciliogenesis.
So, cilia might have a key role in cell-cycle control,
and alterations in this process predisposes to cancer.
Nevertheless, most mutations in cystoproteins do not
predispose to renal cancer, indicating that additional
effects are required.
Conclusions and discussion
Cilia are highly complex organelles that are involved in
numerous functions, from movement of the fertilized
ovum to airway clearance. Their dysfunction has been
implicated in several disorders; this number is expected
to increase. Recent work indicates that cilia assembly and
disassembly is closely related to cell-cycle control,
and so might attract increased attention in terms of the
mechanisms involved in oncogenesis. This knowledge
might also aid in the development of novel therapeutic
strategies to fight human cancer. The identification of
REVIEWS
new molecular disease mechanisms is also likely to
facilitate the development of novel therapeutic strategies
in disorders in which, so far, only organ replacement
therapy can be offered (for example, cystic kidney
disease) (BOX 2).
In this Review, we have discussed how current
models and hypotheses cannot completely explain
the clinical findings observed in some cilia-related dis
orders (for example, laterality defects), and we therefore
anticipate that further research will provide increased
insight into the molecular mechanisms involved in
these disorders.
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2.
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Acknowledgements
We thank E. Davis and G. Pazour for critical evaluation of the
manuscript. H.O. and M.F. are supported by the Deutsche
Forschungs-Gemeinschaft. We are grateful for the collaboration with the patient support group ‘PCD und Kartagener
Syndrom e.V.’. We thank H. Olbrich and N.T. Loges for help
with figure preparations.
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/sites/
entrez?db=gene
Bbs1| Bbs4 | DNAH5 | DNAI1 | Ift88 | Mkks | OFD1 | Pkd1 | RPGR
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM
ADPKD | Alstrom syndrome | ARPKD | BBS | Kartagener’s
syndrome | Meckel–Gruber syndrome | NPHP |
orofacialdigital syndrome | PCD | Usher syndrome
UniProtKB: http://ca.expasy.org/sprot
GLI1 | GLI2 | GLI3 | patched-1 | polycystin‑2 | SMO | SUFU
FURTHER INFORMATION
Heymut Omran’s homepage: http://www.uniklinik-freiburg.
de/kinderklinik/live/forschung/omran.html
Chlamydomonas Flagellar Proteome:
http://labs.umassmed.edu/chlamyfp/index.php
Cilia Proteome database: http://www.ciliaproteome.org
Ciliomics: http://www.sfu.ca/~leroux/ciliome_home.htm
Primary Cilia Resource: www.bowserlab.org/primarycilia/
cilialist.html
SUPPLEMENTARY INFORMATION
See online article: S1 (movie) | S2 (movie) | S3 (movie) |
S4 (movie) | S5 (movie) | S6 (movie) | S7 (movie) | S8 (movie) |
S9 (movie)
All links are active in the online pdf

When cilia go bad: cilia defects and ciliopathies

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