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First published online 4 October 2006
doi: 10.1242/dev.02595
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Review |
Huntsman Cancer Institute Center for Children, Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA.
* Author for correspondence (e-mail: joseph.yost{at}hci.utah.edu)
SUMMARY
Cilia are highly conserved organelles that have diverse motility and sensory functions. Recent discoveries have revealed that cilia also have crucial roles in cell signaling pathways and in maintaining cellular homeostasis. As such, defects in cilia formation or function have profound effects on the development of body pattern and the physiology of multiple organ systems. By categorizing syndromes that are due to cilia dysfunction in humans and from studies in vertebrate model organisms, molecular pathways that intersect with cilia formation and function have come to light. Here, we summarize an emerging view that in order to understand some complex developmental pathways and disease etiologies, one must consider the molecular functions performed by cilia.
Introduction
Cilia and flagella, which are distinguished by distinctive patterns of
movement, are identical in structure and composition. Cilia project from most
eukaryotic cell surfaces with the notable exceptions of cells of higher plants
and fungi (Mitchell and Nakatsugawa,
2004
). Although cilia are almost ubiquitously present in
vertebrate cells, in invertebrates they are restricted to sensory neurons
(Wheatley et al., 1996).
Ciliary motility is important for moving fluids and particles over epithelial
surfaces, and for the cell motility of vertebrate sperm and unicellular algae
Chlamydomonas. Sensory cilia function in the detection of light, odor
and fluid flow in vertebrates; and in sensing osmolarity changes,
chemoattractants and repellants, and sound in invertebrates
(Evans et al., 2006).
Recent discoveries have revealed that cilia have crucial roles in the signal transduction pathways that regulate intracellular Ca2+ levels, as well as in the Hedgehog (Hh) and planar cell polarity (PCP) pathways. Studies that link cilia/basal body/centrosome proteins to human genetic disorders have also shown that compromised ciliary function can have profound consequences for cellular homeostasis. Dysfunction of ciliary proteins gives rise to phenotypes that range from being organ specific (e.g. some polycystic kidney diseases) to broadly pleiotropic (e.g. Bardet-Biedl syndrome). Emerging from this complex spectrum of disease and developmental mutant phenotypes are a set of phenotypic indicators of ciliary dysfunction, including such seemingly disparate phenotypes as cystic disease of the kidney, liver and pancreas, neural tube defects, postaxial polydactyly, situs inversus, and retinal degeneration. The affected genes and the molecular and cellular basis for these phenotypes are now beginning to be elucidated.
In this review, we focus on recent reports that have greatly expanded our appreciation of the diverse functional roles cilia play in cell signaling pathways and in maintaining cellular homeostasis, and discuss how this new information provides new insights into developmental and disease phenotypes.
Cilia structure and function
Structure of the ciliary apparatus
The cilium consists of a microtubule-based axoneme covered by a specialized
plasma membrane that extends from the cell surface into the extracellular
space (Fig. 1A). The axoneme is
a highly ordered structure of nine peripheral microtubule doublets arranged
around a central core that may or may not contain two central microtubules
(9+2 or 9+0 axoneme, respectively) (Fig.
1B). 9+2 cilia usually have dynein arms that link the microtubule
doublets and are motile, while most 9+0 cilia lack dynein arms and are
non-motile. Ciliary motility is accomplished by dynein motor activity, which
slides the microtubule doublets relative to one another. At its base, the
ciliary axoneme extends from the nine triplet microtubules of the basal body,
a microtubule-organizing center (MTOC) derived from the older of the pair of
centrioles. The transition zone at the junction of the basal body and the
ciliary axoneme is demarcated by Y-shaped fibers, which extend from the
microtubule outer doublets to the ciliary membrane. The transition zone, in
combination with the internal structure of the basal body, is thought to
function as a filter for the cilium, regulating the molecules that can pass
into or out of the cilium. The distal tips of cilia are structurally and
functionally complex microtubule-capping structures, which link the ends of
the axonemal microtubules to the ciliary membrane
(Sloboda, 2005).
Intraflagellar transport
During ciliogenesis, cilia elongate from the basal body by the addition of
new axonemal subunits to the distal tip. As protein synthesis does not occur
in cilia, axonemal and membrane components are conveyed in non-membrane-bound
macromolecular particles by intraflagellar transport (IFT) along the axonemal
doublet microtubules (Fig. 1)
(Kozminski et al., 1993). IFT
particles are organized into two complexes: complex A contains four or five
polypeptides, whereas complex B contains at least 12 polypeptides
(Cole et al., 1998;
Piperno et al., 1998).
Because axonemal microtubules are oriented with their plus-ends at the tip of
the cilia, and microtubule motors typically move in only one direction, two
types of motors are needed for IFT. Anterograde transport is driven by
heterotrimeric kinesin 2, which is composed of motor subunits Kif3a and Kif3b
and a non-motor subunit (Cole et al.,
1993). In C. elegans, anterograde IFT to the proximal and
middle part of the cilium is accomplished cooperatively by heterotrimeric
kinesin 2 and homodimeric kinesin 2 (OSM-3), while IFT to the distal cilium
relies only on OSM-3 (Snow et al.,
2004). In addition to moving axonemal components, these kinesins
also move ciliary membrane proteins and help form signaling complexes within
the ciliary membrane (Qin et al.,
2005; Wang et al.,
2006). It is unknown whether the vertebrate ortholog of OSM-3
(Kif17) functions in axonemal construction, although it is localized to the
cilia of cultured canine kidney (MDCK) cells and functions in the transport of
membrane proteins (Jenkins et al.,
2006). Retrograde transport back to the cell body is accomplished
by cytoplasmic dynein 1B (Pazour et al.,
1998; Signor et al.,
1999). The transition between anterograde and retrograde transport
of IFT particles occurs at the ciliary tip. IFT cargo loading and unloading,
and motor protein regulation, at the tip of the cilium are poorly understood
but likely include proteins that are involved in the control of microtubule
stability, such as the plus-end-binding protein, Eb1
(Pedersen et al., 2003).
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). Mutations in a Drosophila pericentrin-like-homolog
also cause malformations in sensory neuron cilia and sperm, indicating that
the pericentrin-mediated interaction between centrosomal and IFT proteins is
evolutionarily conserved (Martinez-Campos
et al., 2004).
Types of cilia
In mammals, motile 9+2 cilia normally concentrate in large numbers on the
cell surface, beat in an orchestrated wavelike fashion, and are involved in
fluid and cell movement. In contrast to motile cilia, primary cilia
(monocilia) project as single immotile organelles from the cell surface.
Primary cilia are found on nearly all cell types in mammals
(Wheatley et al., 1996) (see
also
http://members.global2000.net/bowser/cilia.html),
and many are highly adapted to serve specialized sensory functions. For
example, photoreceptor proteins in the vertebrate retina are housed in a
highly elaborated 9+0 cilium connected to the cell body by a second
`connecting' cilium that emerges from the basal body
(Rohlich, 1975). Although
cilia are broadly classified as 9+2 motile cilia or 9+0 immotile sensory
cilia, there are examples of 9+2 sensory cilia and 9+0 motile cilia (reviewed
by Afzelius, 2004;
Ibanez-Tallon et al., 2003).
For example, odorant receptors have been found on the immotile 9+2 sensory
cilia of specialized olfactory neurons
(Menco, 1994), and 9+0 motile
cilia, which have dynein arms associated with the outer microtubule doublets
and rotate, are present on mouse embryonic node cells
(Nonaka et al., 1998;
Sulik et al., 1994).
Disorders of motile cilia
Primary cilia dyskinesia
A link between ciliary function and human disease was discovered when
individuals suffering from syndromes with symptoms including respiratory
infections, anosmia, male infertility and situs inversus, were shown to have
defects in ciliary structure and function (primary cilia dyskinesia or PCD)
(Afzelius, 1976;
Eliasson et al., 1977).
Individuals with PCD also occasionally suffer from retinal degeneration,
cystic kidney disease and hydrocephalus. The ciliary structural defects
observed in such individuals include abnormal or missing dynein arms, radial
spokes and central microtubule doublets. Dynein arm defects are associated
with mutations in the genes encoding the dynein heavy chain subunits DNAH5,
and DNAH11, and the intermediate chain subunit DNAI1
(Table 1)
(Bartoloni et al., 2002;
Olbrich et al., 2002;
Pennarun et al., 1999).
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). Loss of ciliary function in the respiratory tract results
in impaired mucociliary transport that leads to chronic respiratory
infections. Male infertility results when immature sperm fail to move to the
vas deferens, owing to loss of ciliary motility in the efferent ductules, or
from a loss of sperm flagellar motility. Hydrocephalus has been proposed to
result from impaired fluid flow in the brain ventricles because of loss of
ependymal cilia motility (Eley et al.,
2005). This has been confirmed from studies of mice with mutations
in the axonemal dynein heavy chain Mdnah5, or a hypomorphic mutation in the
IFT protein polaris (Ift88) as well as in zebrafish larvae in which IFT
proteins including polaris are disrupted by mutation or antisense morpholinos
(Banizs et al., 2005;
Bisgrove et al., 2005;
Ibanez-Tallon et al., 2004;
Kramer-Zucker et al., 2005).
In mice with hypomorphic mutations in Ift88, increased cerebrospinal
fluid Cl- levels, arising from altered cellular functions caused by
loss of primary cilia, may also be a contributing factor for hydrocephalus
(Banizs et al., 2005).
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). This link was strengthened by the discovery that the mouse
iv (inversus viscerum) mutant results from a mutation in a ciliary
dynein gene, left-right dynein (lrd; Dnahc11), which is
expressed specifically in monociliated cells of the embryonic node
(Supp et al., 1997), the
region that organizes the primary anteroposterior body axis during
gastrulation. In normal mouse embryos, node monocilia rotate in a clockwise
direction and generate an asymmetric flow of extracellular fluid that moves
across the node in a leftward direction
(Nonaka et al., 1998)
(reviewed by Shiratori and Hamada,
2006). In lrd embryos, node cilia are immotile and there
is no nodal fluid flow, indicating that nodal flow is necessary for
establishing the LR axis (Okada et al.,
1999; Supp et al.,
1999). Studies of mouse embryos in flow chambers have shown that
leftward fluid flow across the node correctly specifies the LR axis of
iv mice, whereas rightward flow can reverse the LR axis in wild-type
embryos, suggesting that fluid flow per se is sufficient for specifying the LR
axis (Nonaka et al., 2002). In
other vertebrates, rotational ciliary beating also causes leftward flow across
structures that are analogous to the mouse node including the posterior
notochordal plate in rabbits and the Kupffer's vesicle (KV) in zebrafish and
medaka fish (Essner et al.,
2005; Kramer-Zucker et al.,
2005; Okada et al.,
2005).
Left-right asymmetry provides an important paradigm for both the challenges
and rewards of studying the multiple roles of cilia in development. Studies of
cilia mutants have revealed, for example, that a correlation exists between
loss of monocilia structure or movement, alterations in nodal flow and
aberrant LR development, as is seen in mice with mutations in microtubule
motors and IFT proteins, including Kif3a, Kif3b, dynein 2 light intermediate
chain, polaris, wimple and Rfx3, a regulator of ciliogenic genes
(Bonnafe et al., 2004;
Huangfu et al., 2003;
Marszalek et al., 1999;
Murcia et al., 2000;
Nonaka et al., 1998;
Rana et al., 2004;
Takeda et al., 1999). LR
defects are also caused by mutations in other ciliary or basal body proteins
including polycystin 2 (Pc2) and inversin (Invs)
(Mochizuki et al., 1998;
Pennekamp et al., 2002). In
zebrafish embryos, interfering with KV cilia structure or function through
knockdown of polaris, Invs, Lrd or Pc2 causes alterations in LR patterning
similar to those reported for the corresponding mouse mutants
(Bisgrove et al., 2005;
Essner et al., 2005;
Kramer-Zucker et al., 2005;
Otto et al., 2003;
Sun et al., 2004). The
challenge is to determine which phenotypes are due to loss of cilia structure,
motility and flow, and loss of mechanosensory and/or signal transduction
functions. It is likely that some LR phenotypes are compound defects that
eliminate not only the function of the targeted protein, but also functions of
other cilia proteins. In addition, some proteins that have functions in cilia
might also have functions in other cellular compartments that are important
for LR development (Wagner and Yost,
2000).
How do rotating nodal cilia cause laminar leftward flow of fluid and not
local vortices? Fluid dynamics studies suggest a linear directional flow might
be achieved if the rotation axis of the cilia has a posterior tilt
(Fig. 2)
(Buceta et al., 2005;
Cartwright et al., 2004)
(reviewed by Shiratori and Hamada,
2006). Microscopy studies confirmed that nodal cilia in mouse and
rabbit embryos are tilted posteriorly and that laminar flow moves particles in
a leftward direction (Nonaka et al.,
2005; Okada et al.,
2005). Cilia on the dorsal surface of KV in zebrafish embryos also
appear to be tipped toward the posterior, indicating that the mechanism of
creating laminar flow is evolutionarily conserved
(Kramer-Zucker et al.,
2005).
Two models have been proposed to explain how fluid flow confers LR
asymmetry (Fig. 2). The
`morphogen flow' model proposes that signaling proteins, such as sonic
hedgehog (Shh), fibroblast growth factor (Fgf) or Nodal are swept to the left
side of the node where they initiate downstream signaling pathways
(Nonaka et al., 1998;
Okada et al., 1999). Although
flow in rabbit and mouse embryos can concentrate fluorescently labeled
proteins on the left side of the node
(Okada et al., 2005), and
small extracellular vesicles (so-called `nodal vesicular particles') have been
reported to be swept to the left periphery of the node where they fragment,
releasing their cargo and triggering downstream signaling events including an
elevation of Ca2+ levels
(Tanaka et al., 2005),
asymmetric distribution of endogenous proteins has not been detected. The
mechanosensory or `two cilia' model proposes that two populations of primary
cilia exist in the mouse node (McGrath et
al., 2003): central Lrd-expressing motile cilia and peripheral
non-motile cilia. The model proposes that leftward fluid flow generated by
motile cilia is detected by mechanical bending of non-motile mechanosensory
cilia, which initiate downstream Ca2+ mediated signaling events. In
support of this, asymmetric Ca2+ transients are not detected in
Pkd2 (Pc2) mouse mutant embryos, which lack functional
ciliary mechanosensitive cation channels (see below), or in zebrafish in which
Ipk1, an important mediator of intercellular Ca2+ signaling, is
knocked down by morpholinos (McGrath et
al., 2003; Sarmah et al.,
2005). Further studies will be necessary to clarify the mechanism
that determines how LR axis patterning information is conveyed by fluid flow
and, perhaps, sensed by specialized cilia.
Disorders of primary cilia
The immotile primary cilia that are associated with many cell types were
thought to be of little physiological importance until recently when homologs
of ciliary proteins in Chlamydomonas and C. elegans were
linked to polycystic kidney diseases (PKDs) in humans and mice, and were also
found to localize to renal primary cilia
(Barr and Sternberg, 1999;
Pazour et al., 2002b;
Yoder et al., 2002a;
Yoder et al., 2002b). The
list of proteins that localize to the primary cilium/basal body/centrosome
complex and are linked to PKDs, nephronophthisis (NPHP) and several other
human syndromes such as Bardet-Biedl Syndrome (BBS) continues to grow (see
Table 1,
Fig. 3). Primary cilia also
play essential roles as signal transducers in the Shh and non-canonical Wnt
developmental pathways (see Huangfu et
al., 2003; Simons et al.,
2005), as discussed in more detail below. In the following
sections we describe recent advances in the study of development and human
disease that shed light on the importance of the ciliary apparatus and its
role in controlling cell fate and homeostasis.
Cilia as mechanosensors and PKD
In humans, inherited PKDs include autosomal dominant PKD (ADPKD), autosomal
recessive PKD (ARPKD) and NPHP. During disease progression, renal epithelial
cells proliferate to form fluid-filled cysts that replace normal renal tissue.
The mechanisms responsible for cyst development are unclear, but cysts appear
to arise due to a combination of factors, including increased cell
proliferation, loss of cell polarity and failure of cell differentiation.
Intracellular Ca2+ levels, which are important for the regulation
of cell proliferation and apoptosis, and for other properties of nephron
function (including ion reabsorption rates), probably play a central role in
cyst formation (Boletta and Germino,
2003).
The gene disrupted in ARPKD encodes a transmembrane protein known as
fibrocystin or polyductin, which localizes to renal primary cilia and basal
bodies, the function of which remains enigmatic
(Wang et al., 2004;
Ward et al., 2002). By
contrast, ADPKD, which results from mutations in PKD1 or
PKD2 (encoding polycystin 1 (Pc1) and polycystin 2 (Pc2),
respectively), is better understood. Pc1 and Pc2 are multi-pass integral
membrane proteins that interact to form Ca2+ permeable cation
channels at the plasma membrane and in primary cilia of human and mouse renal
epithelial cells (Hanaoka et al.,
2000; Pazour et al.,
2002b; Yoder et al.,
2002a). Bending the primary cilia of MDCK cells in culture causes
Ca2+ to influx through these channels in the ciliary membrane
(Praetorius and Spring,
2001). These Ca2+ transients can be blocked by
antibodies that block the function of Pc1 or Pc2
(Nauli et al., 2003),
indicating that these proteins function in detecting mechanical stress and can
probably detect fluid flow.
Several additional studies indicate that ciliary localization is important
for the function of the Pc1/Pc2 channel and that IFT and Pc2 functions are
linked. In mammals, polaris localizes to the basal body and axoneme of motile
and immotile cilia (Taulman et al.,
2001) and colocalizes with Pc1 and Pc2 and a novel murine PKD
protein, cystin, in the primary cilia of kidney cells
(Hou et al., 2002;
Pazour et al., 2002b;
Yoder et al., 2002a). In
cultured renal epithelial cells from polaris mutant mice, high levels of Pc2
accumulate in stunted cilia and in the apical membrane, concomitant with
elevated sub-apical Ca2+ levels
(Pazour et al., 2002b;
Siroky et al., 2006).
Similarly, in C. elegans, Pc1, Pc2 and polaris homologs colocalize in
sensory neuron cilia and accumulate in the stunted cilia of osm-5
(polaris) mutants (Barr et al.,
2001; Barr and Sternberg,
1999; Haycraft et al.,
2001; Qin et al.,
2001). Together, these results indicate that polaris-dependant IFT
is required to regulate ciliogenesis and Pc2 levels in cilia, and that
misregulation of Pc2 levels might perturb Ca2+ entry and contribute
to cyst development.
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;
Low et al., 2006). Because Pc1
is localized to multiple sites in the cell, it is unclear whether any of these
processes are related to ciliary function. Cessation of renal fluid flow,
however, has been found to induce intramembrane proteolysis of Pc1. This
generates a Pc1 C-terminal fragment that can translocate to the nucleus in a
complex with STAT6 and P100 and activate the AP-1 pathway
(Chauvet et al., 2004). These
data suggest that transduction of ciliary mechanosensory information might
also involve changes in gene transcription mediated through proteolysis of
Pc1.
Cilia-mediated mechanoreception of fluid flow may also play a role in the
development and function of other organ systems. Individuals with ADPKD and
ARPKD, and several murine models of PKD that are associated with ciliary
proteins also frequently have cysts in the liver and pancreas
(Cano et al., 2004;
Igarashi and Somlo, 2002;
Lager et al., 2001). Although
the direct causes of these cystic diseases have not been determined, it is
tempting to speculate that a loss of ciliary mechanosensory functions in
hepatic and pancreatic ducts may be involved in these pathologies.
NPHP: PKD proteins that link mechanoreception and cell cycle regulation?
Nephronophthisis (NPHP) encompasses a polygenic group of autosomal
recessive cystic kidney diseases in children and young adults. Individuals
with this disease can also have pancreatic and hepatic fibrosis, situs
inversus, retinal degeneration (in Senior-Loken syndrome and Joubert
syndrome), complex brainstem malformation and mental retardation (Joubert
syndrome). Mutations in six genes (NPHP1-NPHP6) have been
linked to NPHP (Hildebrandt et al.,
1997; Mollet et al.,
2002; Olbrich et al.,
2003; Otto et al.,
2002; Otto et al.,
2005; Otto et al.,
2003; Saunier et al.,
1997; Sayer et al.,
2006; Valente et al.,
2006). NPHP proteins contain multiple protein-protein interaction
domains and appear to function as a complex in the primary cilia/basal
body/centrosome apparatus (Morgan et al.,
2002a; Olbrich et al.,
2003; Otto et al.,
2005; Otto et al.,
2003; Watanabe et al.,
2003). Disruption of Nphp2 (Invs) in mice or zebrafish does not
alter cilia structure (Okada et al.,
1999; Otto et al.,
2003). C. elegans homologs of human NPHP1 and NPHP4
(NPH-1 and NPH-4) localize to the transition zone of ciliated sensory neurons.
Although C. elegans nph-4 mutants have no ciliary structural defects,
cilia-mediated sensory functions, including chemotaxis and male mating
behavior are abnormal, suggesting a role for the proteins in ciliary signaling
(Winkelbauer et al., 2005;
Wolf et al., 2005). NPHP
proteins could also provide a link between sensory cilia and cell cycle
regulation. Invs (Nphp2) contains two predicted calmodulin-binding domains and
can bind Ca2+ (Morgan et al.,
2002b). It also associates with the anaphase promoting complex
protein Apc2 and has a dynamic pattern of expression during mitosis, cycling
between localization at the centrosomes, mitotic spindle poles and the midbody
between cells (Morgan et al.,
2002a; Nurnberger et al.,
2002). Nphp6 also has a dynamic pattern of expression, alternating
between the nucleus and centrosome in a cell cycle-dependant manner
(Sayer et al., 2006). From
these observations, it is tempting to speculate that NPHP proteins provide a
link between Pc1/Pc2, Ca2+ and the cell cycle, and that primary
cilia serve as environmental sensors for the centrosome in the regulation of
the cell cycle.
Cilia dependent signaling and cell cycle coordination
Centrioles play dual roles in the cell: as basal bodies, they influence
cilia function; as centrioles in centrosomes, they mediate cell division.
Centriole duplication is coordinated with the cell cycle. Thus, entry into the
cell cycle is preceded by cilium resorption, whereas exit from mitosis and
cellular differentiation is accompanied by cilium assembly. If cilia provide
information that helps cells remain in a differentiated state, then defects in
cilia-dependent signaling would be predicted to cause proliferative disorders
and alterations in cellular differentiation.
Cell proliferation, migration and apoptosis in mammalian tissues are
controlled, in part, by platelet-derived growth factor receptor 
(Pdgfr), which localizes to primary cilia in primary cultures of mouse
embryonic fibroblasts (Schneider et al.,
2005). In response to ligand, Pdgfr homodimerizes and
activates the Akt and Mek1/2-Erk1/2 pathways. Mek1/2 phosphorylation occurs
within the cilium and basal body. Fibroblasts derived from a hypomorphic
polaris mutant mouse (polaris functions in IFT) form abnormal stunted cilia
and fail to activate the Mek1/2-Erk1/2 pathway in response to ligand,
indicating that activation of Pdgfr and its cellular functions depend
on its localization to normal cilia
(Schneider et al., 2005).
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Studies of mouse PKD models also implicate two members of a cell-cycle
kinase family (NIMA-related kinases or Nek kinases) as links between ciliary
function and cell cycle control. kat (Nek1) mutant mice have
autosomal recessive PKD (Upadhya et al.,
2000). Nek1 interacts with several proteins, including Kif3a, and
localizes to the centrosomes (Mahjoub et
al., 2005; Surpili et al.,
2003). Nek8 mutant mice have juvenile cystic kidney (JCK)
disease (Liu et al., 2002).
Nek8 localizes to the proximal region of the primary cilia during interphase
and is undetectable during mitosis
(Mahjoub et al., 2005). A
kinase domain mutation of Nek8 affects cell cycle progression and
overexpression of mutant forms of Nek8 lead to multinucleate cells
(Bowers and Boylan, 2004;
Liu et al., 2002). As Nek8 is
not required for cilia assembly (Mahjoub
et al., 2005), these observations suggest that it functions to
link cilia and cycle regulation.
Cilia-dependent signaling and developmental disorders
Wnt signaling
Several recent studies implicate cilia in two important developmental
signaling pathways: Wnt signaling and Shh signaling. During early kidney
development, canonical Wnt signaling is required for metanephric mesenchyme
induction and cell proliferation during branching morphogenesis
(Perantoni, 2003). Later in
development, signaling through the non-canonical Wnt, or PCP, pathway (see
Box 1)
(Park et al., 2005;
Veeman et al., 2003;
Wallingford and Habas, 2005)
is required to align the mitotic orientation of proliferating cells of the
renal tubules to allow the tubules to lengthen without substantially
increasing their diameter (Fischer et al.,
2006).
Observations that the kidney phenotype of Invs mutants resembled
that of mice with dysregulated canonical Wnt signaling suggested that Invs
(Nphp2) functions in Wnt signaling
(Simons et al., 2005). In
MDCK cells, Invs forms a protein complex with dishevelled (Dvl1) and Apc2.
This complex targets Dvl1 for proteosomal degradation, resulting in a loss of
ß-catenin stabilization and inhibition of the canonical Wnt pathway
(Simons et al., 2005).
Subsequently, as MDCK cells become confluent and polarized, Invs and Dvl1
translocate to the plasma membrane, where they are in a position to associate
with other PCP proteins and activate the non-canonical Wnt pathway. Although
Invs-mediated PCP signaling in the kidney remains to be formally demonstrated,
morpholino-mediated knockdown of Invs impairs PCP-dependant convergent
extension movements in frog embryos, indicating that Invs functions in the PCP
pathway (Simons et al.,
2005). Invs has also been implicated in PCP processes that orient
ciliated cells of the mouse node along the AP axis
(Okada et al., 2005).
In PKD, cyst formation is associated with increased numbers of cells in the
circumference of renal tubules. In mice with renal-specific inactivation of
Tcf2, and in the pck rat, which has reduced expression of
Pkd2 and/or Pkhd1, mitotic alignments along the axis of the
tubules are significantly distorted, indicating a loss of PCP
(Fischer et al., 2006). In the
pck rat, this distortion precedes tubular distension, suggesting a
correlation between PCP loss and PKD development. As several proteins
implicated in PKD, including Invs, fibrocystin, Pc1 and Pc2 localize to the
cilium and/or basal body, one could speculate that cilia sense primary tubular
urine flow and terminate canonical Wnt signaling, which facilitates
non-canonical Wnt signaling necessary for tubulogenesis. Ciliary bending might
also provide a vectorial cue that orients cell division. Consistent with this
model, when ciliated inner medullary collecting duct cells are exposed to
flow, Invs levels increase and ß-catenin levels decrease
(Simons et al., 2005).
Structural defects in ciliogenesis are correlated with defective PCP. In
Xenopus, the PCP proteins inturned (Xint) and fuzzy (Xfy) accumulate
at the apical surface of ciliated cells
(Park et al., 2006). Xint and
Xfy morphants have convergent extension defects that correlate with a loss of
cilia from cells of the ventral neural tube. In epidermal cells of morphant
embryos, the apical actin network is less dense and contains fewer actin foci
than in control embryos, and microtubules are not organized into apically
projecting cilia. Thus, Xint and Xfy control an apical actin network essential
for the orientation of microtubules and their assembly into
cilia.
| Box 1. Ciliary involvement in Wnt signaling pathways The cilium might act as a switch between canonical Wnt signaling and the non-canonical Wnt/planar cell polarity (PCP) pathway. Canonical and non-canonical Wnt pathways both require Wnt ligands, frizzled (Fz) receptors and dishevelled (Dvl1) but they diverge downstream of Dvl1. In the canonical pathway, Wnt binding to Fz leads to the activation of Dvl1 and the stabilization of cytoplasmic ß-catenin, through inhibition of the Apc2/axin/Gsk3ß degradation complex. ß-Catenin translocates to the nucleus, where it activates Lef/Tcf-mediated gene transcription. In response to an environmental stimulus such as fluid flow, a ciliary signal mediates a switch from canonical to noncanonical Wnt signaling. Dvl1 and possibly inversin (Invs) localize to the plasma membrane, where they interact with PCP proteins; ß-catenin is degraded by the Apc/axin/Gsk3ß complex. PCP signaling influences gene transcription and regulates cytoskeletal rearrangements. The PCP pathway in vertebrates directs several processes, including mitotic spindle orientation, epithelial cell polarization and convergent extension movements during gastrulation and neurulation.
|
Shh signaling
In vertebrates, cilia also function in the Shh-dependent patterning of the
developing neural tube and limb (see Box
2) (reviewed by Huangfu and
Anderson, 2006). Mouse mutants with defects in ciliogenesis
resulting from mutations in IFT protein-encoding genes, such as polaris,
wimple (Ift172), Ngd5 (Ift52) and the gene encoding the retrograde motor
Dnchc2, have neural tube defects and preaxial polydactyly phenotypes similar
to mutants with defects in Shh pathway proteins
(Haycraft et al., 2005;
Huangfu et al., 2003;
Liu et al., 2005;
May et al., 2005;
Zhang et al., 2003). Cilia
are present in the forebrain neurectoderm and in ectodermal and mesenchymal
cells of the limb, but are aberrant or absent from these tissues in IFT
mutants (Haycraft et al.,
2005; May et al.,
2005).
|
Shh expression in the notochord and limb buds of IFT mutants is normal, but
expression of downstream targets, including patched 1 (Ptch1) and
Gli1, in responding tissues (the neural tube and limb bud,
respectively) is reduced, consistent with a loss of Shh signal transduction.
Interestingly, IFT mutants display a loss-of-function Shh phenotype in the
neural tube, where Gli activators (Gli1, Gli2) have a major role in pattern
formation, and a gain-of-function Shh phenotype in the limb, where the Gli3
repressor (Gli3R) plays the main role
(Huangfu and Anderson, 2005).
A series of elegant studies (including epistasis analyses of mouse IFT and Shh
pathway protein mutants, and analysis of Gli3 activator to Gli3R in IFT
mutants) and studies of Gli1 and Gli3R construct expression in cells derived
from IFT mutants indicate that IFT is required downstream of Ptch1 and
smoothened (Smo). These studies also show that IFT proteins are required for
Gli activation and for the proteolytic processing of Gli3 into Gli3R, but not
for the trafficking of Gli proteins to the nucleus
(Haycraft et al., 2005;
Huangfu and Anderson, 2005;
Huangfu et al., 2003;
Liu et al., 2005;
May et al., 2005).
Smo plays a crucial role in the Shh pathway controlling both Gli activation
and, in the absence of Shh signaling, the proteolytic processing of Gli3 to
Gli3R, making it a likely candidate for the component of the pathway directly
affected in IFT mutants. Smo accumulates in the cytoplasm and also on the
primary cilia of MDCK cells and of mouse embryonic node cells
(Corbit et al., 2005;
May et al., 2005). In MDCK
cells, the ciliary localization of Smo increases in response to Shh; blocking
the Shh pathway with cyclopamine eliminates the localization of an activated
Smo protein (Corbit et al.,
2005). Cyclopamine treatment also reduces Smo levels in mouse
nodal cilia (Corbit et al.,
2005). Deletion of a C-terminal hydrophobic motif, common to other
cilia-localized proteins (Brailov et al.,
2000; Dwyer et al.,
2001; Handel et al.,
1999) prevents the truncated Smo protein from localizing to cilia,
even in the presence of Shh, and abolishes Smo activity
(Corbit et al., 2005). These
observations indicate that mobilization of Smo to primary cilia is a key event
in the activation of the Shh pathway (see
Box 2). However, it is unclear
whether IFT function is directly required in Shh signaling to localize Smo to
the ciliary membrane or whether it is required indirectly for the formation of
a structurally and functionally intact cilium. Several other components of the
Shh pathway are also localized to cilia, including suppressor of fused and the
three full-length Gli transcription factors
(Corbit et al., 2005;
Haycraft et al., 2005;
May et al., 2005). How Smo
activates Shh signaling at the cilium is unclear, but the cilium might provide
a specialized microtubule-associated domain that coordinates Smo and other
components of the pathway to facilitate the activation of Gli transcription
factors and the proteolytic processing of Gli3 to generate
Gli3R.
| Box 2. Role of cilia in vertebrate hedgehog signaling
Primary cilia play a crucial role in mediating hedgehog (Hh) signaling in
vertebrates. The transcription of sonic hedgehog (Shh) target genes is
regulated by the nuclear ratio of Gli activators (GliA) to Gli3 repressors
(Gli3R). (A) Upon Shh ligand binding to patched 1 (Ptch1), inhibition
of smoothened (Smo) is eliminated and it localizes to the cilium, where it
controls the activation of Gli transcription factors and their translocation
to the nucleus where they activate transcription of Shh target genes.
(B) In the absence of Shh, the cilium-mediated proteolytic processing
of Gli3 generates a N-terminal fragment that functions in the nucleus as a
transcriptional repressor (Gli3R). (C) In intraflagellar transport
(IFT) mutants with aberrant or absent cilia, the formation of both GliA and
GliR is impaired, which can lead to Shh loss- or gain-of-function phenotypes
in different tissues. Interestingly, in Drosophila, Hh-responsive
tissues lack cilia, and activation of the Hh pathway is associated with
Ptch1-mediated mobilization of Smo from intracellular stores to the cell
surface [for a review comparing vertebrate and invertebrate Hh signaling, see
Huangfu and Anderson (Huangfu and
Anderson, 2006
|
Bardet-Biedl syndrome: microtubule transport connecting ciliary and cellular function?
Bardet-Biedl syndrome (BBS) is a genetically heterogeneous pleiotropic
disorder with symptoms including kidney abnormalities, retinal degeneration,
mental retardation, obesity, diabetes, polydactyly and situs inversus. The
syndrome has been linked to mutations in 11 loci that produce clinically
indistinguishable phenotypes, indicating that the encoded proteins participate
in a common cellular process. With the exceptions of BBS4 and BBS8, which
contain multiple protein-protein interaction domains, BBS6 and BBS10, which
encode chaperonin-like proteins, and BBS11, which encodes an E3 ubiquitin
ligase, most show limited homology to other proteins of known function
(Ansley et al., 2003;
Badano et al., 2003;
Chiang et al., 2006;
Chiang et al., 2004;
Fan et al., 2004;
Katsanis et al., 2000;
Kim et al., 2005;
Li et al., 2004;
Mykytyn et al., 2001;
Mykytyn et al., 2002;
Nishimura et al., 2001;
Nishimura et al., 2005;
Slavotinek et al., 2000;
Stoetzel et al., 2006;
Stone et al., 2000).
BBS proteins localize to the cilia/basal body/centrosome complex. In C.
elegans, BBS::GFP fusion proteins localize to the base of cilia and move
along the ciliary axoneme in sensory neurons
(Ansley et al., 2003;
Blacque et al., 2004;
Fan et al., 2004;
Li et al., 2004). In mammals,
BBS4, BBS5, BBS6 and BBS8 localize to the centrosome, pericentriolar region
and basal body, but are absent from the ciliary axoneme in most cell types,
except in the photoreceptor connecting cilium
(Ansley et al., 2003;
Kim et al., 2004;
Kim et al., 2005;
Li et al., 2004).
Studies in humans and several model systems indicate that BBS proteins
function in microtubule-based cellular processes. In C. elegans,
mutations in bbs-7 or bbs-8 result in loss of the distal
ciliary segment, and dissociation of IFT particles into A and B complexes, as
seen in osm-3 mutants (Blacque et
al., 2004; Ou et al.,
2005), indicating that BBS-7 and BBS-8 are required to keep IFT
particles intact and may regulate their association with the kinesin motors.
In humans, BBS4, BBS6 and BBS8 associate with PCM1, a centrosomal protein
involved in centriolar replication (Ansley
et al., 2003; Kim et al.,
2004; Kim et al.,
2005). BBS4 also interacts with dynactin, a protein that modulates
cargo binding to dynein. RNAi silencing of BBS4 causes PCM1 mislocalization to
the cytosol, the de-anchoring of microtubules at the centrosome and arrested
cell division, indicating that BBS4 functions in association with dynein to
transport PCM1 to the centrosome (Kim et
al., 2004). RNAi knockdown of BBS6, which assembles at the
centrosome in a microtubule-independent manner, also causes cytokinesis
defects. Futhermore, patient-derived mutant variants of BBS4 and BBS6 fail to
associate with centrosomes, suggesting that loss of this function may cause
BBS (Kim et al., 2004;
Kim et al., 2005). BBS6 and
PCM1 do not colocalize in all cell types, which may explain the
cell-type-specific phenotypes associated with this disease.
Loss of BBS function in humans often leads to retinal degeneration and
anosmia. Bbs2 and Bbs4 mutant mice have normal retinas and
normal photoreceptor-connecting cilia early in life, but subsequently undergo
progressive retinal degeneration (Mykytyn
et al., 2004; Nishimura et
al., 2004). Retinal degeneration results from reduced anterograde
transport of rhodopsin across the connecting cilium of the photoreceptors,
which leads to rhodopsin accumulation in the cell body, triggering cell death
(Nishimura et al., 2004).
Similar defects are seen in photoreceptors of mice with mutant IFT proteins
(Marszalek et al., 1999;
Pazour et al., 2002a),
indicating that BBS proteins interact with IFT proteins in some cell types.
Like retinal degeneration, anosmia in Bbs1 and Bbs4 mutant
mice, results from the depletion of olfactory proteins from the ciliary layer
of the olfactory neurons and the accumulation of these proteins in the cell
bodies (Kulaga et al., 2004).
Olfactory cilia of the mutant mice are also depleted of stable microtubules.
Interestingly, the neighboring respiratory epithelium has normal cilia,
indicating that BBS proteins are not required in all ciliated cells. In
zebrafish, morpholino knockdown of ubiquitously expressed BBS genes delays
retrograde intracellular transport of melanosomes within melanophores
(Yen et al., 2006),
consistent with a role for these proteins in microtubule-based transport
processes.
In addition to characteristic BBS phenotypes, some Bbs4 mutant
mice also exhibit anterior neural tube defects similar to those seen in mouse
PCP gene mutants (Ross et al.,
2005). Cochlear stereocilia bundles (actin-enriched microvilli)
are misoriented in Bbs1, Bbs4 and Bbs6 mouse mutants and in
double heterozygotes of Bbs6 and the PCP mutant
looptail/Vangl2, indicating that BBS proteins function in the PCP
pathway. In zebrafish embryos, disruption of Bbs4 or Bbs6 enhances the
convergent extension defects in the zebrafish PCP mutant trilobite
(vangl2). In mammalian epithelial cells, Vangl2 localizes to the
ciliary axoneme and basal bodies. Although it is unclear whether this
localization is necessary for its function, it is consistent with a link
between BBS and PCP protein function, and provides additional evidence that
cilia or basal bodies are intrinsically involved in PCP processes.
In conclusion, although BBS proteins do not appear to be required for the function of the ciliary/basal body/centrosome complex in all cell types, several cellular functions are compromised by mutations in these genes. Present studies indicate that these proteins play crucial roles in other microtubule-based protein transport processes associated with the centrosomes and other intracellular microtubule arrays.
Other cilia-related human disease syndromes
Alstrom syndrome (ALMS) is caused by mutations in a novel gene of unknown
function (ALMS1) (Collin et al.,
2002; Hearn et al.,
2002) and is characterized by several phenotypes reminiscent of
BBS, including retinal degeneration, obesity and diabetes. ALMS1 protein
localizes to centrosomes and to the base of cilia. In fibroblasts with
disrupted ALMS1, primary cilia and the microtubule cytoskeleton appear to be
normal, suggesting that the ALMS phenotype results from impaired ciliary
function rather than from abnormal ciliary structure
(Hearn et al., 2005).
Affected individuals with oral-facial-digital type I syndrome (OFD1) have
craniofacial abnormalities, postaxial polydactyly and polycystic kidneys. Ofd1
localizes to the centrosome and basal body of primary cilia
(Ferrante et al., 2001;
Romio et al., 2004). Mouse
Ofd1 mutants lack cilia on the luminal surface of kidney glomerular
and tubular cells, lack nodal cilia and have LR patterning defects. In
addition, they have morphological and Shh target gene expression defects in
the neural tube and limb bud that recapitulate those seen in Ift172,
Kif3a and polaris mutants (Ferrante
et al., 2006). Protein localization data and mutant phenotypes
indicate that Ofd1 is associated with the IFT machinery and that the human
disease syndrome results from a loss of primary cilia function.
Meckel-Gruber syndrome (MKS) is associated with mutations in at least three
loci and is characterized by renal and hepatic cysts, CNS malformations,
polydactyly and occasionally hydrocephalus. MKS1 has little homology to other
proteins and no known function. Its phenotypes and the fact that a
Chlamydomonas ortholog is present in the flagella/basal body
proteomes suggest a ciliary function
(Keller et al., 2005;
Kyttala et al., 2006;
Li et al., 2004). MKS3 encodes
a seven-transmembrane receptor protein and is syntenic to the wpk
locus in rat, which is associated with PKD and neural tube defects, including
hydrocephalus (Smith et al.,
2006). The lack of a Chlamydomonas ortholog suggests that
it is not part of the basal IFT machinery, but the existence of an ortholog in
the C. elegans ciliary data set
(Blacque et al., 2005)
implicates it in ciliary function. Continued research into the mechanistic
basis for diseases such as those mentioned above will greatly expand our
understanding of the many essential roles played by ciliary function in
developmental disorders and disease.
Conclusions
Ciliary function has recently been shown to be crucial in an increasingly
diverse range of biological functions. The location of the cilium on the cell
surface effectively positions it to function as an environmental sensor and
vehicle to transfer information that affects important developmental
decisions. The ciliary membrane and cytoplasm are relatively isolated from the
cell body, which offers the advantage of compartmentalization, while the IFT
machinery allows for rapid, and presumably regulated, transport of proteins
between the cilium and the cell body. Recent discoveries indicate that cilia
provide both inside-out signaling, such as the positioning of cilia to control
the direction of extracellular fluid flow, and outside-in signaling, which
modulates hedgehog signaling, Pdgf signaling and intracellular
Ca2+ levels.
We now have considerable insight into the molecular and cellular basis of several ciliopathies, including cystic kidney disease, polydactyly and situs inversus. In others, such as the formation of hepatic and pancreatic cysts associated with some forms of PKD, the role of cilia is less well understood but may reflect similar mechanisms as those seen in the generation of renal cysts. A role for ciliary dysfunction in other pathologies, such as obesity and diabetes seen in BBS and ALMS remain enigmatic, but raises the intriguing possibility that ciliary/basal body function is involved in the regulation of glucose metabolism.
Almost all mammalian cells have a primary cilium, and although the functions of these cilia in some organs are now understood, the roles of cilia on many cell types and organs remain obscure. Although it is clear that the primary cilium has multiple functions, there does not yet seem to be a common denominator for all tissues. Ciliary functions are partially overlapping, but different phenotypes present in PKD, BBS, NPHP and other diseases indicates that many roles of cilia are compartmentalized and that specific diseases are a result of defects in specific aspects of cilia function. The diverse phenotypes caused by ciliary dysfunction could reflect variable expression of ciliary proteins in different cell and tissue types. Some ciliary proteins also appear to play multiple functional roles in cells. For example, Invs has been implicated in the coordination of the cell cycle, and as a molecular switch that regulates the canonical and non-canonical Wnt signaling pathways. Much remains to be carried out in order to understand fully the contribution of each of these factors to the spectrum of ciliary function. One of the challenges is to understand which of the many functions of cilia, or which combinations of functions are responsible for each developmental process. Many additional roles for ciliary proteins are likely to be uncovered and will greatly contribute to our understanding of developmental mechanisms and disease.
ACKNOWLEDGMENTS
We thank A. B. Cadwallader, B. L. McMahan and A. L. Parks for their assistance and thoughtful comments on the manuscript. The authors research was supported by the NHLBI and the Huntsman Cancer Foundation (H.J.Y.).
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