THE ANATOMICAL RECORD 291:1074–1078 (2008)
Primary Cilia: Cellular Sensors
for the Skeleton
CHARLES T. ANDERSON,1* ALESHA B. CASTILLO,2 SAMANTHA A. BRUGMANN,3
JILL A. HELMS,3 CHRISTOPHER R. JACOBS,2,4,5 AND TIM STEARNS1,5
1
Department of Biological Sciences, Stanford University, Stanford, California
2
Department of Bioengineering, Stanford University, Stanford, California
3
Department of Surgery, Stanford University, Stanford, California
4
Department of Mechanical Engineering, Palo Alto Veterans Administration
Medical Center, Palo Alto, California
5
Department of Genetics, Stanford University Medical School, Stanford, California
ABSTRACT
The primary cilium is a solitary, immotile cilium that is present in
almost every mammalian cell type. Primary cilia are thought to function
as chemosensors, mechanosensors, or both, depending on cell type, and
have been linked to several developmental signaling pathways. Primary
cilium malfunction has been implicated in several human diseases, the
symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the
development and homeostasis of the skeleton. In this review, we discuss
the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus
on skeletal biology. We also raise several unanswered questions regarding
the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions. Anat Rec,
291:1074–1078, 2008. Ó 2008 Wiley-Liss, Inc.
Key words: primary cilium; kidneys, cilia
The primary cilium is a solitary, immotile cilium that
is present in almost every mammalian cell type
(Wheatley et al., 1996). Multiple developmental signaling pathways have been linked to primary cilia
(reviewed in Christensen et al., 2007), and they are
thought to function as chemosensors, mechanosensors,
or both, depending on cell type. Primary cilium malfunction has been implicated in several human diseases, the
symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys (reviewed in Tobin and
Beales 2007; Yoder 2007). Recently, primary cilia have
also been implicated in the development (Zhang et al.,
2003; Xiao et al., 2006; Haycraft et al., 2007; Koyama
et al., 2007; McGlashan et al., 2007) and homeostasis
(Malone et al., 2007) of the skeleton. In this review, we
discuss the structure and formation of the primary
cilium and some of the mechanical and chemical signals
to which it could be sensitive, with a focus on skeletal
biology. We also raise several unanswered questions
regarding the role of primary cilia as mechanosensors
and chemosensors and identify potential research avenues to address these questions.
Ó 2008 WILEY-LISS, INC.
PRIMARY CILIUM STRUCTURE
The structural core of the primary cilium is an axoneme of nine doublet microtubules that extends from a
basal body (also known as a mother centriole; Fig. 1).
Unlike the motile cilia that exist in cells lining the airway and other tissues, primary cilia lack a central pair
of microtubules and are thus called 910 cilia. The primary cilium is enclosed in a specialized membrane
(Vieira et al., 2006) to which receptors for Sonic hedgehog (Shh), platelet-derived growth factor (PDGF), serotonin, and somatostatin localize (Handel et al., 1999;
Brailov et al., 2000; Schneider et al., 2005; Rohatgi
et al., 2007). The ciliary membrane forms a compart*Correspondence to: Charles T. Anderson, Department of
Biological Sciences, Stanford University, Stanford, California.
E-mail: [email protected]
Received 25 June 2008; Accepted 30 June 2008
DOI 10.1002/ar.20754
Published online in Wiley InterScience (www.interscience.wiley.
com).
CELLULAR SENSORS FOR THE SKELETON
1075
vesicle of unknown origin (Sorokin 1962; Alieva and Vorobjev 2004). This docking event precedes the extension
of nine doublet microtubules from the nine triplet microtubules of the centriole. Further axoneme growth is
accomplished by a process called intraflagellar transport
(IFT), wherein cargo is transported up and down the
axoneme via complexes of IFT adaptor proteins by the
motor proteins kinesin-2 and cytoplasmic dynein 1b,
respectively (reviewed in Scholey and Anderson 2006).
The processes by which cargo is acquired and released
and the regulation of motor attachment to the axonemal
microtubules are unclear, as is the mechanism that
determines the final length of the axoneme, which varies
from a few mm to over 100 mm, depending on cell type
(unpublished observations). The membrane surrounding
the axoneme then fuses with the plasma membrane,
allowing the cilium to protrude into the extracellular
space. In cycling cells, primary cilia appear early in G1
(Alieva and Vorobjev 2004) and are disassembled prior
to mitosis by an Aurora A-dependent mechanism (Pugacheva et al., 2007).
PRIMARY CILIA AS MECHANOSENSORS
IN BONE
Fig. 1. Schematic of primary cilium structure. The primary cilium is
enclosed in a specialized membrane (dark green) that is continuous
with the plasma membrane (light green). Membranes are shown in
cutaway view. The axoneme of the primary cilium (dark blue) extends
from the mother centriole/basal body (purple), which possesses subdistal appendages (orange) that are connected to a subset of cytoplasmic microtubules (red). The mother centriole/basal body is part of
the centrosome, which includes a daughter centriole (light blue) and
pericentriolar material (light gray) from which other cytoplasmic microtubules (red) are nucleated). The mother centriole/basal body is connected to the plasma membrane via transition fibers (yellow) that separate the ciliary compartment from the cytoplasm. Cytoplasmic microtubules are reduced in number and shortened for simplicity; they actually
extend far beyond the boundaries of the image. Image not to scale.
ment that is separated from the cytoplasm by transition
fibers (Fig. 1) (Sorokin, 1962; Deane et al., 2001), which
connect the distal end of the basal body/mother centriole
to the plasma membrane. The basal body/mother centriole also possesses subdistal appendages on its sides
that directly contact cytoplasmic microtubules (Sorokin
1962), linking the primary cilium to the microtubule cytoskeleton. The basal body/mother centriole is part of
the centrosome, which organizes the interphase microtubule cytoskeleton in many cell types and exists at the
poles of mitotic spindles (reviewed in Bettencourt-Dias
and Glover 2007). Centrosomes also contain a daughter
centriole that lacks a cilium, and the two centrioles are
surrounded by pericentriolar material from which microtubules are nucleated (Fig. 1).
PRIMARY CILIUM GROWTH
Primary cilium growth begins when the distal end of
the mother centriole docks with a 300 nm diameter
The skeleton is primarily a mechanical organ whose
function is to support the body against gravitational
force and aid in locomotion. The skeleton adapts to mechanical stimuli: increased loading results in greater
bone formation, whereas reduced loading results in bone
loss. The ability of bone to sense and respond to mechanical stimuli depends on bone cells, including osteocytes,
osteoblasts, and osteoclasts. Osteocytes have been proposed as candidate mechanosensors because of their
location within the bone matrix. Osteocytes are housed
in small pockets, or lacunae, which are connected by a
network of channels, or canaliculi (Lanyon 1993). Osteocytes extend projections throughout this canalicular
network and communicate with one another and with
preosteoblasts on bone surfaces through gap junctions
on these projections.
Osteocytes respond to mechanical stimuli both in vivo
(Robling et al., 2006) and in vitro (Klein-Nulend et al.,
1995). Osteoblasts and mesenchymal stem cells (MSCs),
the main source of osteoblast progenitors, have also
been shown to respond to mechanical stimuli and appear
to play an important role in bone mechanotransduction
(Reich and Frangos 1993; Meinel, Karageorgiou et al.
2004). However, the relative importance of different
types of local, mechanical stimuli to which bones cells
respond is unclear. One such mechanical stimulus is oscillatory fluid flow. During dynamic loading of the skeleton, bending moments are created, causing bone matrix
deformation and intramedullary pressure gradients.
These pressure gradients result in flow through the canalicular network from regions of high pressure to
regions of low pressure (Knothe Tate et al., 1998). Flowinduced osteocyte surface shear stress is estimated to be
in the range of 8–30 dynes/cm2 under normal loading
conditions (Weinbaum et al., 1994). Fluid flow may also
cause drag force on cell processes resulting in a hoop
strain of >0.5% (Han et al., 2004); however, this model
assumes that osteocyte processes are tethered to the
lacunar wall, which has not been clearly established.
Osteocytes may also experience cytoskeletal strain or de-
1076
ANDERSON ET AL.
formation as the lacunar spaces in which they reside are
deformed.
The mechanism by which bone cells sense and respond
to these mechanical stimuli is not known, but recent evidence (Malone et al., 2007) demonstrates that primary
cilia are mechanically sensitive in bone cells. Primary
cilia are known to act as flow sensors in renal epithelial
cells (Liu et al., 2005), in which bending of the cilium
results in an increase of intracellular calcium (Schwartz
et al., 1997). They have also been observed in osteocytes
and osteoblasts (Matthews and Martin, 1971; Tonna and
Lampen, 1972; Xiao et al., 2006) and suggested as potential bone mechanical sensors (Whitfield, 2003; Xiao
et al., 2006). Primary cilia projecting from osteocytes
might experience fluid flow shear stress, mechanical deformation, or pressure gradients depending on their size
and orientation. Mechanical stimuli that could affect primary cilia on preosteoblasts are less well defined, as
these cells reside on bone surfaces rather than within
the bone matrix. Determining the size, orientation and
properties of primary cilia on different bone cell types in
situ will be important for understanding their possible
roles in vivo. Interestingly, chondrocytes in cartilage
also have primary cilia, which possess integrins in their
membranes and contact the extracellular matrix (Jensen
et al., 2004; McGlashan et al., 2006). Since osteocytes
likely experience fluid flow in the confined matrix-filled
spaces between the cell surface and the lacunar wall, cilium-matrix interactions might also play a role in mechanosensation in bone.
PRIMARY CILIA AS CHEMOSENSORS
IN BONE
Primary cilia may also function as specialized regions
of the cell membrane that are equipped with receptors
that transmit chemical or molecular signals from the
environment into the cell. One of the best known of
these molecular signaling pathways is mediated by
secreted growth factors in the Hedgehog family. Hedgehog signaling is critical throughout embryonic development and adulthood: disruption in Hedgehog signaling
has been associated with multiple birth defects and
human cancers (reviewed in Helms et al., 2007). Much
of our current knowledge of Hedgehog signaling comes
from studies conducted in the fruit fly, Drosophila.
Collectively, these studies indicate that the Hedgehog
receptor complex is composed of two trans-membrane
proteins, Smoothened and Patched. In the absence of
Hedgehog, Patched represses pathway activity by inhibiting the function of Smoothened, but when Hedgehog is
present and binds to Patched, Smoothened is relieved
from this inhibition and activates target gene transcription. It is thought that a similar mechanism is responsible for Hedgehog signaling in mammalian cells, where
Shh is one of the Hedgehog family ligands.
Recent evidence from a number of laboratories demonstrates that Shh signaling occurs at the primary cilium
(Huangfu et al., 2003; Rohatgi et al., 2007). These
groups showed that in the absence of Shh, Patched occupies the primary cilium, which may exclude Smoothened
from this structure. Because Patched functions as a Shh
receptor, its presence makes the primary cilium a receptacle for any secreted Shh in the vicinity. When Shh
binds to its receptor, Patched leaves the cilium and
Smoothened instead accumulates in the ciliary membrane (Rohatgi et al., 2007). Mutations that affect formation of the primary cilia thus lead to disruption in Shh
signaling.
The connection between Shh signaling and primary
cilia in bone was first hinted at by the finding that mice
with mutations in IFT components have skeletal patterning defects that are similar to Shh mutants
(Huangfu et al., 2003; Zhang et al., 2003). Null mutation
of IFT components results in embryonic lethality (Marszalek et al., 1999; Murcia et al., 2000), precluding studies of complete skeletogenesis in these mutants, but
tissue-specific mutation of these same IFT components
causes a more restricted range of embryonic defects. For
example, mice lacking the kinesin-2 subunit Kif3a in
retinal photoreceptors display abnormal accumulation of
opsin in the inner segment, causing photoreceptor death
(Marszalek et al., 1999), and elimination of the IFT component Ift88 (also known as polaris) in the central nervous system of mice causes cerebellar disruptions (Chizhikov et al., 2007). Likewise, loss of Ift88/polaris or
Kif3a in the developing skeleton leads to a plethora of
skeletal malformations (Haycraft et al., 2007; Koyama
et al., 2007) for reasons that are not yet clear. Analysis
of additional tissue- and cell type-specific knockouts in
mice and other experimental animals should provide
insight into how primary cilia function as molecular
detectors in bone. It will be important to compare skeletal structure and growth dynamics in conditional
mutants lacking components required for primary cilium
structure (e.g., Kif3a) against those lacking specific ciliary signaling pathway components (e.g., Patched) to
determine the contribution of each signaling pathway to
overall primary cilium-mediated signaling during bone
development and homeostasis.
UNANSWERED QUESTIONS
How are Mechanical Signals Sensed by
Primary cilia?
As observed in time-lapse movies of cultured kidney
tubule cells, primary cilia exposed to unidirectional fluid
flow bend in the direction of flow. However, it is not
known whether mechanosensitive proteins are distributed asymmetrically around the cilium, which would
maximize the sensitivity to this bending. Although the
primary cilium has been modeled as a cantilevered
beam (Schwartz et al., 1997), the minimum degree to
which it must be deflected in order to elicit a response is
not known. Furthermore, it is not known whether the
cilium functions as a switch or a rheostat: is there a
threshold level of mechanostimulation that toggles an
on–off signal from the cilium, or is the ciliary signaling
output graded in proportion to the degree of mechanical
stimulation? If the signaling activity is switch-like, there
might also be a recalcitrant period immediately after
activation during which the cilium cannot re-respond to
stimulation. Combining high-resolution imaging of primary cilia and a signaling output (e.g. calcium flux)
with careful control of the timing and strength of an experimental mechanical stimulus should allow definition
of some of these parameters. In the case of bone cells,
combining current models of fluid flow in bone with
CELLULAR SENSORS FOR THE SKELETON
these parameters would provide useful constraints on
models for how primary cilia function in vivo.
Also unknown is the extent to which the cilium’s connection to cytoplasmic microtubules might play a role in
its function as a mechanosensor. The only characterized
mechanosensory mechanism in the cilium involves the
polycystins, which form a stretch-sensitive membrane
channel complex (Nauli et al., 2003), but as noted above,
the basal body is directly connected to a subset of cytoplasmic microtubules via its appendages. These connections could serve to anchor the cilium in place and provide
it with structural support, much like the roots of a tree
support its trunk. However, they might also play a role in
mechanical signaling; bending of the primary cilium could
translate into movement of the attached microtubules,
which could then lead to downstream signaling activity or
changes in the transport of materials along the cytoplasmic microtubules. The cilium and the basal body could
thus act as an extracellular extension of the cytoplasmic
microtubule cytoskeleton and provide it with information
about the extracellular environment, much as integrins
link extracellular matrix proteins to the actin cytoskeleton. Because the microtubules of the primary cilium and
centrioles are less sensitive to low concentrations of
microtubule-destabilizing drugs, such as nocodazole, than
cytoplasmic microtubules (unpublished observations), it
should be possible to study the mechanosensitivity of the
primary cilium in the presence or absence of cytoplasmic
microtubules. Furthermore, imaging of cytoplasmic
microtubules during primary cilium deformation may
give insight into the strength and extent of the connections between these two microtubule systems.
Is there Feedback Between Mechanical
Stimulus and Ciliary Structure?
In human umbilical cord cells, fluid flow has been
shown to cause loss of primary cilia from the endothelial
surface (Iomini et al., 2004). It is possible that mechanical stimuli could lead to remodeling of ciliary structure,
either through changes in its length or membrane composition. As mentioned above, application of Shh to cells
causes the relocalization of Smoothened to the primary
cilium (Corbit et al., 2005; Rohatgi et al., 2007), indicating that trafficking of proteins to the cilium in response
to external signals occurs. Analysis of primary cilium
components before and after exposure to fluid flow or
other chemical or molecular signals might uncover a
mechanism by which bone cells remodel their primary
cilia in response to mechanical or chemical stimulation.
What are the Connections Between
Mechanical Stimuli and Cellular
Behavior in Bone Cells?
In bone cells, primary cilia are required for cytokine
release and changes in gene transcription in response to
dynamic flow (Malone et al., 2007). However, the connections between mechanical stimulation of the cilium and
cellular responses are not fully known. The best-studied
case of a downstream signal resulting from mechanical
stimulation of the primary cilium is that of calcium influx
into kidney tubule cells after exposure to fluid flow, which
leads to intracellular calcium release (Praetorius and
Spring, 2001). Although calcium influx might also be
1077
involved in other settings, it is apparently only one of multiple mechanically activated second messenger systems in
bone (Malone et al., 2007). There is growing evidence that
the primary cilium can be coupled to other signaling pathways. Adenylyl cyclases have also been found in the primary cilium (Masyuk et al., 2006), suggesting that cAMP
could be a second messenger in some forms of ciliary signaling. In addition, IFT could traffic activated signaling
proteins from the ciliary compartment to the cytoplasm, as
has been proposed for Shh signaling (May et al., 2005).
An important unanswered question is the extent to
which primary cilium-mediated mechanosensation leads
to changes in cellular communication and proliferation
in bone. In kidney tubules, loss of primary cilia leads to
overproliferation of tubule cells and the formation of
cysts (Yoder et al., 2002), and primary cilia have been
linked to cell cycle progression (Morgan et al., 2002; Pan
and Snell, 2007). Although osteocytes themselves do not
divide, it is possible that mechanical stimulation of
osteocyte primary cilia could lead to cell proliferation at
the bone surface, which would require cell–cell communication via the canalicular network. Conditional knockout of genes required for primary cilium in specific bone
cell populations, which has already begun (Haycraft
et al., 2007), should allow the dissection of primary cilium function in osteocytes, osteoblasts, and osteoclasts.
An important caveat of these experiments is that
primary cilia might play different signaling roles (chemosensory, mechanosensory, or both) depending on
developmental stage, and the ability to control ciliogenesis temporally as well as in a tissue- or cell type-specific
fashion would allow more detailed characterization of
primary cilium function in bone.
The primary cilium has emerged as an important
organelle for sensing environmental signals, and its
function in diverse tissues is just beginning to be understood. One research goal that lies ahead is to integrate
ciliary signaling with other cellular sensory systems to
provide a comprehensive picture of how cells sense and
respond to environmental stimuli, and bone, with its
architectural complexity and plasticity, provides a challenging but exciting arena in which to accomplish it.
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