Am J Physiol Cell Physiol 308: C198–C208, 2015.
First published November 26, 2014; doi:10.1152/ajpcell.00336.2014.
Themes
The primary cilium as sensor of fluid flow: new building blocks to the model.
A Review in the Theme: Cell Signaling: Proteins, Pathways and Mechanisms
Helle A. Praetorius
Department of Biomedicine-Physiology, Aarhus University, Aarhus, Denmark
Submitted 8 October 2014; accepted in final form 16 November 2014
ATP; calcium; flow; in vivo; primary cilia
Something Weird in the Cell Culture: The Anecdote of the
Cilium as Flow Sensor
Upon my arrival in 2000 in the laboratory of Dr. Kenneth R.
Spring at the National Institutes of Health in Bethesda, MD,
my introduction to the primary cilium was a little backward. In
the Spring laboratory, every microscope was equipped with
ingenious homemade gadgets not found elsewhere, tweaking
the microscopes to the outermost in terms of resolution and
contrast. In this microscopist’s Eldorado, not even the slightest
impurity or membrane bleb of the cultured cells could go
unnoticed. A fellow “postdoc,” who was exceedingly conscientious, was very worried about what appeared to be hyphens
in an overgrown culture of Madin-Darby canine kidney
(MDCK) cells. “No, no, those are primary cilia,” Dr. Spring
answered. I was none the wiser. However, when asked if I, as
a trained patch-clamper, could please manipulate their position,
I naively thought, “no problem.”
So the systematic investigation of one possible function of
the primary cilium was initiated, by chance, by a completely
ignorant postdoc. Normally, such an exercise would be
doomed to failure. In this instance, however, the process was
guided by the most capable supervisor one could ask for: one
who knew exactly when to challenge, when to cheer, and when
Address for reprint requests and other correspondence: H. A. Praetorius,
Dept. of Biomedicine-Physiology, Aarhus Univ., Ole Worms Alle 3, Bldg.
1170, 8000 Aarhus C, Denmark (e-mail: [email protected]).
C198
to stay in the office. Without any genuine expectations of what
was to be found, the process was at least relatively unbiased
and left behind many elaborate theories that never saw the light
of day.
A very crude model of the primary cilium as a flow sensor
(Fig. 1) was ready after about one year. Our data showed that
direct bending of the cilium by application of negative pressure
to a pipette placed near the cilium or bending of the cilium by
increasing the fluid flow over the apical membrane resulted in
an increase in intracellular Ca2⫹ concentration [Ca2⫹]i in type
I MDCK cells (73). This [Ca2⫹]i response was characterized
by bending of the cilium with a micropipette and by an increase
in laminar fluid flow over the cell culture. In both instances, the
response required Ca2⫹ influx from the apical surface, and this
influx could be inhibited by substances that generally inhibit
nonselective cation channels permeable to Ca2⫹, e.g., Gd3⫹
(73). One very characteristic feature of the cilium-dependent
[Ca2⫹]i response was a considerable period of time between
the initial stimulus and the maximal response, in contrast to
direct pipette manipulation of the apical membrane, which
resulted in an instantaneous increase in [Ca2⫹]i. The reason for
this gradual slow response remained unsolved for many years.
The cilium-dependent [Ca2⫹]i response occurred initially in
the cell to which the cilium was attached and then spread to
surrounding cells (73). This transmission to the neighboring
cells was reduced by inhibitors of gap junctions and was
presumed to result from diffusion of inositol 1,4,5-trisphosphate (IP3) through the gap junctions. This issue may not be
0363-6143/15 Copyright © 2015 the American Physiological Society
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
Praetorius HA. The primary cilium as sensor of fluid flow: new building
blocks to the model. A Review in the Theme: Cell Signaling: Proteins,
Pathways and Mechanisms. Am J Physiol Cell Physiol 308: C198 –C208, 2015.
First published November 26, 2014; doi:10.1152/ajpcell.00336.2014.—The
primary cilium is an extraordinary organelle. For many years, it had the full
attention of only a few dedicated scientists fascinated by its uniqueness. Unexpectedly, after decades of obscurity, it has moved very quickly into the limelight with
the increasing evidence of its central role in the many genetic variations that lead
to what are now known as ciliopathies. These studies implicated unique biological
functions of the primary cilium, which are not completely straightforward. In
parallel, and initially completely unrelated to the ciliopathies, the primary cilium
was characterized functionally as an organelle that makes cells more susceptible to
changes in fluid flow. Thus the primary cilium was suggested to function as a
flow-sensing device. This characterization has been substantiated for many epithelial cell types over the years. Nevertheless, part of the central mechanism of signal
transduction has not been explained, largely because of the substantial technical
challenges of working with this delicate organelle. The current review considers the
recent advances that allow us to fill some of the holes in the model of signal
transduction in cilium-mediated responses to fluid flow and to pursue the physiological implications of this peculiar organelle.
Themes
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
quite so simple and is discussed further in the description of
paracrine amplification by ATP (see below). Moreover, we
demonstrated that cells too immature to express primary cilia
(73) and mature MDCK from which the cilium had been
removed chemically (75) were significantly less sensitive to
subtle changes in laminar fluid flow and that the existence of a
primary cilium emerging from the apical surface sensitized the
cells to flow.
Despite our own delight in having resolved this issue, the
road to publication of our finding was long and paved with
cobblestones of rejection letters. After yet another letter explaining the lack of interest to a broader audience, we almost
gave up altogether. In desperation, Dr. Spring proposed that, as
an alternative, we could print a couple thousand copies and
distribute them by throwing them off the roof of Building 10 at
the National Institutes of Health. Today, these findings have
been verified in several studies on many types of epithelia and
even in endothelial cells, which in certain situations may
express primary cilia (40 – 42, 51, 57, 59, 79, 80, 83, 84, 93,
108, 111) and, as it happens, have been observed to be relevant
in many physiological and pathophysiological settings.
Initial Ca2⫹ Entry
The first study on the cilium-dependent flow response established a requirement for Ca2⫹ influx as one of the very early
steps in the signaling mechanism (73). Brief removal of extracellular Ca2⫹ on the apical side of the epithelium was sufficient
to make the cells completely nonresponsive to mechanical
manipulation of the cilium by pipette or an increase in the fluid
flow rate (73). Similar results were achieved by blocking
nonselective Ca2⫹-permeant channels with Gd3⫹. On the basis
of these results, it was concluded that bending of the cilium
activated a mechanosensitive channel permeable to Ca2⫹. It
was assumed that this Ca2⫹ channel was situated in the cilium
or at the base of the cilium, because characteristics of the
mechanoresponse to touching the apical membrane, including
a lack of dependence on extracellular Ca2⫹ (73), were significantly different from those related to the cilium. Despite these
data, it was not possible to detect the initial Ca2⫹ increase in
the cilium. Because of the proportion of the cilium (⬎0.2 ␮m
diameter), demonstration of entry of Ca2⫹ into the cilium is
technically challenging. The membrane-permeable fluorescent
Ca2⫹ probes did not accumulate in the primary cilium in
sufficient amount to allow detection of the postulated ciliary
Ca2⫹ increase.
Recently, there have been more reports on Ca2⫹ signaling in
primary cilia (18, 36, 96). These studies take advantage of
expression of Ca2⫹-sensitive constructs in the given cell type
coupled to one of the proteins that primarily target the primary
cilium. With this strategy, it is possible to accumulate an
amount of Ca2⫹-sensitive fluorophore in the primary cilium
sufficient to detect the ciliary Ca2⫹ concentration ([Ca2⫹])
changes over the background cytosol fluorescence. Su et al.
(96) used a construct of myosin light kinase (M13), a circularly
permuted GFP, and calmodulin, which was localized to primary cilia of NIH-3T3 and mIMCD3 cells with the 5-HT6
receptor, for ciliary targeting. Via this construct, it was possible to successfully detect ciliary changes in [Ca2⫹] in response
to ionomycin and ATP (96). Demonstration of a flow-induced
increase in intraciliary [Ca2⫹] is, however, complicated by the
simultaneous displacement of the cilium. This was solved by
coexpression of mCherry-GFP attached to 5-HT6, which is
insensitive to changes in [Ca2⫹]. From the ratio of backgroundnormalized GFP to background-normalized mCherry signal
intensities, Su et al. showed that changes in fluid flow rate
displaced the cilium and caused an immediate rise in intraciliary [Ca2⫹].
Ca2⫹ signaling in the primary cilium is generally interesting,
because it truly is a very localized compartment that empties in
a very localized area around the centriole. Because the cilium
is free of other membrane compartments/organelles, it is reasonable to assume that the Ca2⫹-buffering capacity is relatively
limited. When the ciliary volume is restricted, influx of only a
few Ca2⫹ ions is likely to change [Ca2⫹] dramatically (75). As
the cilium only communicates with the cytosol in an area
around the centriole, this particular location will be a hot spot
for Ca2⫹ signaling, which is likely to be essential in ciliary
signal transduction. Local Ca2⫹ signaling has been known for
decades, and initiation through hot spots has been suggested to
direct the intracellular Ca2⫹ signaling in many cell types, e.g.,
cardiomyocytes (14). In this context, it is interesting that the
ciliary Ca2⫹ signaling has been suggested to be confined to the
cilium itself, and not to cause pancellular changes in [Ca2⫹]
(18). In the study of Delling et al. (18), mCherry-GCaMP3
linked to smoothened was used to study intraciliary [Ca2⫹],
whereas fluo 4-AM loading was used to monitor cytosolic
[Ca2⫹]. Using this method, they showed that when the tip of
the cilium was ruptured by a laser pulse, the increase in
intraciliary [Ca2⫹] did not spread to the cytosol, except from a
minor event at the base of the cilium. This potentially suggests
a diffusion barrier from the cilium to the cytosol for Ca2⫹,
which, in principle, could be created by the alar sheets, which
are transitional fibers radiating from the basal body toward the
plasma membrane (104). Another possible explanation is that
the cytosolic buffering capacity of the cytosol for Ca2⫹ is
magnitudes higher than the buffering capacity of the cilium for
Ca2⫹ and that cytosolic buffers immediately sequester any
Ca2⫹ arriving at the cilium base. The latter explanation is very
conceivable, in that Ca2⫹, uncaged in the cytosol, had no
problem entering the cilium (18) in situations where the cytosolic Ca2⫹ capacity was temporarily saturated. This explana-
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
Fig. 1. First sketch of the model for the cilium-dependent flow-response in
Madin-Darby canine kidney cells. ER, endoplasmic reticulum.
C199
Themes
C200
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
The Ciliary Channel, Giving Rise to the Ciliary Ca2⫹
Increase
One could ask why it is still is necessary to discuss which
channel gives rise to the cilium-dependent Ca2⫹ response. As
mechanoinduced increases in the ciliary [Ca2⫹] were only very
recently demonstrated, there is only one study that directly
implicates a particular channel in mechanically induced Ca2⫹
influx in the cilium (36). However, the transient receptor
potential (TRP) channel polycystin 2 has long been a strong
candidate for the central channel involved in the Ca2⫹ entry
triggered by cilium bending. Nauli et al. (57) showed that it
was primarily deficiency of the binding partner of polycystin 2,
i.e., polycystin 1, that was important for normal cilium-dependent flow sensing. They also provide data for a substantial
reduction of the flow-induced [Ca2⫹]i response by antibodies
directed against polycystin 2 (57). Despite pending direct proof
that polycystin 2 is the ciliary influx channel, several studies in
support of the concept followed (1, 40, 42, 44, 51, 54, 57, 97).
This evidence was particularly demanded, because polycystin 2 has been shown to serve other functions within the cell.
Many of the above-mentioned results are challenged by data
suggesting that polycystin 2 is acting as a Ca2⫹ release channel
situated on the ER (43). The function of polycystin 1 is
apparently similarly essential to the function of polycystin 2 in
the ER (56), where it interacts with the IP3 receptor to manifest
its effects (47, 85). Since the cilium-dependent flow response
in many cases has been shown be potentiated by downstream
activation of IP3 receptors/PKC activation (40, 73) or ryanodine receptors (57), studies implicating polycystin 2 in the
cilium-dependent flow response could be explained by polycystin 2 acting as a Ca2⫹-permeant channel on the ER.
Nonetheless, the primary ciliary membrane has been shown
to express polycystin 2. This was initially demonstrated in
Caenorhabditis elegans, where normal sensory behavior of the
male depends on functional expression of the homolog of
polycystin 2 in the cilia of the sensory neurons (7, 8). Moreover, polycystin 2 must be expressed in the flagella of the
spermatozoa to sustain normal fertility in Drosophilia (26,
102). The presence of polycystin 2 has also been confirmed in
mammalian cells by immunostaining (66, 110), as well as
functionally (17, 78). There was, however, a marked time gap
before evidence was provided by Kotsis et al. (41) that a
reduction of polycystin 2 expression in the cilium itself would
abrogate the flow response. In the most recent work on the
topic, Nauli’s group (36) is the first to show that polycystin 2
is needed for influx of Ca2⫹ in the cilium. Knockdown of
PKD2 with siRNA reduced polycystin 2 expression by ⬎90%
Fig. 2. Model for changes in ciliary Ca2⫹ concentration ([Ca2⫹]) in response to flow-mediated bending of the cilium.
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
tion is supported by Su et al. (96), who demonstrated that, upon
stimulation of ciliated NIH-3T3 cells by an agonist, a Ca2⫹
wave will begin at the base of the cilium and propagate to the
tip. If the ability of a ciliary [Ca2⫹] signal to become a
pancytosolic event depends on the cytosolic Ca2⫹-buffering
capacity, it is likely to be cell type-specific and involve the
proximity of the endoplasmic reticulum (ER) and mitochondria
to the cilium base. Most of the data support the model illustrated in Fig. 2.
It must be stressed that the cilium-dependent flow response
was primarily measured as changes in [Ca2⫹]i (40, 57, 73).
Thus, at least in some cells, it is likely to become a robust
global [Ca2⫹]i increase. This idea is supported by a recent
study that primarily confirms a flow-induced increase in intraciliary [Ca2⫹] (36). In this study, GCaMP3 is directed to the
primary cilium of LLC-PK1 cells by the ciliary-targeting
domain of fibrocystin (36). Using this construct, Jin et al. (36)
showed that it was possible to measure changes in intraciliary
[Ca2⫹] and cytosolic [Ca2⫹]. They demonstrated that low flow
rates caused a bending of the cilium, followed by an increase
in intraciliary [Ca2⫹] and, immediately thereafter, an increase
in cytosolic [Ca2⫹] (36).
It is, however, feasible that there will be cellular differences
in whether a ciliary Ca2⫹ response will generate a cytosolic
Ca2⫹ response. It was initially proposed that early Ca2⫹ influx
was potentiated by Ca2⫹-induced Ca2⫹ release from intracellular stores. Later, it became clear that not all cell types show
this pattern. It has been demonstrated that, in MLO-Y4 osteocyte-like cells, the cilium-dependent flow response requires
Gd3⫹-sensitive Ca2⫹ entry, which reduces the cAMP levels in
the cells via inhibition of adenylyl cyclase 6 by a mechanism
that apparently does not require a global increase in [Ca2⫹]i
(45). In these cells, it is quite feasible that the bending-induced
[Ca2⫹] increase never occurs outside the cilium and, thus, is
not converted to a pancytosolic Ca2⫹ response but, apparently,
is sufficient to inhibit adenylyl cyclase 6. This illustrates that
there may very well be a difference in the cellular response
downstream from the cilia-dependent part of the flow response.
Themes
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
Amplification of the Initial Signal
2⫹
Ca entry into the primary cilium is a very subtle signal
that arrives at the base of the cilium, where it may or may not
expand into a global [Ca2⫹]i increase. It is certainly very likely
that Ca2⫹ influx into the cilium is important only as a robust
Ca2⫹ event at the cilium base similar to the discrete Ca2⫹
sparks, which are known to produce very precise subcellular
signaling. One possibility is that condensed [Ca2⫹] would be
sufficient for Ca2⫹-induced Ca2⫹ release, as it is classically
described in cardiac and skeletal muscle as requiring ryanodine
receptors. This type of response is apparently seen in several
cell types (1, 36, 57, 58). In other cell types/tissues, there is a
distinct dependence on IP3 generation (73, 111). It was primarily suggested to be a consequence of Ca2⫹-induced Ca2⫹
release, which, in principle, is also a possibility for the IP3
receptor, although it would be more likely that dependence on
the IP3 receptor results from 7TM receptor stimulation. This
would, however, require stimulation by an extracellular molecule of sorts. One of the regular extracellular signaling molecules associated with mechanotransduction is ATP. Interestingly, ATP release and P2 receptor signaling have been shown
to be involved in the cilium-dependent flow response (33, 72,
76, 109). In this model (Fig. 3), the subtle cellular response
initiated by cilium bending is amplified by ATP release, and
the purinergic response amounts to a substantial part of the
overall response. This model is appealing in tissues working in
a coordinated fashion, where it is not necessary that all cells
experience the same degree of stimulation. Even though only a
few cells reach the threshold for response, paracrine factors
will reinforce a collective coordinated tissue response. It may
also very easily explain the lateral signal transmission from the
cell to which the cilium is attached to surrounding cells during
manipulation of a single cilium.
It is peculiar, but very interesting, that the ciliary current
carried by polycystin 2 is augmented by extracellular P2
receptor activation by ATP (17). One could easily envision that
activation of a P2X receptor could contribute to overall ciliary
current and even [Ca2⫹] in the structure. This is, however, not
the case. Apparently, UTP and ADP have an impact on current
Fig. 3. Model for the flow-mediated, cilium-dependent [Ca2⫹] response. IP3,
inositol 1,4,5-trisphosphate.
similar to that of ATP, which suggests that a P2Y receptor is
mediating the response (17). These findings would certainly
explain why ATP release is such an effective amplification
system for cilium-mediated flow response.
A consequence of ATP-mediated amplification of the cilium-mediated flow response is that the response can appear
slightly different in various tissues, depending on the specific
P2 receptor expression pattern. For example, the aforementioned flow response in osteocytes has been suggested to be
cilium-dependent; however, the downstream events from the
initial Ca2⫹ entry do not implicate Ca2⫹ release from intracellular Ca2⫹ stores but, rather, adenylyl cyclase activation,
cAMP generation, and PKA activation. This could suggest that
osteocytes merely have a different P2 receptor expression
pattern, which alters the flavor of the downstream signaling. In
this connection, it is interesting that osteocytes are known to
express P2X7 receptors, which would contribute to Ca2⫹ influx, rather than Ca2⫹ mobilization, from intracellular Ca2⫹
stores. In this context, it is noteworthy that the P2X7 receptor
knockout mouse has reduced bone density (63), and singlenucleotide polymorphism variants in P2X7 have been proposed
to identify women at greater risk of developing osteoporosis
(27, 37, 103).
The Primary Cilium Increases the Cell’s Sensitivity for
Subtle Flow Changes
The primary cilium is not a sensor of shear stress per se but,
rather, is subjected to beam stress because of its structure. The
mechanical properties of the primary cilium have been modeled on several occasions (81, 84, 87). The primary cilium
sensitizes cells to changes in laminar fluid flow, which means
that particular attention must be paid to the way cells are
mechanically stimulated to isolate the cilium-dependent flow
response (70). But is the primary cilium the only flow sensor?
The short answer to this question is no. As pleasant as it is
when answers are completely black and white, important
information from the gray tones tends to be missed. In this
case, it was known from the beginning from the abundance of
shear stress data from nonciliated endothelial cells that the
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
and completely abolished the flow-induced increase in ciliary
and cytosolic [Ca2⫹]. This effect is unlikely to be caused by a
reduced ability of intracellular Ca2⫹ stores to release Ca2⫹,
because when the store-mediated response was blocked with
caffeine, only cytosolic [Ca2⫹] was affected, whereas the
ciliary [Ca2⫹] response was intact (36). Inhibition of the
LLC-PK1 cells’ IP3 receptor pathway did not affect the ciliumdependent flow response (36); therefore, involvement of the IP3
receptor and polycystin 2 in Ca2⫹ stores is unlikely. Thus, polycystin-mediated Ca2⫹ release from intracellular Ca2⫹ stores may
contribute to, but is not responsible for, polycystin 2 dependence
in the flow-induced [Ca2⫹] response of ciliated cells.
Polycystin 2’s property in terms of mechanosensation has
been queried, and Kottgen et al. (42) tested whether it works in
complex with TRPV4, which has these features. This study, in
addition to confirming that cilia sense discrete flow rates, also
shows that the cilium-dependent flow response requires
TRPV4. TRPV4 is permeable to Ca2⫹ (101), which would
potentially contribute to Ca2⫹ entry in the cilium. This, however, remains to be established.
C201
Themes
C202
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
The Primary Cilium as Flow Sensor in Renal Epithelia
All renal tubules and the parietal layer of Bowman’s capsule
(5, 46, 69) express primary cilia; however, in the more distal
part of the renal tubules, cilia are seen only in a subfraction of
the cells (5, 50, 69). Interestingly, the role of the primary
cilium as a flow sensor is still heavily debated in the renal
community. This is noteworthy, as the primary cilium was
initially proposed to be the sensory mechanism. In cultured
renal epithelial cells, where particular attention has been paid
to isolation of subtle changes in laminar flow, there have been
no problems with isolation of the primary cilium as an important sensitizer for detection of these changes (40 – 42, 44, 73,
74, 83, 84, 90, 93). Recently, Raghavan et al. (76) demonstrated that LLC-PK1 cells have a robust cilium-dependent
[Ca2⫹]i response that modulated endocytotic activity in a
clathrin- and dynamin-dependent manner. They showed that a
cilium-dependent flow response gave rise to increased endocytosis of albumin and dextran as a fluid phase marker ⬃15–30
min after the flow rate was increased (76). In addition to
showing absolute cilium dependence, Raghavan et al. reported
that both the flow-induced [Ca2⫹] response and endocytosis
were abolished by intracellular Ca2⫹ chelation and by scavenging of extracellular ATP by apyrase. Moreover, the entire
system could be short-circuited by extracellular addition of 100
␮M ATP. Given the dependence of ryanodine-sensitive stores,
the effect of extracellular ATP is unlikely to be mediated via
P2Y receptors, which classically cause G␣q-dependent IP3
generation (38, 67). This idea is consistent with a report that
LLC-PK1 cells functionally express primarily P2X receptors
(25). Thus the dependence of intracellular Ca2⫹ release apparently is purely a result of Ca2⫹-induced Ca2⫹ release, where
the Ca2⫹ may, in turn, be a combination of Ca2⫹-permeable
channels in the cilium and P2X receptors in the entire membrane. Nevertheless, this study confirms the established model
for a cilium-dependent flow response and adds to the long list
of reports about cilium-dependent, flow-induced responses in
renal epithelial cells.
The consistent findings from cell cultures are not easily
translated into isolated renal tissue. This is clearly a disappointment, because isolated tissue systems usually are a closer
approximation of the in vivo situation than are cells in culture.
When a finding from cell culture cannot be immediately
replicated in freshly isolated tissue, it is often considered to be
an artifact. In this case, one should be very careful in dismissing such a finding, because the laminar flow properties are
easier to replicate and control in cell culture models. In isolated
perfused renal tubules, it has been immensely difficult to
isolate the cilium-dependent flow response, because changes in
fluid flow rate in the tubular lumen also create a pressure
gradient from the lumen to the bath. These pressure changes
have been mimicked in a cell culture model, where the transepithelial pressure can be changed without a change in the net
flow rate in the cell chamber. Data from the study of Praetorius
et al. (71) show that the primary cilium has no role in the
mechanical response caused by lateral stretching of the epithelium. The flow-induced response in the perfused renal tubules
clearly has an element of stretch (35, 107); thus the epithelial
cells will be subjected to two types of mechanical stimuli at the
same time. Nonetheless, some cilium dependence of the flowinduced changes in tubular structures could be detected (49,
51). This tubular stretch is not necessarily unphysiological. It is
very likely that some changes in tubular diameter will occur
during papillary contractions (20, 86). Such changes are just more
severe mechanical perturbation, which would occur under different circumstances from subtle changes in laminar flow rates.
The real controversy of the meaning of cilium-dependent
flow response resides in the connecting tubule and the collecting duct. These segments consist of several distinct cell types;
among these, the intercalated cells do not express primary cilia,
whereas the principal cells do (5, 50). Both of these cell types
have been proposed to be the main cell type that senses fluid
flow in the collecting duct. Since the intercalated cells do not
have primary cilia, the cilium can hardly be responsible for
flow sensing by this cell type. Arguments for flow sensing by
the intercalated cells are compelling. The slight protrusion of
the apical membrane of the intercalated cells into the lumen
(50) potentially exposes them to mechanical stress. The ␤-intercalated cells express connexin30 (Cx30) in the apical membrane (52), which suggests that these connexins not only form
gap junctions but may form hemichannels in the apical membrane. Since these hemichannels are large unselective pores
that allow passage of larger molecules such as ATP, they have
been promoted as a major pathway for mechanically induced
ATP release in these cells (52, 92). This leads to a model where
an increase in fluid flow is sensed by the intercalated cells,
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
primary cilium could not be the only flow-sensing device (4,
19, 28, 88, 89). The syncytium of endothelial cells is the
primary organ in which an increase in vascular flow and
transfer of various signals for vasorelaxation, including nitric
oxide release, were detected (for overview see Ref. 77). The
flow-induced Ca2⫹ response in endothelial cells does, however, require quite high laminar flow rates, corresponding to
a shear stress of 8 –10 dyn/cm2 (4, 19, 28, 89). The term
shear stress is more appropriate in endothelial cells, considering the higher Reynolds number in the vascular system,
where the entire luminal membrane of the endothelial cell is
regarded as the flow-sensing apparatus (106). Interestingly,
a few studies have shown that, under pathological conditions where flow is significantly reduced, endothelial cells
do express primary cilia (34).
The cilium-dependent flow response is maximal at flow rates
that are magnitudes lower (9, 73), i.e., shear stress of 0.1– 0.2
dyn/cm2; thus the stress on the luminal membrane in these
situations is too low to be detected by the nonciliated version
of the same cell type (73, 74). Even in renal epithelial cells,
primary cilia are not required for detection of changes in shear
forces, if the shear stress is increased sufficiently. This is
illustrated quite clearly in a study where the MDCK renal
epithelial cell line, which is known to produce quite stable
cilium-dependent flow responses, was subjected to shear stress
far above that needed to trigger the cilium-dependent flow
response (82). In that case, the shear-mediated response was
similar whether or not the cells possessed a cilium, and there
was only a minor contribution from Gd3⫹-sensitive Ca2⫹
influx (82). Ergo, the primary cilia are not required to sense
fluid flow on a larger scale, but the structure makes cells susceptible to minor changes in fluid flow that would otherwise go
unnoticed. Thus the physiological importance of the primary
cilium as a sensory organelle can only be evaluated in situations
where the in vivo flow pattern is mimicked meticulously.
Themes
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
Moreover, ATP generally acts as an effective paracrine
factor by laterally spreading via ATP-induced ATP release (3,
15). ATP-induced ATP release is unlikely to be supported by
the connexin hemichannels, which are not activated by an
increase in [Ca2⫹]. A subfraction of the hemichannels is,
however, stimulated by minor increases in [Ca2⫹]i but inhibited when [Ca2⫹]i exceeds 300 – 400 nM (16). Moreover, some
of the connexin hemichannels are inhibited by PKC (30),
which is downstream of the P2Y receptors following G␣q
activation. In addition, many connexin hemichannels are
mainly activated by low extracellular Ca2⫹. In the kidney,
[Ca2⫹] can only fall substantially in the luminal compartment
because of the tight regulation of [Ca2⫹] in the blood. In the
urine, [Ca2⫹] is normally 3–5 mM (100) and, thus, not low
enough to cause stimulation of connexin hemichannels (6).
However, after massive water load, it is possible to achieve ⬍1
mM Ca2⫹ in the urine (100). Interestingly, a study that addresses the effects of luminal [Ca2⫹] on tubular K⫹ secretion
in microperfused distal tubules of rats shows that K⫹ secretion
is significantly greater when tubular [Ca2⫹] is decreased to
⬍0.2 mM (62). In accordance with the overall model, these
findings could be explained by opening of Cx30 hemichannels,
Fig. 4. Cilium-dependent flow response during normal heart development. A: angle of
the cilium as the heart of the zebrafish starts
to produce flow in the aorta [from no flow at
24 h postfertilization (hpf) to a substantial
oscillatory flow (100 –300 ␮m/s) after 28 h].
B: effect of 1) absence of flow lacking troponin T2a (tnnt2MO) or silent heart (sih), 2)
absence of polycystin 2 (photo-pkd2⫹UV,
pkd2MO, pkd2MO ATG, and pkd2MO
Ex3), or 3) absence of erythrocytes (gata1MO)
on [Ca2⫹]i. Values are expressed as GCaMP3
fluorescence (Kaede protein) intensity. C:
overall model proposed on the basis of the
data. [Reproduced from Goetz et al. (29)
with permission.]
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
causing ATP release, which is then responsible for an autocrine- and paracrine-mediated increase in [Ca2⫹]i. The notion
that flow is sensed by the intercalated cells is supported by
studies of flow-mediated K⫹ secretion in the same segment.
This flow-dependent K⫹ secretion is mediated by the Ca2⫹sensitive K⫹ channel KCa1.1 (24, 48), which is functionally
expressed preferentially in intercalated cells (64, 65).
There are very few arguments against this model. One
should, however, consider that the connexin hemichannel is not
a selective ATP release mechanism. The notion that ATP is
released through a channel partially defies the idea that ATP
release is needed to cause K⫹ secretion. The opening of
connexin hemichannels itself would create an immense efflux
of K⫹. This would be exceedingly effective, because connexin
hemichannels, as opposed to the selective K⫹ channel, would
not clamp the membrane at the reversal potential for K⫹. If
there is a vast abundance of Cx30 hemichannels in the apical
membrane, which is activated by mechanical stress, Cx30 will,
by itself, be an efflux pathway for K⫹, particularly because it
simultaneously depolarizes the apical membrane and, thus,
allows for prolonged, unhindered K⫹ secretion. This would, in
principle, render the KCa1.1 channel redundant.
C203
Themes
C204
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
The Primary Cilium as Flow Sensor In Vivo
In living animals, the flow of the various solutions is usually
laminar under physiological conditions. In humans, the Reynolds number is only high enough to cause turbulence in the
aortic arch (95), although turbulence also has been suggested to
occur immediately downstream of arterial branching and bifurcations in models of the vascular system at Reynolds numbers that normally are consistent with laminar flow (55, 94).
Cells are, in general, sensitive to a large variety of mechanical
manipulations, such as deformation and stretch. In this context,
it is interesting that the primary cilium apparently makes cells
particularly sensitive to subtle changes in laminar flow and,
thus, would not be required for more pronounced perturbation
of the cells. This was to be expected because of the tissue
distribution of the expression of primary cilia. Primary cilia are
generally expressed by epithelial cells facing a lumen containing solution passing at modest speed, such as the pancreatic
(112) and bile (51) ducts and the renal tubular system (5, 46,
69). Endothelial cells that experience a higher degree of shear
stress do not regularly express primary cilia (105).
However, there has been sporadic mention of cilia on endothelial cells (aorta decendens, arcus aortae, aorta thorasica,
arteria carotis communis, arteria brachiocephalica, and truncus
pulmonalis) in rabbit (21, 22) and bovine (10) aortic endothelial cells in culture, human aorta (12), capillaries around human
teeth (23), and capillaries of the spinal cord in 7-wk-old human
embryos (11). This has, however, not been a consistent finding
(105), and human umbilical vein endothelial cells are devoid of
primary cilia (105). Interestingly, primary cilia have been
found to develop in areas of low perfusion induced by atherosclerotic plaques, and cilia in endothelia (31) have been shown
Fig. 5. Flow sensing in the node of polycystin 2 knockout mice and wild-type controls. PKD2 is necessary for sensing of nodal flow. A: nodal flows in wild-type
and Pkd2⫺/⫺ embryos were examined by particle image velocimetry analysis at embryonic day 8. Flow is maintained in the Pkd2⫺/⫺ embryo. Each arrowhead
denotes direction and speed of the flow at the indicated position. Color scale indicates direction and magnitude of flow velocity: yellow and red (leftward) and
blue (rightward). White lines indicate outlines of the node. L, left; R, right. Scale bar, 10 mm. B: embryonic day 8 Pkd2⫹/⫹ and Pkd2⫺/⫺ embryos harboring
the ANE-LacZ transgene were stained with X-gal. Scale bar, 50 mm. C: summary of left-right patterns of ANE-LacZ activities in embryos of the indicated
genotypes in situ or under conditions of artificial rightward flow in vitro. Numbers of embryos showing each pattern are indicated. D: Pkd2⫹/⫹ embryos with
ANE-LacZ were cultured under the influence of a rightward artificial flow from early headfold to 6-somite stage. Artificial rightward flow reversed the pattern
of Pitx2 expression in the lateral plate mesoderm (left, red arrowhead) and that of ANE activity in crown cells, as detected by in situ hybridization of LacZ mRNA
(middle) and X-gal staining (right). Scale bars, 100 ␮m. [Reproduced from Yoshiba et al. (111) with permission.]
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
allowing release of ATP and K⫹ into the luminal compartment.
This model could certainly explain why transepithelial potential is lower at 0 than 0.8 mM luminal Ca2⫹ (62).
Nonetheless, there is compelling evidence for the primary
cilium as a flow sensor in renal epithelial cells. As mentioned
above, this has been confirmed in renal epithelial cells in cell
culture when particular care is taken to control laminar flow
conditions. In this context, it must be stressed that isolated
changes in laminar flow are not possible in the perfused renal
tubules when the standard technique for renal perfusion is
applied in the split-open tubule. In the split-open tubule, an
increase in superfusion flow rate will create turbulence in the
proximity of the edges of the glued-down tissue and very likely
lift the edges of the tissue, resulting in mechanical stimulation
of the tissue quite far from the site at which mechanical
stimulation occurs in vivo. Moreover, the tissue is easily
damaged by the procedure, making the more vulnerable cells
more prone to release paracrine factors, such as ATP. It is
therefore interesting to investigate the dependence of the cilium in vivo in tissue exposed to inartificial flow.
Themes
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
(54). However, this could, in principle, result from a defect in
nodal flow development in PKD2 knockout mice.
This defect in nodal flow development in PKD2 knockout
mice was recently addressed very convincingly by Hamada’s
group (111) in an investigation of the two-cilia model of nodal
flow. Although this is not strictly an in vivo model, it primarily
uses the naturally generated flow by the motile cilia in the
node. Hamada’s group confirms the lack of sidedness in the
expression of nodal and Pitx2 in PKD2-deficient mice (Fig. 5).
Importantly, they demonstrate that the PKD2 knockout mice
do not have a reduced nodal flow; thus the lack of polycystin
2 does not alter the ability of the motile cilium to develop nodal
flow (111). Moreover, they showed that the lack of sidedness
of PKD2 knockout mice was mimicked in wild-type mice by
addition of Gd3⫹, thapsigargin, and the IP3 receptor inhibitor
2-aminoethoxydiphenylborate, whereas the ryanodine receptor
pathway did not influence the response. It was verified that the
ability of cilia to sense flow requires expression of polycystin
2 in cilia of the crown cells. As an extra refinement, the authors
used Kif3a knockout mice, which lack cilia completely in the
pit and the crown of the node and express the construct
NDE-hsp-Kif3A-IRES-LacZ, which restores cilia in the crown
cells. Comparison of these embryos showed no sidedness in the
Kif3a knockout mice but did show restoration of the sided
expression of the node by rescue of the sensory cilia (as the
Kif3a knockout still showed a minor leftward flow). Moreover,
the sidedness could be reversed by artificial superimposition of
the reverse flow direction over the node. This study convincingly shows that sensory cilia expressing polycystin 2 are
necessary to sense the flow direction in the node to determine
left and right in embryonic development.
Interestingly, Hamada’s group (91) has convincingly shown
that motility of as few as two rotating cilia is sufficient to create
a nodal flow, which may explain the functional difference
between mutations that reduces motility of the cilia to the
mutations that result in lack of cilia altogether.
Conclusion
Expression of a primary cilium allows cells to respond to
subtle changes in laminar flow. This mechanism is essential for
normal function and development of structures selectively
exposed to slight mechanical stress.
ACKNOWLEDGMENTS
The author thanks Andreas Praetorius for constructive criticisms and editing.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author.
AUTHOR CONTRIBUTIONS
H.A.P. prepared the figures; H.A.P. drafted the manuscript; H.A.P. edited and
revised the manuscript; H.A.P. approved the final version of the manuscript.
REFERENCES
1. Abou Alaiwi WA, Takahashi M, Mell BR, Jones TJ, Ratnam S, Kolb
RJ, Nauli SM. Ciliary polycystin-2 is a mechanosensitive calcium
channel involved in nitric oxide signaling cascades. Circ Res 104:
860 –869, 2009.
2. Afzelius BA. Situs inversus and ciliary abnormalities. What is the
connection? Int J Dev Biol 39: 839 –844, 1995.
3. Anderson CM, Bergher JP, Swanson RA. ATP-induced ATP release
from astrocytes. J Neurochem 88: 246 –256, 2004.
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
to disassemble as a function of shear stress (34). This may
explain the inconsistent reports on primary cilia on endothelial
cells. Moreover, primary cilia have been shown to be expressed
in developing murine aortic endothelial cells, where they can
function as sensors of fluid flow in a polycystin 1-dependent
(58) and polycystin 2-dependent (1) manner.
Recently, Goetz et al. (29) expanded these findings in a
zebrafish in vivo model. They reported that the flow rate in
early embryonic cardiovascular development in zebrafish directly depends on heart function. They found that blood flow
increased in an almost linear fashion at 24 –28 h after fertilization, which resulted in an increase in the cilium tilt angle to
a maximum of 42° at 28 h after fertilization. In this period of
early cardiovascular development, 76% of endothelial cells
expressed primary cilia. This percentage decreased as the flow
intensified, and at 48 h postfertilization, cilia were present in
only 4% of the endothelial cells. In morphants lacking troponin
T2a, the myocardium does not show cardiac contractions, and,
as a consequence, there is no flow in the aorta. In these
morphants, the endothelial cells persistently express cilia also
in the later phases of development, and these cilia were not
deflected. Associated in time to the increase in blood flow and
bending of the cilium, the endothelial cells show an increase in
[Ca2⫹]i (Fig. 4), and the flow-induced [Ca2⫹]i was absent in
the endothelial cells lacking the intraflagellar transport protein
88 homolog and, thus, primary cilia. The cilium-dependent,
flow-mediated [Ca2⫹]i response required endothelial expression of PKD2, which did not change the aortic flow rate. Goetz
et al. conclude that the cilium is very sensitive to very subtle
shear forces, because the cilium tilt angle and the [Ca2⫹]i
response are sensitive to the absence of erythrocytes in the
blood. This is, in many aspects, an extraordinary study. Particularly, it demonstrates the functional importance of the
cilium-dependent flow response in vivo.
Similar to the development of the cardiovascular system in
zebrafish, the immotile primary cilia are essential in flow
sensing, leading to the development of left-right asymmetry. In
the node of murine embryos, flow generated by motile 9⫹0
cilia with preserved dynein arms is essential for development
of left-right asymmetry (for overview see Refs. 13 and 53). If
these cilia are lacking (60, 98, 99) or immotile (61), situs
inversus is seen in ⬃50% of the offspring. This is consistent
with the finding that situs inversus is common in Kartagener
syndrome, which is characterized by cilia with reduced mobility (2, 39). There are, however, some differences between lack
of motility of the cilia and complete lack of nodal cilia. In the
case of lack of ciliary motility, some of the genes (e.g., nodal
and lefty) still show asymmetry (for overview see Ref. 53).
These discrepancies have led to the proposal of a cilia-type
model for generation and sensing of nodal flow (53, 54). In the
node, centrally placed motile cilia with 9⫹0 structure make
circular movements and create a left-right laminar flow (60).
At the rim of the node, regular immotile primary cilia with
9⫹0 structure have been proposed to sense the laminar flow of
the node (53, 54). It is striking that the PKD2 knockout mouse
shows a defect in development of left-right asymmetry (68).
Polycystin 2 was shown to be expressed on the motile (situated
on the pit cells) and the immotile (situated on the crown cells)
cilia of the node, and in mice lacking PKD2, the characteristic
flow-induced [Ca2⫹] on the left side of the node was absent
C205
Themes
C206
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
bone mineral density and accelerated bone loss in post-menopausal
women. Eur J Hum Genet 20: 559 –564, 2012.
Geiger RV, Berk BC, Alexander RW, Nerem RM. Flow-induced
calcium transients in single endothelial cells: spatial and temporal analysis. Am J Physiol Cell Physiol 262: C1411–C1417, 1992.
Goetz JG, Steed E, Ferreira RR, Roth S, Ramspacher C, Boselli F,
Charvin G, Liebling M, Wyart C, Schwab Y, Vermot J. Endothelial
cilia mediate low flow sensing during zebrafish vascular development.
Cell Rep 6: 799 –808, 2014.
Hawat G, Baroudi G. Differential modulation of unapposed connexin
43 hemichannel electrical conductance by protein kinase C isoforms.
Pflügers Arch 456: 519 –527, 2008.
Hierck BP, Van der HK, Alkemade FE, Van de PS, Van Thienen JV,
Groenendijk BC, Bax WH, Van der LA, Deruiter MC, Horrevoets
AJ, Poelmann RE. Primary cilia sensitize endothelial cells for fluid
shear stress. Dev Dyn 237: 725–735, 2008.
Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med 364:
1533–1543, 2011.
Hovater MB, Olteanu D, Hanson EL, Cheng NL, Siroky B, Fintha A,
Komlosi P, Liu W, Satlin LM, Bell PD, Yoder BK, Schwiebert EM.
Loss of apical monocilia on collecting duct principal cells impairs ATP
secretion across the apical cell surface and ATP-dependent and flowinduced calcium signals. Purinergic Signal 4: 155–170, 2008.
Iomini C, Tejada K, Mo W, Vaananen H, Piperno G. Primary cilia of
human endothelial cells disassemble under laminar shear stress. J Cell
Biol 164: 811–817, 2004.
Jensen ME, Odgaard E, Christensen MH, Praetorius HA, Leipziger
J. Flow-induced [Ca2⫹]i increase depends on nucleotide release and
subsequent purinergic signaling in the intact nephron. J Am Soc Nephrol
18: 2062–2070, 2007.
Jin X, Mohieldin AM, Muntean BS, Green JA, Shah JV, Mykytyn K,
Nauli SM. Cilioplasm is a cellular compartment for calcium signaling in
response to mechanical and chemical stimuli. Cell Mol Life Sci 71:
2165–2178, 2014.
Jorgensen NR, Husted LB, Skarratt KK, Stokes L, Tofteng CL,
Kvist T, Jensen JE, Eiken P, Brixen K, Fuller S, Clifton-Bligh R,
Gartland A, Schwarz P, Langdahl BL, Wiley JS. Single-nucleotide
polymorphisms in the P2X7 receptor gene are associated with postmenopausal bone loss and vertebral fractures. Eur J Hum Genet 20:
675–681, 2012.
Kastritsis CH, Salm AK, McCarthy K. Stimulation of the P2Y purinergic receptor on type 1 astroglia results in inositol phosphate formation
and calcium mobilization. J Neurochem 58: 1277–1284, 1992.
Kosaki K, Casey B. Genetics of human left-right axis malformations.
Semin Cell Dev Biol 9: 89 –99, 1998.
Kotsis F, Nitschke R, Boehlke C, Bashkurov M, Walz G, Kuehn EW.
Ciliary calcium signaling is modulated by kidney injury molecule-1
(Kim1). Pflügers Arch 453: 819 –829, 2007.
Kotsis F, Nitschke R, Doerken M, Walz G, Kuehn EW. Flow modulates centriole movements in tubular epithelial cells. Pflügers Arch 456:
1025–1035, 2008.
Kottgen M, Buchholz B, Garcia-Gonzalez MA, Kotsis F, Fu X,
Doerken M, Boehlke C, Steffl D, Tauber R, Wegierski T, Nitschke R,
Suzuki M, Kramer-Zucker A, Germino GG, Watnick T, Prenen J,
Nilius B, Kuehn EW, Walz G. TRPP2 and TRPV4 form a polymodal
sensory channel complex. J Cell Biol 182: 437–447, 2008.
Koulen P, Cai Y, Geng L, Maeda Y, Nishimura S, Witzgall R,
Ehrlich BE, Somlo S. Polycystin-2 is an intracellular calcium release
channel. Nat Cell Biol 4: 191–197, 2002.
Kuehn EW, Hirt MN, John AK, Muehlenhardt P, Boehlke C, Putz
M, Kramer-Zucker AG, Bashkurov M, van de Weyer PS, Kotsis F,
Walz G. Kidney injury molecule 1 (Kim1) is a novel ciliary molecule
and interactor of polycystin 2. Biochem Biophys Res Commun 364:
861–866, 2007.
Kwon RY, Temiyasathit S, Tummala P, Quah CC, Jacobs CR.
Primary cilium-dependent mechanosensing is mediated by adenylyl cyclase 6 and cyclic AMP in bone cells. FASEB J 24: 2859 –2868, 2010.
Latta H, Maunsbach AB, Madden SC. Cilia in different segments of
the rat nephron. J Biophys Biochem 11: 248 –252, 2006.
Li Y, Wright JM, Qian F, Germino GG, Guggino WB. Polycystin 2
interacts with type I inositol 1,4,5-trisphosphate receptor to modulate
intracellular Ca2⫹ signaling. J Biol Chem 280: 41298 –41306, 2005.
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
4. Ando J, Komatsuda T, Kamiya A. Cytoplasmic calcium response to
fluid shear stress in cultured vascular endothelial cells. In Vitro Cell Dev
Biol 24: 871–877, 1988.
5. Andrews PM, Porter KR. A scanning electron microscopic study of the
nephron. Am J Anat 140: 81–115, 1974.
6. Arcuino G, Lin JH, Takano T, Liu C, Jiang L, Gao Q, Kang J,
Nedergaard M. Intercellular calcium signaling mediated by point-source
burst release of ATP. Proc Natl Acad Sci USA 99: 9840 –9845, 2002.
7. Barr MM, DeModena J, Braun D, Nguyen CQ, Hall DH, Sternberg
PW. The Caenorhabditis elegans autosomal dominant polycystic kidney
disease gene homologs lov-1 and pkd-2 act in the same pathway. Curr
Biol 11: 1341–1346, 2001.
8. Barr MM, Sternberg PW. A polycystic kidney-disease gene homologue
required for male mating behaviour in C. elegans. Nature 401: 386 –389,
1999.
9. Bjaelde RG, Arnadottir SS, Overgaard MT, Leipziger J, Praetorius
HA. Renal epithelial cells can release ATP by vesicular fusion. Front
Physiol 4: 238, 2013.
10. Briffeuil P, Thibaut-Vercruyssen R, Ronveaux-Dupal MF. Ciliation
of bovine aortic endothelial cells in culture. Atherosclerosis 106: 75–81,
1994.
11. Bruska M, Wozniak W. Fine structure of the capillaries in the spinal
cord of human embryos at 7th week. Folia Morphol (Warsz) 56: 22–30,
1997.
12. Bystrevskaya VB, Lichkun VV, Krushinsky AV, Smirnov VN. Centriole modification in human aortic endothelial cells. J Struct Biol 109:
1–12, 1992.
13. Cartwright JH, Piro O, Tuval I. Fluid-dynamical basis of the embryonic development of left-right asymmetry in vertebrates. Proc Natl Acad
Sci USA 101: 7234 –7239, 2004.
14. Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary
events underlying excitation-contraction coupling in heart muscle. Science 262: 740 –744, 1993.
15. Cotrina ML, Lin JH, ves-Rodrigues A, Liu S, Li J, zmi-Ghadimi H,
Kang J, Naus CC, Nedergaard M. Connexins regulate calcium signaling by controlling ATP release. Proc Natl Acad Sci USA 95: 15735–
15740, 1998.
16. De Vuyst E, Wang N, Decrock E, De Bock M, Vinken M, Van
Moorhem M, Lai C, Culot M, Rogiers V, Cecchelli R, Naus CC,
Evans WH, Leybaert L. Ca2⫹ regulation of connexin 43 hemichannels
in C6 glioma and glial cells. Cell Calcium 46: 176 –187, 2009.
17. DeCaen PG, Delling M, Vien TN, Clapham DE. Direct recording and
molecular identification of the calcium channel of primary cilia. Nature
504: 315–318, 2013.
18. Delling M, DeCaen PG, Doerner JF, Febvay S, Clapham DE. Primary
cilia are specialized calcium signalling organelles. Nature 504: 311–314,
2013.
19. Dull RO, Davies PF. Flow modulation of agonist (ATP)-response
(Ca2⫹) coupling in vascular endothelial cells. Am J Physiol Heart Circ
Physiol 261: H149 –H154, 1991.
20. Dwyer TM, Schmidt-Nielsen B. The renal pelvis: machinery that
concentrates urine in the papilla. News Physiol Sci 18: 1–6, 2003.
21. Edanaga M. A scanning electron microscope study on the endothelium
of the vessels. I. Fine structure of the endothelial surface of aorta and
some other arteries in normal rabbits. Arch Histol Jpn 37: 1–14, 1974.
22. Edanaga M. A scanning electron microscope study on the endothelium
of vessels. II. Fine surface structure of the endocardium in normal rabbits
and rats. Arch Histol Jpn 37: 301–312, 1975.
23. Ekblom A, Hansson P. A thin-section and freeze-fracture study of the
pulp blood vessels in feline and human teeth. Arch Oral Biol 29:
413–424, 1984.
24. Estilo G, Liu W, Pastor-Soler N, Mitchell P, Carattino MD, Kleyman
TR, Satlin LM. Effect of aldosterone on BK channel expression in
mammalian cortical collecting duct. Am J Physiol Renal Physiol 295:
F780 –F788, 2008.
25. Filipovic DM, Adebanjo OA, Zaidi M, Reeves WB. Functional and
molecular evidence for P2X receptors in LLC-PK1 cells. Am J Physiol
Renal Physiol 274: F1070 –F1077, 1998.
26. Gao Z, Ruden DM, Lu X. PKD2 cation channel is required for
directional sperm movement and male fertility. Curr Biol 13: 2175–2178,
2003.
27. Gartland A, Skarratt KK, Hocking LJ, Parsons C, Stokes L, Jorgensen NR, Fraser WD, Reid DM, Gallagher JA, Wiley JS. Polymorphisms in the P2X7 receptor gene are associated with low lumbar spine
Themes
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
70. Praetorius HA. Measuring cilium-induced Ca2⫹ increases in cultured
renal epithelia. Methods Cell Biol 91: 299 –313, 2009.
71. Praetorius HA, Frokiaer J, Leipziger J. Transepithelial pressure pulses
induce nucleotide release in polarized MDCK cells. Am J Physiol Renal
Physiol 288: F133–F141, 2005.
72. Praetorius HA, Leipziger J. Released nucleotides amplify the ciliumdependent, flow-induced [Ca2⫹]i response in MDCK cells. Acta Physiol
(Oxf) 197: 241–251, 2009.
73. Praetorius HA, Spring KR. Bending the MDCK cell primary cilium
increases intracellular calcium. J Membr Biol 184: 71–79, 2001.
74. Praetorius HA, Spring KR. Removal of the MDCK cell primary cilium
abolishes flow sensing. J Membr Biol 191: 69 –76, 2003.
75. Praetorius HA, Spring KR. The renal cell primary cilium functions as
a flow sensor. Curr Opin Nephrol Hypertens 12: 517–520, 2003.
76. Raghavan V, Rbaibi Y, Pastor-Soler NM, Carattino MD, Weisz OA.
Shear stress-dependent regulation of apical endocytosis in renal proximal
tubule cells mediated by primary cilia. Proc Natl Acad Sci USA 111:
8506 –8511, 2014.
77. Raitakari OT, Celermajer DS. Flow-mediated dilatation. Br J Clin
Pharmacol 50: 397–404, 2000.
78. Raychowdhury MK, McLaughlin M, Ramos AJ, Montalbetti N,
Bouley R, Ausiello DA, Cantiello HF. Characterization of single channel currents from primary cilia of renal epithelial cells. J Biol Chem 280:
34718 –34722, 2005.
79. Resnick A. Chronic fluid flow is an environmental modifier of renal
epithelial function. PLos One 6: e27058, 2011.
80. Resnick A. Use of optical tweezers to probe epithelial mechanosensation. J Biomed Opt 15: 015005, 2010.
81. Resnick A, Hopfer U. Force-response considerations in ciliary mechanosensation. Biophys J 93: 1380 –1390, 2007.
82. Rodat-Despoix L, Hao J, Dandonneau M, Delmas P. Shear stressinduced Ca2⫹ mobilization in MDCK cells is ATP dependent, no matter
the primary cilium. Cell Calcium 53: 327–337, 2013.
83. Rydholm S, Frisk T, Kowalewski JM, Andersson Svahn H, Stemme
G, Brismar H. Microfluidic devices for studies of primary cilium
mediated cellular response to dynamic flow conditions. Biomed Microdevices 10: 555–560, 2008.
84. Rydholm S, Zwartz G, Kowalewski JM, Kamali-Zare P, Frisk T,
Brismar H. Mechanical properties of primary cilia regulate the response
to fluid flow. Am J Physiol Renal Physiol 298: F1096 –F1102, 2010.
85. Sammels E, Devogelaere B, Mekahli D, Bultynck G, Missiaen L,
Parys JB, Cai Y, Somlo S, De Smedt H. Polycystin-2 activation by
inositol 1,4,5-trisphosphate-induced Ca2⫹ release requires its direct association with the inositol 1,4,5-trisphosphate receptor in a signaling
microdomain. J Biol Chem 285: 18794 –18805, 2010.
86. Schmidt-Nielsen B, Graves B. Changes in fluid compartments in hamster renal papilla due to peristalsis in the pelvic wall. Kidney Int 22:
613–625, 1982.
87. Schwartz EA, Leonard ML, Bizios R, Bowser SS. Analysis and
modeling of the primary cilium bending response to fluid shear. Am J
Physiol Renal Physiol 272: F132–F138, 1997.
88. Schwarz G, Callewaert G, Droogmans G, Nilius B. Shear stressinduced calcium transients in endothelial cells from human umbilical
cord veins. J Physiol 458: 527–538, 1992.
89. Shen J, Luscinskas FW, Connolly A, Dewey CF Jr, Gimbrone MA
Jr. Fluid shear stress modulates cytosolic free calcium in vascular
endothelial cells. Am J Physiol Cell Physiol 262: C384 –C390, 1992.
90. Shiba D, Takamatsu T, Yokoyama T. Primary cilia of inv/inv mouse
renal epithelial cells sense physiological fluid flow: bending of primary
cilia and Ca2⫹ influx. Cell Struct Funct 30: 93–100, 2005.
91. Shinohara K, Kawasumi A, Takamatsu A, Yoshiba S, Botilde Y,
Motoyama N, Reith W, Durand B, Shiratori H, Hamada H. Two
rotating cilia in the node cavity are sufficient to break left-right symmetry
in the mouse embryo. Nat Commun 3: 622, 2012.
92. Sipos A, Vargas SL, Toma I, Hanner F, Willecke K, Peti-Peterdi J.
Connexin 30 deficiency impairs renal tubular ATP release and pressure
natriuresis. J Am Soc Nephrol 20: 1724 –1732, 2009.
93. Siroky BJ, Ferguson WB, Fuson AL, Xie Y, Fintha A, Komlosi P,
Yoder BK, Schwiebert EM, Guay-Woodford LM, Bell PD. Loss of
primary cilia results in deregulated and unabated apical calcium entry in
ARPKD collecting duct cells. Am J Physiol Renal Physiol 290: F1320 –
F1328, 2006.
94. Stehbens WE. Turbulence of blood flow. J Exp Physiol 44: 110 –117,
1958.
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
48. Liu W, Morimoto T, Woda C, Kleyman TR, Satlin LM. Ca2⫹
dependence of flow-stimulated K secretion in the mammalian cortical
collecting duct. Am J Physiol Renal Physiol 293: F227–F235, 2007.
49. Liu W, Murcia NS, Duan Y, Weinbaum S, Yoder BK, Schwiebert E,
Satlin LM. Mechanoregulation of intracellular Ca2⫹ concentration is
attenuated in collecting duct of monocilium-impaired orpk mice. Am J
Physiol Renal Physiol 289: F978 –F988, 2005.
50. Madsen KM, Clapp WL, Verlander JW. Structure and function of the
inner medullary collecting duct. Kidney Int 34: 441–454, 1988.
51. Masyuk AI, Masyuk TV, Splinter PL, Huang BQ, Stroope AJ,
LaRusso NF. Cholangiocyte cilia detect changes in luminal fluid flow
and transmit them into intracellular Ca2⫹ and cAMP signaling. Gastroenterology 131: 911–920, 2006.
52. McCulloch F, Chambrey R, Eladari D, Peti-Peterdi J. Localization of
connexin 30 in the luminal membrane of cells in the distal nephron. Am
J Physiol Renal Physiol 289: F1304 –F1312, 2005.
53. McGrath J, Brueckner M. Cilia are at the heart of vertebrate left-right
asymmetry. Curr Opin Genet Dev 13: 385–392, 2003.
54. McGrath J, Somlo S, Makova S, Tian X, Brueckner M. Two populations of node monocilia initiate left-right asymmetry in the mouse. Cell
114: 61–73, 2003.
55. Meisner JE, Rushmer RF. Eddy formation and turbulence. Circ Res 12:
455–463, 1963.
56. Mekahli D, Sammels E, Luyten T, Welkenhuyzen K, van den Heuvel
LP, Levtchenko EN, Gijsbers R, Bultynck G, Parys JB, De Smedt H,
Missiaen L. Polycystin-1 and polycystin-2 are both required to amplify
inositol-trisphosphate-induced Ca2⫹ release. Cell Calcium 51: 452–458,
2012.
57. Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia
AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J. Polycystins 1
and 2 mediate mechanosensation in the primary cilium of kidney cells.
Nat Genet 33: 129 –137, 2003.
58. Nauli SM, Kawanabe Y, Kaminski JJ, Pearce WJ, Ingber DE, Zhou
J. Endothelial cilia are fluid shear sensors that regulate calcium signaling
and nitric oxide production through polycystin-1. Circulation 117: 1161–
1171, 2008.
59. Nauli SM, Rossetti S, Kolb RJ, Alenghat FJ, Consugar MB, Harris
PC, Ingber DE, Loghman-Adham M, Zhou J. Loss of polycystin-1 in
human cyst-lining epithelia leads to ciliary dysfunction. J Am Soc
Nephrol 17: 1015–1025, 2006.
60. Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y, Kido
M, Hirokawa N. Randomization of left-right asymmetry due to loss of
nodal cilia generating leftward flow of extraembryonic fluid in mice
lacking KIF3B motor protein. Cell 95: 829 –837, 1998.
61. Okada Y, Nonaka S, Tanaka Y, Saijoh Y, Hamada H, Hirokawa N.
Abnormal nodal flow precedes situs inversus in iv and inv mice. Mol Cell
4: 459 –468, 1999.
62. Okusa MD, Velazquez H, Ellison DH, Wright FS. Luminal calcium
regulates potassium transport by the renal distal tubule. Am J Physiol
Renal Fluid Electrolyte Physiol 258: F423–F428, 1990.
63. Orriss I, Syberg S, Wang N, Robaye B, Gartland A, Jorgensen N,
Arnett T, Boeynaems JM. Bone phenotypes of P2 receptor knockout
mice. Front Biosci 3: 1038 –1046, 2011.
64. Pacha J, Frindt G, Sackin H, Palmer LG. Apical maxi K channels in
intercalated cells of CCT. Am J Physiol Renal Fluid Electrolyte Physiol
261: F696 –F705, 1991.
65. Palmer LG, Frindt G. High-conductance K channels in intercalated
cells of the rat distal nephron. Am J Physiol Renal Physiol 292: F966 –
F973, 2007.
66. Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Witman GB.
Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in
orpk mice with polycystic kidney disease. Curr Biol 12: R378 –R380,
2002.
67. Pearce B, Murphy S, Jeremy J, Morrow C, Dandona P. ATP-evoked
Ca2⫹ mobilisation and prostanoid release from astrocytes: P2-purinergic
receptors linked to phosphoinositide hydrolysis. J Neurochem 52: 971–
977, 1989.
68. Pennekamp P, Karcher C, Fischer A, Schweickert A, Skryabin B,
Horst J, Blum M, Dworniczak B. The ion channel polycystin-2 is
required for left-right axis determination in mice. Curr Biol 12: 938 –943,
2002.
69. Pfaller W, Klima J. A critical reevaluation of the structure of the rat
uriniferous tubule as revealed by scanning electron microscopy. Cell
Tissue Res 166: 91–100, 1976.
C207
Themes
C208
NEW BLOCK TO PRIMARY CILIUM AS FLOW SENSOR
104. Wheatley DN. The Centriole: A Central Enigma of Cell Biology. New
York: Elsevier, 1982.
105. Wheatley DN. Primary cilia in normal and pathological tissues. Pathobiology 63: 222–238, 1995.
106. Wiesner TF, Berk BC, Nerem RM. A mathematical model of the
cytosolic-free calcium response in endothelial cells to fluid shear stress.
Proc Natl Acad Sci USA 94: 3726 –3731, 1997.
107. Woda CB, Leite M Jr, Rohatgi R, Satlin LM. Effects of luminal flow
and nucleotides on [Ca2⫹]i in rabbit cortical collecting duct. Am J Physiol
Renal Physiol 283: F437–F446, 2002.
108. Xu C, Rossetti S, Jiang L, Harris PC, Brown-Glaberman U, Wandinger-Ness A, Bacallao RL, Alper SL. Human ADPKD primary cyst
epithelial cells with a novel, single codon deletion in the PKD1 gene
exhibit defective ciliary polycystin localization and loss of flow-induced
Ca2⫹ signaling. Am J Physiol Renal Physiol 292: F930 –F945, 2007.
109. Xu C, Shmukler BE, Nishimura K, Kaczmarek E, Rossetti S, Harris
PC, Wandinger-Ness A, Bacallao RL, Alper SL. Attenuated, flowinduced ATP release contributes to absence of flow-sensitive, purinergic
Cai2⫹ signaling in human ADPKD cyst epithelial cells. Am J Physiol
Renal Physiol 296: F1464 –F1476, 2009.
110. Yoder BK, Hou X, Guay-Woodford LM. The polycystic kidney
disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are
co-localized in renal cilia. J Am Soc Nephrol 13: 2508 –2516, 2002.
111. Yoshiba S, Shiratori H, Kuo IY, Kawasumi A, Shinohara K, Nonaka
S, Asai Y, Sasaki G, Belo JA, Sasaki H, Nakai J, Dworniczak B,
Ehrlich BE, Pennekamp P, Hamada H. Cilia at the node of mouse
embryos sense fluid flow for left-right determination via Pkd2. Science
338: 226 –231, 2012.
112. Zeigel RF. On the occurrence of cilia in several cell types of the chick
pancreas. J Ultrastruct Res 7: 286 –292, 1962.
AJP-Cell Physiol • doi:10.1152/ajpcell.00336.2014 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 14, 2017
95. Stein PD, Sabbah HN. Turbulent blood flow in the ascending aorta of
humans with normal and diseased aortic valves. Circ Res 39: 58 –65,
1976.
96. Su S, Phua SC, DeRose R, Chiba S, Narita K, Kalugin PN, Katada
T, Kontani K, Takeda S, Inoue T. Genetically encoded calcium
indicator illuminates calcium dynamics in primary cilia. Nat Methods 10:
1105–1107, 2013.
97. Takao D, Nemoto T, Abe T, Kiyonari H, Kajiura-Kobayashi H,
Shiratori H, Nonaka S. Asymmetric distribution of dynamic calcium
signals in the node of mouse embryo during left-right axis formation. Dev
Biol 376: 23–30, 2013.
98. Takeda S, Yonekawa Y, Tanaka Y, Okada Y, Nonaka S, Hirokawa
N. Left-right asymmetry and kinesin superfamily protein KIF3A: new
insights in determination of laterality and mesoderm induction by
kif3A⫺/⫺ mice analysis. J Cell Biol 145: 825–836, 1999.
99. Taulman PD, Haycraft CJ, Balkovetz DF, Yoder BK. Polaris, a
protein involved in left-right axis patterning, localizes to basal bodies and
cilia. Mol Biol Cell 12: 589 –599, 2001.
100. Thode J. The calcium ion activity and the standardized excretion rate of
calcium in urine of healthy adults. Scand J Clin Lab Invest 45: 327–334,
1985.
101. Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B.
Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature 424: 434 –438, 2003.
102. Watnick TJ, Jin Y, Matunis E, Kernan MJ, Montell C. A flagellar
polycystin-2 homolog required for male fertility in Drosophila. Curr Biol
13: 2179 –2184, 2003.
103. Wesselius A, Bours MJ, Henriksen Z, Syberg S, Petersen S, Schwarz P,
Jorgensen NR, van Helden S, Dagnelie PC. Association of P2X7 receptor
polymorphisms with bone mineral density and osteoporosis risk in a
cohort of Dutch fracture patients. Osteoporos Int 24: 1235–1246, 2013.