Am J Physiol Renal Physiol 298: F1096–F1102, 2010.
First published January 20, 2010; doi:10.1152/ajprenal.00657.2009.
Mechanical properties of primary cilia regulate the response to fluid flow
Susanna Rydholm,1 Gordon Zwartz,1 Jacob M. Kowalewski,1 Padideh Kamali-Zare,1 Thomas Frisk,1
and Hjalmar Brismar1,2
1
Division of Cell Physics, Department of Applied Physics, Royal Institute of Technology; and 2Department of Women’s
and Children’s Health, Karolinska Institutet, Stockholm, Sweden
Submitted 13 November 2009; accepted in final form 20 January 2010
calcium; frequency; delayed membrane stress; modeling
of interacting
cellular processes that take input from external stimuli. One
class of external stimuli is the physical factors, including
temperature, pressure, stretch, and flow. The primary cilium
has been described as an organelle involved in fluid flow
sensing. It is ubiquitously present on nearly all mammalian
cells, with the exception of cells of myeloid and lymphoid
origin (21). It is expressed only once per cell and originates
from the distal end of the mother centriole, which migrates
near the apical plasma membrane upon cilia formation (22).
Structurally, it is composed of nine pairs of circularly arranged
microtubules (9 ⫹ 0) without dynein arms and is not motile.
A cilium structure is comprised of more than 650 proteins
(3) with different functions, such as temperature sensitivity
[transient receptor potential vanilloid 4 (TRPV4) (6)], protein
binding [polycystin-1; (10)], ion channels [TRPV4, TRRP2
(12)], and protein trafficking (28). When cilia or ciliary signaling molecules are absent or inhibited, various pathological
disorders can occur, giving symptoms such as impaired brain
development (1), obesity, hypertension, hearing impairment,
blindness, diabetes, lack of muscle control (20), or cystic
disorders in liver (19) and pancreas (3). However, most of the
disorders coupled to primary cilia have one symptom in com-
NORMAL CELL FUNCTION INVOLVES A COMPLEX ARRAY
Address for reprint requests and other correspondence: H. Brismar, Cell
Physics, AlbaNova University Center, Royal Institute of Technology, SE-106
91 Stockholm, Sweden ([email protected]).
F1096
mon, which is renal failure due to kidney cyst formation.
Understanding the pathology of these diseases from a molecular or mechanistic level may give insights that can lead us to
new treatment strategies.
In 2001, Praetorius and Spring (24) first observed that
primary cilia expressed on kidney epithelial cells could generate intracellular calcium in response to fluid flow. Since then,
a protein complex between polycystin-1 and polycystin-2 was
found to be localized on the cilium (35) and was suggested to
function as a calcium channel in the ciliary membrane (18),
initiating calcium influx upon cilium bending. Malfunction of
either polycystin-1 or polycystin-2 disrupts the cilia-induced
calcium response and leads to autosomal dominant polycystic
kidney disease, indicating a direct coupling between the calcium signal and renal cyst formation. Another study showed
that polycystin-2 and TRPV4 also colocalize on cilia and form
a coupled complex that responds to fluid flow (12). How these
molecular sensors detect flow and generate signaling, such as
intracellular calcium response, is not fully known. Several
studies have also revealed that the flow-induced calcium signal
shows a significant lag between the onset of fluid flow and
calcium increase (16, 25). This lag differs between cell types,
but in Madin-Darby canine kidney (MDCK) and rabbit-collecting duct cells it was found that the peak occurs ⬃30 s after
a stimulus.
To understand how cilia motion is integrated into biological
processes, cilia in flow have been studied using fluid dynamical
modeling (2, 4, 5, 27). These models describe cilia movement
in the presence of forces using biophysical parameters such as
length, diameter, bending, and membrane resistance (5). They
have shown that with basic cilia parameterization, complex
cilia motion and ciliary stress can be described for both motile
and nonmotile cilia. What is not known is whether ciliary
motion can directly translate into intracellular signaling, e.g.,
mediated by calcium, or is incidental, and molecular sensors
alone are responsible for fluid flow mechanical signal transduction. There are several hundred proteins that comprise a
cilium structure (3), and some of these proteins are sensitive to
extracellular changes such as fluid flow. Whether these molecular sensors detect flow and generate signaling aided directly or
indirectly by cilium bending is not fully known. Any model
that describes an intracellular calcium response due to flow will
also need to describe the time delay between the start of fluid
flow and when a cell generates a signal (8, 23).
We have modeled ciliary response to bending using basic
cilium parameters such as length (8 ␮m, although in vitro, cilia
can grow to lengths of 55 ␮m) (24), bending rigidity (32, 34),
and membrane stiffness. The model describes an elastic cilium
surrounded and covered by a viscoelastic membrane that was
simulated using finite element methods (FEMs). The simulations show that the actual bending of the cilium is fast, whereas
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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. First
published January 20, 2010; doi:10.1152/ajprenal.00657.2009.—The
primary cilium is a ubiquitous organelle present on most mammalian
cells. Malfunction of the organelle has been associated with various
pathological disorders, many of which lead to cystic disorders in liver,
pancreas, and kidney. Primary cilia have in kidney epithelial cells
been observed to generate intracellular calcium in response to fluid
flow, and disruption of proteins involved in this calcium signaling lead
to autosomal dominant polycystic kidney disease, implying a direct
connection between calcium signaling and cyst formation. It has also
been shown that there is a significant lag between the onset of flow
and initiation of the calcium signal. The present study focuses on the
mechanics of cilium bending and the resulting calcium signal. Visualization of real-time cilium movements in response to different types
of applied flow showed that the bending is fast compared with the
initiation of calcium increase. Mathematical modeling of cilium and
surrounding membrane was performed to deduce the relation between
bending and membrane stress. The results showed a delay in stress
buildup that was similar to the delay in calcium signal. Our results
thus indicate that the delay in calcium response upon cilia bending is
caused by mechanical properties of the cell membrane.
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MECHANICAL PROPERTIES REGULATE CILIA’S RESPONSE TO FLOW
METHODS
Cell culture. All experiments were performed on MDCK cells
(purchased from ECACC). The cells were cultured in modified Eagle’s medium (MEM; Gibco, Grand Island, NY) supplemented with
10% fetal bovine serum, 100 IU/ml penicillin, 100 ␮g/ml streptomycin, 2 mM L-glutamine, and 0.1 mM nonessential amino acids (Sigma,
St. Louis, MO) at 37°C and in an atmosphere containing 5% CO2.
After culture to confluence, the cells were treated with 0.25% trypsin
for 2 min at 37°C and harvested by gentle cell scraping. Two
milliliters of cell suspension (105 cells/ml) was seeded onto 28-mm
coverslips in 35-mm petri dishes, giving an initial density of 200
cells/mm2, and was then cultured for 3–7 days before experiments.
Intracellular calcium. Intracellular calcium (Ca2⫹) responses were
measured using the fluorescent calcium indicator fluo-4 (ex 494
nm/em 516 nm; Invitrogen, Carlsbad, CA). The dye was loaded by
incubation for 30 min at 37°C with 2.5 ␮M Fluo-4-AM and 5 mM
probenecid (Sigma, St. Louis, MO). All experiments were performed
using phosphate-buffered saline (PBS), which contained (in mM) 110
NaCl, 4.0 KCl, 1.5 CaCl2, 1.2 MgCl2, 20.0 HEPES, 1.0 NaH2PO4,
and 10 D-glucose, and the pH was adjusted to 7.4 at 37°C.
Immunocytochemistry. Fixation and immunocytochemistry were
performed using cells cultured on coverslips. Cells were fixed in 4%
paraformaldehyde solution containing 0.1% Triton X-100 for 10 min
at room temperature. After rinsing with PBS, the cells were stained
with a monoclonal mouse antibody against acetylated ␣-tubulin
(Sigma), followed by an anti-mouse antibody labeled with Alexa fluor
488 (Invitrogen). The coverslips were mounted on glass slides with
Immunomount and kept dark at room temperature for 3 h and stored
at 5°C until used.
Transfection. Cilia of living cells were imaged by transfection with
cDNA coding for full-length rat dopamine D5 receptor and subcloned
into an enhanced green fluorescent protein (GFP) vector. Transfection
was performed using Effectene transfection reagent (Qiagen, Hilden,
Germany) according to the manufacturer’s protocol.
Cilia lengths. Lengths of cilia from culture day 3 to day 6 were
measured by confocal microscopic analysis of cells immunostained
for acetylated ␣-tubulin. Two to four plates from each day were
AJP-Renal Physiol • VOL
analyzed and revealed that the cilia lengths were 3.5 ⫾ 0.75 ␮m on
day 3, 4.8 ⫾ 0.67 ␮m on day 4, 6.7 ⫾ 1.6 ␮m on day 5, and 11.6 ⫾
2.0 ␮m at day 6.
Decilliation. Removal of primary cilia was done by supplementing
cultured MEM with 4 mM aqueous chloral hydrate (Sigma) for 72 h.
The supplemented medium was changed twice daily. After 72 h the
cells were washed two times with PBS, and fresh medium was added
for 24 h before cells were used in flow experiments.
Microscopy. All imaging of intracellular calcium, transfected, and
immunostained cells were performed using confocal microscopy
(LSM 510; Carl Zeiss) with a 63⫻ 1.4 NA objective. Fluo-4, Alexa
fluor 488, and GFP fluorescence was detected using 488 nm excitation
and a long-pass 505-nm filter. Calcium images were recorded every 5
s for ⱕ1 h, and the whole image as well as individual cells were
analyzed.
Shear. Laminar flow can be generated by different means; in a
previous study we used a microfluidic device in silicon (30). Here we
have used a different approach. Laminar flow was generated by
controlling the rotation of a 2-cm diameter Plexiglas cylinder, which
was positioned 200 ␮m above and parallel to a confluent layer of
MDCK cells cultured on a coverslip. Cells were immersed in 900 ␮l
of PBS solution supplemented with probenecid (5 mM) to inhibit the
leakage of fluo-4. A schematic of the experimental setup is shown in
Fig. 1. Positioning of the cylinder above the cells was aided by
200-␮m spacers attached to the end cap of the cylinder. Cylinder
rotation was controlled by a DC motor (ELFA Micromotors) and a
power supply/function generator (Wavetek).
Cells were exposed to either continuous or pulsate (step function,
on and off) flow from 5 s up until 5 min. Several consecutive flow
cycles were applied in each experiment, with an ⬃5-min resting
period between each cycle. Cells were imaged in a region 3 mm from
the cylinder center, as the flow here can be considered to be linear (not
rotating), and there is also enough distance to avoid interference from
the spacers. All experiments were performed at 37°C. The flow device
uses an open chamber configuration, which eliminates the risk of
oxygen deprivation and suffocation. Temperature was maintained
using a microscope heating insert, and the cell buffer remained
unchanged during the course of an experiment.
The fluid flow field above the ciliated cells can be described by a
planar flow model, where the upper and lower boundaries (i.e., the
cylinder and the coverslip) have nonslip conditions. If the rotational
period of the cylinder is T and the distance from the center of the
cylinder to the cells of interest is r, the rotational velocity of the
cylinder is
␯cyl ⫽
2␲r
T
.
(1)
The two nonslip boundaries and zero pressure gradient result in a
constant velocity gradient described as
d␯
dz
⫽
␯cyl
d
,
(2)
Fig. 1. Left: cross-section of flow setup with coverslip holder and rotating
cylinder. Cells are imaged ⬃3 mm from center of cylinder. Right: schematic
drawing of linear flow profile.
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the ciliary membrane stress gradually builds up in the vicinity
of the cilium’s base and requires ⱖ20 s before reaching a
maximal value after the application of force. We have also
measured intracellular calcium signaling from MDCK cells
and observed a similar time delay. It was also noted that the
longer a force (fluid flow) was applied to cilia the stronger the
intracellular calcium signal, which reached a maximum value
30 s after the start of flow.
To deduce the position of cilia stress areas, we observed
primary cilia motion in fluid flow using confocal microscopy.
We observed that the distal parts of cilia, ⬃5 ␮m above the
base, move in time with fluid flow but have no effect on
intracellular calcium response. The basal part, on the other
hand, shows limited deflection upon application of flow. This
suggests that the lower part of cilia is the region responsible for
flow sensitivity. These data, together with results from the cilia
stress model, suggest that the delayed intracellular calcium
response to fluid flow is due to the length of time for stress to
build up on the cell’s membrane, resulting from cilium bending, before signaling can begin. Since polycystin-1 and -2 and
TRPV4 are at least two possible coupled molecular flow
sensors that colocalize around the base of cilia, the localization
of stress at the base of the ciliary membrane could be a
mechanical means of altering the conformation of these sensors. Changes in conformation could lead to opening of calcium channels and signaling pathways.
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MECHANICAL PROPERTIES REGULATE CILIA’S RESPONSE TO FLOW
where d ⫽ 200 ␮m is the distance between the cells and the cylinder;
see Fig. 1. The shear in the fluid is calculated by
␶⫽␩
d␯
dz
⫽␩
␯cyl
d
⫽␩
2␲r
Td
,
(3)
冉 冊
E⫽EI/I⫽EI/
␲a4
4
(4)
␴mises
⫽ 兹␴2x ⫹ ␴2y ⫹ ␴2z ⫺ ␴x␴y ⫺ ␴y␴z ⫺ ␴x␴z ⫹ 3␶2xy ⫹ 3␶2yz ⫹ 3␶2xz ,
(5)
where the normal stress components ␴x, ␴y, and ␴z (in Pa) are
defined as
(1 ⫺ ␯)␧x ⫹ ␯␧y ⫹ ␯␧z
(1 ⫹ ␯)(1 ⫺ 2␯)
(6)
(with symmetrical expressions for ␴y and ␴z), and the shear stress
components ␶xy, ␶yz, ␶xz (in Pa) as
␶xy ⫽ Em
␩m
k1
冉 冊
1⫹
k1
k2
,
(8)
where k1 and k2 are elastic constants and ␩m is the viscosity of a
viscoelastic material. The values listed by Lim et al. (15) result in time
constants ranging from 0.6 to 150 s. In our simulations Rayleigh
stiffness damping was set to 10 s, well within the span of literature
values. Other parameters are listed in Table 1.
Simulations were run to calculate the displacement and stress of the
system as a function of time with pulsed and continuous flow for ⱕ3
min. Shear was applied to the axoneme in the form of a horizontal
drag force per unit length caused by fluid flow, as published previously by Resnick and Hopfer (26):
fdrag(z) ⫽
4␲␩␯(z)
1
2
⫺ ␥ ⫺ ln
冉 冊
(9)
a␳␯共z兲
4␩
at the cylindrical part of the cilium, where ␥ is Euler’s constant and ␳
the density of the fluid, and at the semispherical tip of the cilium:
fdrag(z) ⫽ 3␲␩a␯共z兲.
(10)
A boundary condition in the model is that the outer edge of the
membrane in the cell surface has zero displacement. Stress and
displacement in the membrane and inner structure were calculated
using the built-in tools in Comsol Multiphysics and the scripting
interface between Comsol Multiphysics and Matlab (The Mathworks,
Natick, MA).
RESULTS
⫽ 178 kPa,
where EI is the bending rigidity of the cilium and a is its radius.
Young’s modulus of the apical membrane was calculated as Em ⫽
Kml/A, where Km is the membrane spring constant (17, 33), l is the
radius of the membrane’s geometry in the model, and A is an order of
magnitude estimate of the membrane cross-sectional area along a
plane perpendicular to the drag force. Young’s modulus for the ciliary
membrane was assumed to be the same as for the apical membrane.
Membrane stress in each mesh element was calculated according to
the von Mises definition as
␴x ⫽ Em
tdamp ⫽
␧xy
1⫹␯
(7)
(with symmetrical expressions for ␶yz and ␶xz). ␯ ⫽ 0.33 is Poisson’s
ratio. The normal strains εx, εy, and εz and the shear strains εxy, εxz,
AJP-Renal Physiol • VOL
Modeled delay in membrane stress is similar to measured
calcium delay as well as response amplitude. Modeling of
cilium bending and membrane stress was performed in Comsol
Multiphysics. According to the model, the rate of stress building up in the cell membrane is significantly slower compared
with the actual bending or deflection of the cilium (Fig. 2A).
The delay in stress buildup is due predominantly to mechanical
membrane properties such as spring constant and damping.
Flow experiments performed on MDCK cells cultured between 3 and 7 days and stained with the calcium-sensitive dye
fluo-4 showed similar results. Immunostain for acetylated ␣-tubulin shows that cells at this stage of culture have wellTable 1. Parameters and values used in the model
Parameter
r
d
T
a
␳
␩
EI
l
A
Km
Definition
Radius of cylinder in device
3 mm
Thickness of fluid layer
0.2 mm
Rotational period of device
1.5 s
Radius of cilium (32)
100 nm
Density of fluid
1,000 kg/m3
Viscosity of fluid (water at 37°C)
6.97 ⫻ 10⫺4 Pa·s
Flexural rigidity of cilium (7, 16, 32)
1.4 ⫻ 10⫺23 Nm2
Geometrical radius of membrane structure
2 ␮m
Estimated cross-sectional area of membrane
10⫺14 m2
Membrane spring constant (17)
5 ⫻ 10⫺5 N/m
Cilium parameters.
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where ␩ is the viscosity of the fluid and was taken to be that of water
at 37°C. The resulting shear was between 10 and 60 mPa.
Ca2⫹ analysis. Calcium responses were quantified as relative change
in fluo-4 intensity. Intensity measurements were compared within the
same experiment and presented as relative change in response. Each pulse
frequency condition was repeated at least two times.
Modeling. A three-dimensional, structural, mechanical model for
the apical part of the cell, including the primary cilium, was created.
On the basis of the geometry, the boundary conditions, and the
physical and mechanical properties described below, the stress and
displacement as a function of time were calculated using a FEM with
the structural mechanics module in Comsol Multiphysics (Comsol,
Stockholm, Sweden). The model consists of three parts: the cilium
microtubule structure, the ciliary membrane, and the apical plasma
membrane. The microtubule structure was depicted as a single elastic
beam extending 6 ␮m into the extracellular space forming the ciliary
axoneme and 2 ␮m into the cell cytoplasm forming the basal body.
The surface of the axoneme part of the beam forms the ciliary
membrane, thickness 10 nm, which transcends into the perpendicular
apical plasma membrane at the base of the cilium. At the tip of the
cilium the membrane forms a semispherical cap. The diameter of the
cilium is 200 nm. The apical membrane is modeled as a circle with a
radius of 2 ␮m and a thickness of 10 nm. The FEM simulation model
was constructed using the shell and solid structure descriptions in
Comsol Multiphysics. The two-dimensional mesh of both membranes
consists of in total 7,476 mesh elements, and the microtubule beam
consists of 6,502 elements (Fig. 5A).
The physical and mechanical properties for the separate parts in the
model were found in literature. Cilia microtubule structure is elastic
with a Young’s modulus calculated from parameters previously published by Schwartz et al. (32) and Liu et al. (16) as
and εyz are components of the dimensionless mesh element deformation calculated in the FEM simulation.
A Rayleigh model with stiffness damping as a membrane parameter was included. Lim et al. (15) have listed parameters from
references therein using viscoelastic linear solid models. In this kind
of model, a time constant, corresponding well within an order of
magnitude to a Rayleigh stiffness damping, can be calculated as (31)
MECHANICAL PROPERTIES REGULATE CILIA’S RESPONSE TO FLOW
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developed cilia (24). Experiments revealed an approximate
time delay of 20 s before the calcium signal was initiated (Fig.
2B and Supplemental Video S1; Supplemental Material for this
article can be found on the AJP-Renal Physiology web site).
Furthermore, a single pulse with a duration of 30 s was able to
generate a calcium response with equal amplitude as continuous flow (2 min), whereas a single pulse of ⱕ5 s generated
little or no response (Fig. 2C).
Removal of cilia abolishes calcium signal. To verify that
flow-induced calcium increase depends on the presence of
cilia, deciliation with 4 mM of chloral hydrate for 72 h was
performed. This process removed the cilia and abolished the
calcium increase in response to flow (Fig. 2B).
Modeled stress buildup due to pulsate flow corresponds well
with calcium response. The model predicts that, with multiple
short pulses of flow, membrane stress is gradually built up, and
at higher frequencies (⬎0.1 Hz) maximum stress reaches
⬃60% of the stress compared with continuous flow (Fig. 3A
and Supplementary Video S2). In theory, several pulses should
thus be able to generate enough stress to trigger a calcium
response even when a single pulse does not. Experiments with
continuous flow as well as pulsate flow with frequencies
ranging from 0.1 to 2 Hz were performed. In each experiment,
several consecutive flow cycles were applied (Fig. 3C), and
calcium responses were compared between different flow treatments within the same experiment. All experiments included at
least two separate flow cycles with continuous flow and two
cycles with pulsate flow. Analysis revealed that the cells did
respond to multiple flow pulses but with a calcium increase that
was significantly lower compared with continuous flow for all
frequencies. However, between the various frequencies, no
statistically determined differences were found.
To analyze whether the reduced calcium signal depends on
fewer responding cells or decreased signal from each cell, 256
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areas in each image were analyzed separately with regard to
response or no response for each flow cycle. This analysis
revealed a statistically significant decrease in both number of
responding cells as well as response amplitude with 30 –50%
for pulsed flow compared with continuous flow (Fig. 3B). Also,
there were no significant differences found between the separate frequencies.
Cilium bending is fast compared with calcium signal. To
visualize primary cilia of living cells and to perform real-time
measurements of deflection rates, MDCK cells were transfected with a dopamine 5 receptor construct conjugated to
GFP, which predominantly targeted cilia. Transfected cells
were then exposed to continuous, pulsate, or oscillatory flows
(forward-backward) with different frequencies and flow rates.
Figure 4A shows an en-face view time series of a cilium
exposed to 1 Hz frequency oscillating flow with 19 mPa shear.
The cilium tip rapidly switches direction in accordance with
flow, thus within 0.5 s. For real-time cilia bending, see Supplementary Video S3. Modeled cilium bending shows similar
results; displacement of the cilium after application of 44 mPa
shear is a process that occurs within seconds (Fig. 2A, thin
curve).
To examine whether prolonged flow led to a gradual increase in bending, three-dimensional imaging of cells exposed
to continuous flow from 0 to 5 min was performed. No
difference in amount of bending was possible to detect, indicating that cilia rapidly reach a maximal deflection that does
not change with time. Slow bending of cilia is thus not likely
to explain the time delay for calcium signaling.
Membrane stress is likely localized near the base of the
cilium. Figure 5B shows the color-coded stress as modeled in
the ciliary membrane (left and middle) and the microtubular
structure (right). In the membrane, the stress is located distinctly near the base of the cilium (Fig. 5B, left and middle).
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Fig. 2. A: thin trace shows displacement of tip
vs. time of 8-␮m-long cilium subjected to a
drag force corresponding to 44 mPa of shear,
applied as a step function, as modeled in
Comsol Multiphysics. Thick curve shows
corresponding ciliary membrane stress near
the cilium’s base. The stress gradually builds
up and requires ⬎20 s to reach maximal value
after application of force. B: calcium curves
of cells exposed to 59 mPa continuous shear.
Whole curve is obtained with cells deciliated
with 4 ⫻ 10⫺3 M chloral hydrate (CH) for 48
and 6 h of recovery. Dotted curve shows
corresponding wild-type (WT) cells. C, left:
calcium response of cells exposed to a single
pulse of 5 s followed by continuous (120 s)
flow. Virtually no response is seen during the
short pulse, whereas a strong calcium response is seen during continuous flow. C,
right: calcium response of cells exposed to a
single 30-s-long pulse followed by continuous flow. The 30-s pulse generates equally
strong calcium signal as continuous force.
MDCK, Madin-Darby canine kidney; AU,
arbitrary units.
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MECHANICAL PROPERTIES REGULATE CILIA’S RESPONSE TO FLOW
The low stress in the rest of the ciliary membrane is likely due
to the tight connection between the membrane and the more
rigid microtubules, which will take up the largest part of the
applied force. The stress in the microtubules is more spread,
albeit still localized toward the cilium’s base. These results
indicate that it is the lower part of the cilium that is most likely
to be involved in calcium signaling.
Figure 4B shows z sections of a cilium bending in response
to increasing levels of continuous shear from no flow up to 55
mPa. The figure shows that the top part of the cilium is
increasingly bent with higher force, whereas the lowest part
seems to remain in almost the same tilted angle as it had before
any flow was applied. Comparing this bending to the modeled
bending (Fig. 5), where the cilium has the same flexural
rigidity along the entire length, suggests that there is a difference in rigidity between the lower 3–5 ␮m and the top of the
cilium. This effect is seen in most cilia we have studied and is
more pronounced in longer cilia (Fig. 4B, right). This also
points in the direction that the basal part is able to take up most
stress.
Slow frequency flow pulses generate multiple calcium peaks.
One single flow pulse with 30 s duration was able to generate
Fig. 4. A: en-face views showing bending of
cilium exposed to forward-backward flow
(arrow) with 1 Hz frequency and 19 mPa
shear (step function). Image series shows the
cilium tip (*) rapidly changing direction
(within 0.5 s) in response to the fluctuating
flow. Cilium is ⬃8 ␮m long and is visualized
by transfection with dopamine 5 receptorgreen fluorescent protein. B, left 4 images: z
section view of cilium (length ⬃12 ␮m) after
5 min of exposure to different levels of
continuous shear force. B, right: 28-␮m-long
cilium exposed to 55 mPa continuous force
for 1 min. Dashed lines indicate slope of
basal part of cilium.
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Fig. 3. A: maximum membrane stress at different pulsate frequencies relative to continuous flow, as modeled in Comsol Multiphysics. B: bar diagram showing both percentage
of responding areas (16 ⫻ 16 square grid)
and the mean calcium signal in a pulsate flow
compared with a continuous flow with the
same shear force (19 –59 mPa). Pulsate flow
results in both decreased number of responding cells as well as reduced amplitude of the
calcium signal. Comparisons were made only
between different flow cycles within the
same experiment. C: curve showing intracellular calcium level in AU, averaged over the
whole image area, of cells exposed to 37- to
59- mPa force with constant or 0.5 Hz pulsed
flow. Intracellular calcium was visualized
with the calcium-sensitive fluorescent dye
fluo-4. C, bottom: time series of calcium
images from cells exposed to 59 mPa continuous and 0.5 Hz pulsed flow.
MECHANICAL PROPERTIES REGULATE CILIA’S RESPONSE TO FLOW
F1101
maximum calcium response. Thus several pulses with a frequency of 0.017 Hz (cycle time 60 s) were tested. As shown in
Fig. 6, cells responded by generating multiple peaks according
to the applied flow pulsations.
DISCUSSION
It is well described in the literature that primary cilia have a
flow-sensing function (24). Several studies have focused on the
molecular mechanisms involved in cilia calcium signals as well
as the location of the proteins involved (18). Those studies
suggest protein complexes located near the base of the cilium
Fig. 6. Representative figure showing relative Ca2⫹ concentration of a single
cell as fluo-4 intensity during the time course of an experiment. The cell is
exposed to 30-s-long pulses of flow. All pulses except the last one give rise to
calcium signals.
AJP-Renal Physiol • VOL
and different mechanisms for stretch-activated calcium channel
functions. However, little attention has been given to the
mechanical properties of cilia and how a fluid flow field can
affect the structure to transduce from a mechanical to a chemical signal.
We propose here that the structure of primary cilia, with a
relatively rigid central structure of microtubule bundles covered by an elastic cell membrane, has important dynamic
mechanical properties that serve to decode slow fluctuations in
fluid flow. Our experiments and simulations show that the
primary cilia can function as a low-pass filter, where fast
fluctuations in fluid flow do not result in any signals, whereas
slower and steadier flows initiate intracellular calcium signaling. Furthermore, our data point out that the region where
maximal membrane stress occurs is at the base of the cilia,
where it has been shown that the relevant protein complexes
for calcium signaling are located. The length of the cilia has an
important function; by reaching out into the lumen it becomes
more sensitive to absolute flow, and edge effects from cell
surfaces are reduced.
For the physiological function of a low-pass filtering in flow
sensitivity, one can so far only speculate. A possible function
could be in avoiding spurious signaling due to fluctuations not
resulting in any net flow. Normal renal function requires that the
flow through the nephron is kept within a narrow range. If it is not,
renal ability to maintain bodily solute and water balance will be
compromised. A mean shear stress during a certain time can be
translated into transport of a minimum volume of liquid through
the nephron. The calcium signaling would thus verify that renal
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Fig. 5. A: mesh of the finite element method
(FEM) model implemented in Comsol Multiphysics. Magnified portion shows the detailed structure of the mesh at the surface of
the cell. B: stress in membrane and ciliary
microtubules as modeled in Comsol Multiphysics. The simulated drag force corresponds to a shear of 44 mPa. Young modulus
was set to 178 kPa for the cilia and 10 kPa for
the plasma membrane. Rayleigh stiffness
damping was set to 10 s. B, middle: stress in
the cilium’s membrane and plasma membrane.
Largest stress appears near the base of the
cilium and in the plasma membrane in close
proximity to the cilium. B, left: magnification
of the membrane stress near the cilium’s base.
B, right: stress in ciliary microtubules, also
largest in the area around the base of the
cilium. Note the different scales of the colored
stress scale bars.
F1102
MECHANICAL PROPERTIES REGULATE CILIA’S RESPONSE TO FLOW
ACKNOWLEDGMENTS
We thank Anita Aperia for valuable input and discussions throughout the
study.
GRANTS
This study was supported by research grants from the Swedish Research
Council, the Knut and Alice Wallenberg Foundation, and the Erling-Perssons
Foundation.
DISCLOSURES
No conflicts of interest are declared by the authors.
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fluidic transport does not fall below a critical value. Such signaling could provide a continuous feedback of nephron flow conditions and participate in the regulation and fine tuning of secretion
and absorption processes.
Interestingly, the delay in calcium signaling is within the
same time domain as the oscillations in the tubuloglomerular
feedback (TGF), a mechanism in macula densa that regulates
glomerular filtration rate (9, 13). Studies using in vivo multiphoton microscopy (11, 29) have revealed that TGF-mediated
oscillations occur with a cycle time of 20 –50 s and that similar
periodicity in tubular flow rates occur with a delay of 5–10 s in
proximal tubule and 25–30 s in distal tubule. The delay in
calcium signal is thus within the same order of magnitude as
the inherent periodicity of normal nephronic flow pulsations,
pulsations that are detectable throughout the nephron.
Our results showed that pulsed flow with a cycle time of 60
s can generate regular calcium peaks in ciliated cells. Thus one
could speculate that regular calcium peaks also occur in vivo in
response to TGF-mediated pulses. Calcium peaks are known to
activate various signaling pathways and transcription factors,
depending on the peak frequency. For example, slow calcium
oscillations (3–5 min between peaks) have been shown to
activate the transcription factor NF-␬B, which in the developing kidney leads to increased cell proliferation and protection
from apoptosis (14).