93

CELL STRUCTURE AND FUNCTION 30: 93–100 (2005)

© 2005 by Japan Society for Cell Biology

Primary Cilia of inv/inv Mouse Renal Epithelial Cells Sense Physiological Fluid Flow: Bending of Primary Cilia and Ca2+ Influx

Dai Shiba1, Tetsuro Takamatsu2, and Takahiko Yokoyama1�

1Department of Anatomy and Developmental Biology and 2Department of Pathology and Cell Regulation,

Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan

ABSTRACT. Primary cilia are hypothesized to act as a mechanical sensor to detect renal tubular fluid flow.

Anomalous structure of primary cilia and/or impairment of increases in intracellular Ca2+ concentration in

response to fluid flow are thought to result in renal cyst formation in conditional kif3a knockout, Tg737 and pkd1/

pkd2 mutant mice.

The mutant inv/inv mouse develops multiple renal cysts like kif3a, Tg737 and pkd1/pkd2 mutants. Inv proteins

have been shown to be localized in the renal primary cilia, but response of inv/inv cilia to fluid stress has not been

examined. In the present study, we examined the mechanical response of primary cilia to physiological fluid flow

using a video microscope, as well as intracellular Ca2+ increases in renal epithelial cells from normal and inv/inv

mice in response to flow stress. Percentages of ciliated cells and the length of primary cilia were not significantly

different between primary renal cell cultures from normal and inv/inv mutant mice. Localization of inv protein

was restricted to the base of primary cilia even under flow stress. Inv/inv mutant cells had similar bending

mechanics of primary cilia in response to physiological fluid flow compared to normal cells. Furthermore, no dif-

ference was found in intracellular Ca2+ increases in response to physiological fluid flow between normal and inv/

inv mutant cells. Our present study suggests that the function of the inv protein is distinct from polaris (the Tg737

gene product), polycystins (pkd1 and pkd2 gene products).

Key words: primary cilia/kidney/Ca2+/inv/fluid stress/inversin

Introduction

Monocilia (primary cilia) used to be considered a vestigial

or remnant structure of no functional importance. However,

recent studies have shown that primary cilia are important

in the establishment of body left-right asymmetry and to

maintain normal renal tubular architecture. During early

developmental stages, primary cilia in the node are motile

and create leftward fluid flow by rapidly rotating them-

selves. Studies of nodal primary cilia are performed in

mutants that show randomization of body situs, such as

kif3a, kif3b, Tg737, iv and pkd2 mutant mice (Supp et al.,

1997; Nonaka et al., 1998; Takeda et al., 1999; Murcia et

al., 2000; Pennekamp et al., 2002). kif3a, kif3b and Tg737

mutants fail to produce nodal primary cilia. iv and pkd2 mu-

tants possess primary cilia, but iv mutant cilia are immotile

(Okada et al., 1999). The pkd2 mutant lacks a Ca2+ response

in the node during development (McGrath et al., 2003). In

addition to randomization of body situs, kif3a, Tg737 and

pkd2 mutants develop multiple renal cysts (Moyer et al.,

1994; Wu et al., 2000; Lin et al., 2003). In contrast to nodal

motile cilia, primary cilia in renal epithelial cells are non-

motile. Renal epithelial cells of kif3a and Tg737 mutants

show a loss or shortened cilia in vivo and in vitro (Pazour

et al., 2000; Yoder et al., 2002; Lin et al., 2003). Renal

cells derived from the pkd1 mutant or cells treated with

polycystin2 (a gene product of pkd2) antibody fail to in-

crease intracellular Ca2+ concentrations in response to fluid

stress (Nauli et al., 2003). Furthermore, isolated renal tubules

in Tg737 mutants displayed blunted increases in intracellular

Ca2+ concentration in response to fluid stress compared to

normal tubules (Liu et al., 2005). Thus, abnormal structures

in primary cilia and/or impairments in increases in intra-

cellular Ca2+ concentration in response to fluid flow are

thought to cause renal cyst formations.

One explanation why loss or truncation of cilia causes

renal cyst formation is that non-motile primary cilia on

*To whom correspondence should be addressed: Takahiko Yokoyama,MD, Department of Anatomy and Developmental Biology, Kyoto Prefec-tural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto602-0841, Japan.

Tel: +81–75–251–5303, Fax: +81–75–251–5304E-mail: tyoko@koto.kpu-m.ac.jp

94

D. Shiba et al.

renal epithelial cells function as a flow sensor (Praetorius

and Spring, 2003; Yokoyama, 2004). Fluid flow can bend

primary cilia of rat kangaroo cells (PtK1) (Schwartz et al.,

1997). Bending a cilium by pipette or fluid flow induced

Ca2+ influx in Madin-Darby canine kidney cells (MDCK)

(Praetorius and Spring, 2001). Nauli et al. showed that fluid

flow increased intracellular Ca2+ in collecting tubule cells

derived from normal mice, but not from pkd1 null mice or

renal cells treated with anti-pkd1/2 protein antibodies (Nauli

et al., 2003). Taken together with a recent report concerning

abundant cation-permeable channel activities in the ciliary

membrane (Raychowdhury et al., 2005), it is hypothesized

that the pkd1/pkd2 complex could function as a molecular

sensor as well as a Ca2+ channel, and that a lack of flow-

sensing in primary cilia could lead to renal cyst formation.

The inv (inversion of embryonic turning) mouse mutant

was discovered in a family of transgenic mice that showed

situs inversus associated with multiple renal cysts (Yokoyama

et al., 1993; Mochizuki et al., 1998). Mutation in the inv

gene in human was later found to cause nephronophthisis

type 2 (NPHP2) (Otto et al., 2003). Recently, primary cilia

in the primitive node of inv/inv mutants were reported to

show aberrant rotation and subsequently made turbulent

nodal flow (Okada et al., 1999, 2005). Artificial leftward

nodal flow rescued situs inversus in inv/inv mutants in vitro,

suggesting that the turbulent nodal flow causes situs ab-

normalities (Watanabe et al., 2003). It is possible that a

dysfunction of the primary cilia machinery is responsible

for the turbulent nodal flow. Scanning electron microscopy

(SEM) analysis of inv/inv kidney sections showed normal

appearing primary cilia at cystic tubules (Phillips et al.,

2004), but response of primary cilia in inv/inv mutants to

fluid stress has yet to be clarified.

In the present study, we first examined subcellular local-

ization of inv proteins and whether flow stimulation affects

localization of inv protein. The main purpose of the present

study was to examine if inv/inv mutant renal cells have any

abnormalities in mechanical response of primary cilia to

physiological fluid flow, or abnormalities in intracellular

Ca2+ increase in response to fluid stress.

Materials and Methods

Reagents

One-�m-diameter polystyrene beads were purchased from Poly-

sciences, Inc. (Warrington, PA). Fura-2 AM and Pluronic F127

gel were from Molecular Probes (Eugene, OR). Fluorescein-

conjugated LTA (LTA-FITC) was obtained from Vector Labora-

tories (Burlingame, CA). Cell culture supplements were obtained

from Invitrogen (Carlsbad, CA). Unless otherwise stated, all

chemicals were purchased from Sigma (St Louis, MO) or Wako

Pure Chemical (Osaka, Japan).

Animals and primary cultures of mouse renal epithelial cells

Normal, inv/inv and inv-GFP transgenic mice (Watanabe et al.,

2003) were maintained in an animal facility according to experi-

mental procedures that were approved by the Committee for

Animal Research, Kyoto Prefectural University of Medicine. Mice

(postnatal day 5) were anesthetized by intraperitoneal administra-

tion of sodium pentobarbital at a dose of 50 mg/kg body weight.

Kidneys were isolated and dissociated with Krebs buffer contain-

ing 10% BSA and 1 mg/ml collagenase for 30 min with gentle

shaking at 37°C. Digested tissue fragments were passed through

125 �m, 105 �m and 45 �m sieves, and centrifuged at 1000×g

for 10 min at room temperature. The pellet was resuspended in

Dulbecco’s modified Eagle’s medium/F-12 medium containing

10% fetal bovine serum, and cells were seeded on plastic dishes

or glass coverslips coated with human collagen IV (50 �g/ml).

Cells were incubated at 37°C, and equilibrated with 5% CO2 in

humidified air. After 24 h of incubation, culture medium was

changed to D-MEM / F-12 medium containing 0.5% fetal bovine

serum, 100 �M MEM non-essential amino acid solution, 5 mg/l

insulin, 5 �g/l sodium selenite, 5 mg/ml transferrin, 400 �g/l

dexamethasone, 10 ng/ml epidermal growth factor, 5 pg/ml 2,3,5-

triido-l-thyronine, 10000 U/l penicillin, 100 mg/l streptomycin, and

250 �g/l Fungizone®. Medium was changed daily.

Imaging of primary cilia under flow stimulation

Primary renal epithelial cells were grown on type IV collagen-

coated glass coverslips for at least 2 days. Cells were placed in a

parallel plate-type perfusion chamber (FSC2 closed system,

Bioptechs, Butler, PA). The flow chamber was set on the stage of

an inverted microscope (IX70, Olympus, Tokyo, Japan) equipped

with a CCD camera (UIC-QE, Molecular Devices Corporation,

Sunnyvale, CA). One end of the chamber was connected to a reser-

voir filled with Hanks balanced salt solution via a silicon tube. The

chamber and reservoir were maintained at 37°C by a temperature

sensor and heater (FCS2 controller, Bioptechs). Fluid flow was

applied to cells by adjusting the height of the reservoir, and aver-

aged volume flow (ml/s) was calculated from changes in weight of

the reservoir. We captured Nomarski images of primary cilia using

MetaFluor (Molecular Devices Corp., Sunnyvale, CA) for Win-

dows every 25 msec. One �m-diameter polystyrene beads were

used for determining linear fluid velocity profiles at the level of

primary cilia. When averaged linear velocity in the chamber was

3.1 mm/s, linear fluid velocity at the level of the primary cilia (at

10 �m) was about 280 �m/s under our experimental conditions

(Fig. 1A). Linear fluid velocity applied in the present study corre-

sponded to an appropriate physiological range of proximal tubular

flow rates (Chou and Marsh, 1987). Percentage of ciliated cells

was assessed by microscope. Length of primary cilia (L, �m) was

determined as follows:

L= (Fig. 1B).

a: the top of the cilium, b: the base of the cilium, c: a point where

ac2 bc2+

Primary Cilia and Ca2+ Influx in inv/inv Mice

95

the apical cell surface and a vertical line from the “a” to the apical

cell surface cross. ac is the height of primary cilia (�m). Since the

cilium was slightly tilted, the height does not correspond to the

length of the cilium. The height (distance between a and c) was

determined by focusing from the apical cell surface (b) to the top

of cilia (a). (n>30).

Localization of inv-GFP

Primary kidney epithelial cells derived from transgenic inv/inv

mice expressing the inv-GFP transgene were grown on type IV

collagen-coated glass coverslips for at least 2 days. GFP fluores-

cent images were obtained with an Olympus microscope (IX70)

and a CCD camera (UIC-QE). Primary cilia were bent by fluid

flow to make their entire length visible.

Detection of intracellular Ca2+ concentrations under flow stimulation

Primary renal epithelial cells were grown on type IV collagen-

coated glass coverslips for at least 2 days. Cells were incubated for

30 min with the Ca2+ sensitive probe Fura-2 AM (at a final concen-

tration of 5 �M) and 0.01% Pluronic F127 at room temperature, in

serum free medium. Cells were washed twice to remove excess

Fura-2 AM, and incubated for 15 min at 37°C for de-esterization.

During de-esterization, cells were co-incubated with LTA-FITC

(diluted 1:1000) for identification of proximal convoluted tubules

(Laitinen et al., 1987). Subsequently, cells were placed in the

chamber described above, and fluid flow was applied to the cells.

Paired fluorescent images were captured using MetaFluor every 5

s at excitation wavelengths of 340 nm and 380 nm with a xenon

light source. The fluorescent ratio (F340/F380) was monitored as

changes in intracellular Ca2+ concentrations. Data were obtained

from 7 regions of 2 to 5 cells.

Data analysis and statistics

Data are expressed as mean±S.E. Data obtained from the two

groups were compared using a t-test. P values of less than 0.05

were considered significant.

Results

Percentage of ciliated cells and lengths of primary cilia

Primary cilia of normal mice were seen as dots at the static

state under Nomarski observation, suggesting that primary

cilia extended perpendicularly to the apical membrane, and

became parallel to the optical axis of the microscope (indi-

cated by arrows in Fig. 2B). When physiological fluid flow

(flow profiles in Fig. 1A) was applied, primary cilia bend

and easily visible as lines (indicated by dashed circles in

Fig. 2C). Successive Z-axis pictures of primary cilia both

static and under fluid flow are available in the Supplemen-

tary Information, Video S1. In confluent cultures, ciliated

cells were 81.3±1.3% in normal cells, and 78.2±3.7% in inv/

inv mutant cells (Table I). Primary cilia were 11.5±0.6 �m

in length in normal and 13.0±0.6 �m in inv/inv mutant cells

(Table I). The percentage of ciliated cells and lengths of

Fig. 1. (A) Analysis of flow velocity and (B) primary cilium length.

(A) Fluid velocities were measured by tracking the displacement of 1-�m polystyrene beads. At least 10 beads were used to characterize fluid velocities.

Representative flow profiles are shown. The velocity profile between the two planes is parabolic. However, the velocity profile in a small distance from the

cell surface (about 30 um) became linear.

(B) Schematic diagram of a primary cilium. Plane � is the apical cell surface. Length of primary cilia (L, �m) was determined as follows: L= a:

the top of the cilium, b: the base of the cilium, c: a point where cell surface and a vertical line from the “a” to apical cell surface cross. ac is the height of

primary cilium (�m). Since the cilium was slightly tilted, the height does not correspond to the length of the cilium. The height (the distance between a and

c) is determined by focusing from the base of the cilium (b) to the top of cilium (a).

ac2 bc2+

96

D. Shiba et al.

primary cilia were not significantly different between nor-

mal and inv/inv mutant cells (P>0.05).

Determination of inv protein localization under physio-logical fluid flow stimulation

We examined the subcellular localization of functional inv

protein in primary renal epithelial cells using transgenic inv/

inv mice expressing the inv-GFP transgene, which rescues

the complete phenotype of inv/inv mice, including kidney

cyst formation and situs inversus. Fig. 3 shows Nomarski

images at the level of cells/primary cilia (Fig. 3A to E) and

corresponding inv-GFP fluorescent images (Fig. 3F to J) in

primary cultured renal epithelial cells. Primary cilia were

not observed as dots at the level of cell nuclei (Fig. 3A and

D) and we could not detect inv-GFP fluorescence in the

nucleus or in the membranes between cells (Fig. 3F and I).

Primary cilia were clearly seen as dots above the cell

nucleus level (Fig. 3B and C). We detected strong inv-GFP

fluorescence in the base of primary cilia (Fig. 3G), but not

in the top (Fig. 3H). Fluid flow was applied to visualize

primary cilia fully (Fig. 3E), and the corresponding inv-

GFP image showed strong GFP fluorescence in the base of

primary cilia (Fig. 3J). Furthermore, the inv-GFP signal in

the base of cilia did not translocate or change in intensity

by physiological fluid flow for 90 min or more (data not

shown).

Analysis of primary cilium bending speed by physiolog-ical fluid flow stimulation

We analyzed the response of renal cilia to physiological

flow stress. As shown in Fig. 2, primary cilia were observed

as dots at the static state. When physiological fluid flow was

applied, primary cilia of normal mice were bent. As soon as

flow stimulation stopped, primary cilia quickly returned to

their previous position. Fig. 4 shows consecutive pictures of

a bending cilium every 25 msec when fluid flow stress was

applied. It took on average 131±10 msec (n=10) from the

beginning of bending to the completely bended state (Fig.

4A). When flow stress was stopped, cilia returned to the

static state within an average time of 160±21 msec (n=10)

(Fig. 4B). See the Supplementary Information, Video S2.

Next, we examined whether inv/inv mouse cilia showed

any abnormality in bending mechanics in response to flow

stress. The inv/inv mouse cilia showed the same bending

mechanics in response to physiological fluid flow as that of

normal mouse cilia (Fig. 5). In inv/inv renal epithelial cells,

it took on average 133±11 msec (n=10) from the beginning

of bending to the completely bended state (Fig. 5A). When

fluid flow was stopped, cilia returned to the static state

within an average time of 160±14 msec (n=10) (Fig. 5B).

See the Supplementary Information, Video S3. No statis-

Fig. 2. Observation of primary cilia in normal mice-derived cells. Normal mice-derived primary kidney epithelial cells were cultured, and primary cilia

were observed. Nomarski images at the level of cell nuclei (A), and at the level of primary cilia (B, C) are shown. Primary cilia were easily observed under

fluid flow. Corresponding successive Z-axis pictures of primary cilia both at static and under fluid flow are available in the Supplementary Information,

Video S1. Scale bars=10 �m.

Table I. PERCENTAGE OF CILIATED CELLS AND LENGTH OF PRIMARY CILIA

OF RENAL CELLS DERIVED FROM NORMAL AND inv MUTANT MICE

Normal cells inv/inv cells

ciliated cells (%) 81.3±1.3 78.2±3.7

cilia length (�m) 11.5±0.6 13.0±0.6

Ciliated cells and cilia length were assessed by microscope in confluent

cultures. Ciliated cells were calculated as the percentage of cells that had

primary cilia. More than 150 cells were examined. Data were obtained

from 2–3 different cultures. Data are expressed mean±S.E.

Primary Cilia and Ca2+ Influx in inv/inv Mice

97

tical difference in bending and reflecting time of primary

cilia between normal and inv/inv mutant cells was observed

(P>0.05).

Physiological fluid flow stimulation and intracellular Ca2+ increase

Cells were loaded with the Ca2+ indicator Fura-2. We select-

ed LTA-positive proximal renal epithelial cells from both

normal and inv/inv mutant mice to examine intracellular

Ca2+ response to fluid flow (Fig. 6A and B). We detected a

rise in intracellular Ca2+ concentration in response to fluid

flow and this increase of intracellular Ca2+ concentration

was maintained while fluid flow was applied. In normal

cells, it took on average 96.4±4.7 sec to reach peak Ca2+ lev-

els from the start of fluid stress. Ca2+ levels were maintained

at higher than basal levels during fluid stimulation. After the

flow was stopped, intracellular Ca2+ concentrations decreased

and returned to basal levels within an average time of

153.6±15.3 sec.

Inv/inv mutant cells also showed a rise in intracellular

Ca2+ concentration in response to fluid flow and this in-

crease of intracellular Ca2+ concentration was maintained

while fluid flow was applied. inv/inv mutant cells took on

average 99.9±4.9 sec to reach peak Ca2+ levels while fluid

stress was applied. Ca2+ levels were maintained at higher

than basal levels during fluid stimulation. After the flow

was stopped, intracellular Ca2+ concentrations decreased,

and returned to basal levels within an average time of

122.9±7.6 sec. There were no statistical differences between

primary cilia of normal and inv/inv mutant cells in the time

to reach the peak and the time to return to basal levels.

Discussion

The present study provides three findings about renal cells

of normal and inv/inv mice in response to fluid flow. First,

functional inv protein was localized at the base of primary

cilia and remained there even under fluid flow stimulation.

Second, primary cilia of primary inv/inv mutant mouse renal

epithelial cells bend in response to physiological fluid

flow in an identical manner as those of normal mouse renal

epithelial cells. Third, renal cells derived from inv/inv mice

increased their intracellular Ca2+ concentration in response

to physiological fluid flow.

Localization of inv protein has been reported to occur in

cell membrane (Nurnberger et al., 2002; Simons et al., 2005),

cytoplasm (Simons et al., 2005), nucleus (Nurnberger et al.,

2002) and cilia (Morgan et al., 2002a; Otto et al., 2003;

Watanabe et al., 2003). Previous reports except Watanabe et

al. used antibodies against inv protein and cultured renal

cell lines. Localization of inv protein using antibodies indi-

cates the place where inv protein exists, but does not deter-

mine the place where the inv protein is functioning. The

inv-GFP fusion construct rescues all the inv phenotypes.

Fig. 3. Localization of functional inv protein to the base of primary cilia of kidney epithelial cells. inv/inv, inv-GFP mouse primary cilia of primary kidney

epithelial cells. Images at the level of cell nuclei (A, D, F and I), and at the level of primary cilia (B, C, E, G, H and J) are shown. GFP fluorescence is

detected at the base of primary cilia (G and J). Black arrows indicate the direction of fluid flow. Scale bars=10 �m.

98

D. Shiba et al.

Thus, localization of the fusion protein indicates the place

where the protein is functioning. In the present study, we

showed that a strong GFP signal was observed at the base of

primary cilia of primary cultured renal cells derived from

inv-GFP mice at static state, and no translocation of the

protein was observed after fluid flow stress, suggesting that

the base of primary cilia is where the inv protein functions.

Primary cilia of mouse primary cultured renal epithelial

cells stood straight and never displayed active beating under

static conditions. In response to physiological fluid flow,

primary cilia were bent, hence were easily visualized. As

soon as the fluid flow stopped, primary cilia were able to

Fig. 4. Primary cilium bending mechanics in response to fluid flow in normal mice-derived cells. Normal mice-derived primary kidney epithelial cells

were cultured, and primary cilia were visualized. Representative consecutive pictures of a bending cilium at every 25 msec under fluid flow stress are

shown. A) From the beginning of bending to the completely bended state. Flow is leftward. B) Primary cilia returned to the static state after the flow has

stopped. Corresponding time-lapse video images (40 frames per second) are available in the Supplementary Information, Video S2. Scale bars=5 �m.

Fig. 5. Primary cilium bending mechanics in response to fluid flow in inv/inv mice-derived cells. inv/inv mice-derived primary kidney epithelial cells

were cultured, and primary cilia were visualized. Representative consecutive pictures of a bending cilium at every 25 msec under fluid flow stress are

shown. A) From the beginning of bending to the completely bended state. Flow is leftward. B) Primary cilia returned to the static state after the flow has

stopped. Corresponding time-lapse video images (40 frames per second) are available in the Supplementary Information, Video S3. Scale bars=5 �m.

Primary Cilia and Ca2+ Influx in inv/inv Mice

99

return to their previous position without overshooting.

These results correlated well with a previous report using

renal cell lines of rat kangaroo (PtK1 cells) (Schwartz et al.,

1997). Mutations in kif3a and Tg737 caused structural

abnormalities of renal primary cilia in vivo and in vitro

(Pazour et al., 2000; Yoder et al., 2002; Lin et al., 2003).

Recently, primary cilia in the primitive node of inv/inv

mutants were reported to show aberrant rotation and sub-

sequently produced turbulent nodal flow, suggesting the

possibility of a structural or functional alteration of pri-

mary cilia in inv mutant mice (Okada et al., 1999, 2005).

However, our study showed that the lengths of primary cilia

were almost identical in both normal and inv/inv mutant

mice, and that irregularities in bending-and-return mechanics

of inv/inv primary cilia were not observed under physiological

fluid flow. Furthermore, ten times faster fluid flow did not

eliminate primary cilium from the cell, indicating that inv/

inv primary cilia are also firmly anchored to the cell (data

not shown). Together with a previous SEM study (Phillips

et al., 2004), it is unlikely that renal primary cilia in inv/inv

mutants have structural abnormalities that cause renal cyst

formation. Bending primary cilia in MDCK was reported to

increase intracellular Ca2+ concentrations (Praetorius and

Spring, 2001). Renal cells of pkd1 mutants or cells treated

with anti-polycystin2 were unable to increase their intra-

cellular Ca2+ concentration in response to physiological

flow stress (Nauli et al., 2003). However, inv/inv cells

showed intracellular Ca2+ increases after physiological flow

stress that could bend primary cilia of renal cells the same

way as normal cells. Although we cannot deny that more

subtle difference may exist between normal and inv cells

in the response or resting level of Ca2+, the present results

strongly suggested that inv renal cells have the same Ca2+

response mechanism to flow stress as normal renal cells have.

Inv protein contains calmodulin-binding motifs, and Ca2+

controls calmodulin-inv binding (Yasuhiko et al., 2001;

Morgan et al., 2002b). The polycystin complex acts as a

Ca2+ channel (Hanaoka et al., 2000). Both inv protein and

polycystin are localized in cilia. Losses of inv protein and

polycystin-2 function lead not only to cyst formation, but

also to situs inversus (Yokoyama et al., 1993; Pennekamp

et al., 2002). Thus, there is a possible relationship between

inv and the polycystin signaling pathway. Importantly, when

mutant cells that lack inv were exposed to fluid flow, we

detected Ca2+ influx. The present results suggest that inv

protein participates in downstream signaling of Ca2+ influx.

Recently, inv protein was shown to act on the Wnt pathway

(Simons et al., 2005). It would be interesting to investigate

whether polycystins also modulate the Wnt signaling path-

way, and share a common pathway with inv.

In summary, inv renal cells show no structural abnormali-

ties of cilia, and intracellular Ca2+ increases in response to

physiological fluid flow are the same as in normal renal

cells. Although the inv protein is localized in the cilia like

polaris, kif3 and polycystins, the present results suggest that

the inv protein has a distinct function.

Acknowledgments. This research was partially supported by the Mitsu-

bishi Foundation and by Grants-in-Aid for Scientific Research from the

Ministry of Education, Science, Sports and Culture (15370095) to T.Y. and

for Young Scientists (17790142) to D.S. We are grateful to Dr. Hiroshi

Hamada (Developmental Genetics Group, Graduate School of Frontier

Biosciences, Osaka University) for providing inv/inv mice expressing the

inv-GFP. We thank Drs. Joji Ando, Kimiko Yamamoto (Dept. of Bio-

medical Engineering, Graduate School of Medicine, University of Tokyo)

and Hideo Tanaka (Department of Pathology and Cell Regulation, Graduate

School of Medical Science, Kyoto Prefectural University of Medicine) for

their valuable suggestions about mechanisms of flow-induced Ca2+ influx.

Fig. 6. Flow-induced Ca2+ responses in lectin-labeled, proximal convoluted tubule cells. Epithelial cells of proximal convoluted tubule origin were

detected using LTA-FITC as markers. Cells loaded with Fura2-AM were exposed to fluid flow. Flow-induced Ca2+ responses in normal (A) and inv/inv (B)

cells were analyzed. Representative data are shown. Detailed procedures are described in ‘Materials and Methods’.

100

D. Shiba et al.

References

Chou, C.L. and Marsh, D.J. 1987. Measurement of flow rate in rat

proximal tubules with a nonobstructing optical method. Am. J. Physiol.,

253: F366–F371.

Hanaoka, K., Qian, F., Boletta, A., Bhunia, A.K., Piontek, K., Tsiokas, L.,

Sukhatme, V.P., Guggino, W.B., and Germino, G.G. 2000. Co-assembly

of polycystin-1 and -2 produces unique cation-permeable currents.

Nature, 408: 990–994.

Laitinen, L., Virtanen, I., and Saxen, L. 1987. Changes in the glycosylation

pattern during embryonic development of mouse kidney as revealed

with lectin conjugates. J. Histochem. Cytochem., 35: 55–65.

Lin, F., Hiesberger, T., Cordes, K., Sinclair, A.M., Goldstein, L.S., Somlo,

S., and Igarashi, P. 2003. Kidney-specific inactivation of the KIF3A

subunit of kinesin-II inhibits renal ciliogenesis and produces polycystic

kidney disease. Proc. Natl. Acad. Sci. USA, 100: 5286–5291.

Liu, W., Murcia, N.S., Duan, Y., Weinbaum, S., Yoder, B.K., Schwiebert,

E., and Satlin, L.M. 2005. Mechanoregulation of intracellular Ca2+

concentration is attenuated in collecting duct of monocilium-impaired

orpk mice. Am. J. Physiol. Renal Physiol., 289: F978–F988.

McGrath, J., Somlo, S., Makova, S., Tian, X., and Brueckner, M. 2003.

Two populations of node monocilia initiate left-right asymmetry in the

mouse. Cell, 114: 61–73.

Mochizuki, T., Saijoh, Y., Tsuchiya, K., Shirayoshi, Y., Takai, S., Taya,

C., Yonekawa, H., Yamada, K., Nihei, H., Nakatsuji, N., Overbeek,

P.A., Hamada, H., and Yokoyama, T. 1998. Cloning of inv, a gene that

controls left/right asymmetry and kidney development. Nature, 395:

177–181.

Morgan, D., Eley, L., Sayer, J., Strachan, T., Yates, L.M., Craighead, A.S.,

and Goodship, J.A. 2002a. Expression analyses and interaction with the

anaphase promoting complex protein Apc2 suggest a role for inversin in

primary cilia and involvement in the cell cycle. Hum. Mol. Genet., 11:

3345–3350.

Morgan, D., Goodship, J., Essner, J.J., Vogan, K.J., Turnpenny, L., Yost,

H.J., Tabin, C.J., and Strachan, T. 2002b. The left-right determinant

inversin has highly conserved ankyrin repeat and IQ domains and inter-

acts with calmodulin. Hum. Genet., 110: 377–384.

Moyer, J.H., Lee-Tischler, M.J., Kwon, H.Y., Schrick, J.J., Avner, E.D.,

Sweeney, W.E., Godfrey, V.L., Cacheiro, N.L., Wilkinson, J.E., and

Woychik, R.P. 1994. Candidate gene associated with a mutation causing

recessive polycystic kidney disease in mice. Science, 264: 1329–1333.

Murcia, N.S., Richards, W.G., Yoder, B.K., Mucenski, M.L., Dunlap, J.R.,

and Woychik, R.P. 2000. The Oak Ridge Polycystic Kidney (orpk)

disease gene is required for left-right axis determination. Development,

127: 2347–2355.

Nauli, S.M., Alenghat, F.J., Luo, Y., Williams, E., Vassilev, P., Li, X.,

Elia, A.E., Lu, W., Brown, E.M., Quinn, S.J., Ingber, D.E., and Zhou, J.

2003. Polycystins 1 and 2 mediate mechanosensation in the primary

cilium of kidney cells. Nat. Genet., 33: 129–137.

Nonaka, S., Tanaka, Y., Okada, Y., Takeda, S., Harada, A., Kanai, Y.,

Kido, M., and Hirokawa, N. 1998. 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.

Nurnberger, J., Bacallao, R.L., and Phillips, C.L. 2002. Inversin forms a

complex with catenins and N-cadherin in polarized epithelial cells. Mol.

Biol. Cell, 13: 3096–3106.

Okada, Y., Nonaka, S., Tanaka, Y., Saijoh, Y., Hamada, H., and Hirokawa,

N. 1999. Abnormal nodal flow precedes situs inversus in iv and inv

mice. Mol. Cell, 4: 459–468.

Okada, Y., Takeda, S., Tanaka, Y., Belmonte, J.C., and Hirokawa, N.

2005. Mechanism of nodal flow: a conserved symmetry breaking event

in left-right axis determination. Cell, 121: 633–644.

Otto, E.A., Schermer, B., Obara, T., O’Toole, J.F., Hiller, K.S., Mueller,

A.M., Ruf, R.G., Hoefele, J., Beekmann, F., Landau, D., Foreman, J.W.,

Goodship, J.A., Strachan, T., Kispert, A., Wolf, M.T., Gagnadoux, M.F.,

Nivet, H., Antignac, C., Walz, G., Drummond, I.A., Benzing, T., and

Hildebrandt, F. 2003. Mutations in INVS encoding inversin cause

nephronophthisis type 2, linking renal cystic disease to the function of

primary cilia and left-right axis determination. Nat. Genet., 34: 413–420.

Pazour, G.J., Dickert, B.L., Vucica, Y., Seeley, E.S., Rosenbaum, J.L.,

Witman, G.B., and Cole, D.G. 2000. Chlamydomonas IFT88 and its

mouse homologue, polycystic kidney disease gene tg737, are required

for assembly of cilia and flagella. J. Cell Biol., 151: 709–718.

Pennekamp, P., Karcher, C., Fischer, A., Schweickert, A., Skryabin, B.,

Horst, J., Blum, M., and Dworniczak, B. 2002. The ion channel

polycystin-2 is required for left-right axis determination in mice. Curr.

Biol., 12: 938–943.

Phillips, C.L., Miller, K.J., Filson, A.J., Nurnberger, J., Clendenon, J.L.,

Cook, G.W., Dunn, K.W., Overbeek, P.A., Gattone VH 2nd, and

Bacallao, R.L. 2004. Renal cysts of inv/inv mice resemble early infantile

nephronophthisis. J. Am. Soc. Nephrol., 15: 1744–1755.

Praetorius, H.A. and Spring, K.R. 2001. Bending the MDCK cell primary

cilium increases intracellular calcium. J. Membr. Biol., 184: 71–79.

Praetorius, H.A. and Spring, K.R. 2003. The renal cell primary cilium

functions as a flow sensor. Curr. Opin. Nephrol. Hypertens, 12: 517–

520.

Raychowdhury, M.K., McLaughlin, M., Ramos, A.J., Montalbetti, N.,

Bouley, R., Ausiello, D.A., and Cantiello, H.F. 2005. Characterization

of single channel currents from primary cilia of renal epithelial cells. J.

Biol. Chem., 280: 34718–34722.

Schwartz, E.A., Leonard, M.L., Bizios, R., and Bowser, S.S. 1997. Analysis

and modeling of the primary cilium bending response to fluid shear. Am.

J. Physiol., 272: F132–F138.

Simons, M., Gloy, J., Ganner, A., Bullerkotte, A., Bashkurov, M., Kronig,

C., Schermer, B., Benzing, T., Cabello, O.A., Jenny, A., Mlodzik, M.,

Polok, B., Driever, W., Obara, T., and Walz, G. 2005. Inversin, the gene

product mutated in nephronophthisis type II, functions as a molecular

switch between Wnt signaling pathways. Nat. Genet., 37: 537–543.

Supp, D.M., Witte, D.P., Potter, S.S., and Brueckner, M. 1997. Mutation

of an axonemal dynein affects left-right asymmetry in inversus viscerum

mice. Nature, 389: 963–966.

Takeda, S., Yonekawa, Y., Tanaka, Y., Okada, Y., Nonaka, S., and

Hirokawa, N. 1999. Left-right asymmetry and kinesin superfamily pro-

tein KIF3A: new insights in determination of laterality and mesoderm

induction by kif3A–/– mice analysis. J. Cell Biol., 145: 825–836.

Watanabe, D., Saijoh, Y., Nonaka, S., Sasaki, G., Ikawa, Y., Yokoyama,

T., and Hamada, H. 2003. The left-right determinant Inversin is a

component of node monocilia and other 9+0 cilia. Development, 130:

1725–1734.

Wu, G., Markowitz, G.S., Li, L., D’Agati, V.D., Factor, S.M., Geng, L.,

Tibara, S., Tuchman, J., Cai, Y., Park, J.H., van Adelsberg, J., Hou, H.

Jr, Kucherlapati, R., Edelmann, W., and Somlo, S. 2000. Cardiac

defects and renal failure in mice with targeted mutations in Pkd2. Nat.

Genet., 24: 75–78.

Yasuhiko, Y., Imai, F., Ookubo, K., Takakuwa, Y., Shiokawa, K., and

Yokoyama, T. 2001. Calmodulin binds to inv protein: implication for

the regulation of inv function. Develop. Growth Differ., 43: 671–681.

Yoder, B.K., Tousson, A., Millican, L., Wu, J.H., Bugg C.E. Jr, Schafer,

J.A., and Balkovetz, D.F. 2002. Polaris, a protein disrupted in orpk

mutant mice, is required for assembly of renal cilium. Am. J. Physiol.

Renal Physiol., 282: F541–F552.

Yokoyama, T. 2004. Motor or sensor: a new aspect of primary cilia

function. Anat. Sci. Int., 79: 47–54.

Yokoyama, T., Copeland, N.G., Jenkins, N.A., Montgomery, C.A., Elder,

F.F., and Overbeek, P.A. 1993. Reversal of left-right asymmetry: a situs

inversus mutation. Science, 260: 679–682.

(Received for publication, October 18, 2005 and accepted December 6, 2005)