1 Department of Cell Biology, 2 High Resolution Imaging Facility, Departments of 3 Medicine, 4 Surgery, and 5 Physiology and Biophysics, and the 6 Birmingham Veterans Affairs Medical Center, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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Cilia are organelles that play diverse roles, from fluid movement to sensory reception. Polaris, a protein associated with cystic kidney disease in Tg737°rpk mice, functions in a ciliogenic pathway. Here, we explore the role of polaris in primary cilia on Madin-Darby canine kidney cells. The results indicate that polaris localization and solubility change dramatically during cilia formation. These changes correlate with the formation of basal bodies and large protein rafts at the apical surface of the epithelia. A cortical collecting duct cell line has been derived from mice with a mutation in the Tg737 gene. These cells do not develop normal cilia, which can be corrected by reexpression of the wild-type Tg737 gene. These data suggest that the primary cilia are important for normal renal function and/or development and that the ciliary defect may be a contributing factor to the cystic disease in Tg737°rpk mice. Further characterization of these cells will be important in elucidating the physiological role of renal cilia and in determining their relationship to cystic disease.
Tg737; ciliogenesis; cell line; polycystic kidney disease; Madin-Darby canine kidney cells; Oak Ridge Polycystic Kidney
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INTRODUCTION |
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CILIA AND FLAGELLA ARE COMPLEX organelles found throughout the animal kingdom, within protozoa, and in some plants, where they perform a broad range of functions (5, 35, 54). Cilia on epithelial cells in the lung or ependymal cells lining the ventricles of the brain are motile and function in the movement of mucus and cerebral spinal fluid, respectively. Motile cilia on the surface of the node, an important early embryonic-stage organizing center, play a critical role in specification of left-right body axis during development (34, 36, 50). In addition, there are primary nonmotile cilia on many cells in the nephron, the bile and pancreatic ducts, on retinal rods in the eye, and on olfactory neurons (3, 8, 32, 43, 53). Although the specialized cilia on the rods and olfactory neurons are involved in reception of extracellular stimuli, the role of the primary nonmotile cilia in the kidney, pancreas, and liver is unknown and has been relatively ignored. The nearly ubiquitous nature (http://www.wadsworth.org/BMS/SCBlinks/cilia1.html) of these solitary cilia has led some to propose that they are vestigial organelles of little consequence to normal tissue function (14, 53). However, recent data have suggested that aberrant formation of cilia on cells in the murine kidney can result in cystic kidney disease (37, 51).
The formation of cysts in the kidney is a pathological entity common to a number of inherited and acquired diseases (16). Of these cystic disorders, polycystic kidney disease (PKD) is the most common, most extensively studied, and one of the leading causes of end-stage renal failure in humans. Most cases of the dominant form of human PKD (ADPKD) arise from mutations in either of two genes, PKD1 and PKD2 (8a, 13a, 22a). The gene responsible for the less common, recessive form of human PKD (ARPKD) has yet to be cloned. Pathological findings in PKD include the formation of epithelia-lined cysts throughout the nephron in ADPKD and predominantly in the collecting duct in ARPKD, changes in extracellular matrix composition, improper epithelial cell differentiation, and alterations in cell polarity. In addition to the lesions in the kidney, abnormalities are often found in other tissues, including the liver and pancreas (30, 31).
The considerable morbidity and mortality resulting from cystic kidney diseases in humans have prompted intense investigative efforts to identify the molecular mechanisms involved in cystogenesis and cystic disease progression. A good deal of knowledge about human PKD has come from the availability of numerous mouse models (4, 6, 19, 33). These models exhibit similar pathology to human PKD with regard to cyst localization, epithelial polarity defects, and extrarenal involvement. Pkd1 and Pkd2 mutant mice have been engineered, and these mice are now being used to study the human disease and the function of their respective protein products (28, 55). In addition to the Pkd1 and Pkd2 mice, several interesting mouse models for PKD have risen spontaneously, through chemical mutagenesis, and through transgenic insertional mutagenesis. Two of the best-studied models are the Oak Ridge Polycystic Kidney disease (orpk; Tg737°rpk) and the congenital polycystic kidney disease (cpk) mutants (6, 33). Both orpk and cpk mice develop renal cysts along with hepatic and pancreatic abnormalities (17, 20, 33).
There are several lines of evidence suggesting a connection between PKD and cilia. First, polaris, the protein associated with the cystic lesions in Tg737°rpk mice, localizes to the basal body and cilia axoneme (51). Because of the defect in polaris function, the cilia are aberrantly formed in orpk mutant mice (37, 51). Studies of the polaris homologues in Caenorhabditis elegans and Chlamydomonas indicate that the homologues function as a component of the intraflagellar transport (IFT) system (21, 37, 41). IFT is a process that describes the kinesin- and dynein-mediated movement of large protein rafts, of which polaris is a component, along the axoneme of cilia and flagella (25). An additional connection between cilia and PKD is seen in cystic lesion of the cpk mouse. A candidate gene responsible for the cpk phenotype has recently been cloned (Guay-Woodford LM, unpublished observations). Although the function of cystin, the protein encoded by the cpk gene, remains to be determined, exogenously expressed epitope-tagged cystin is detected in the ciliary axoneme. Finally, although the function and subcellular localization of mammalian polycystins remain somewhat controversial, it is intriguing that the C. elegans homologues of polycystin-1 (lov-1) and polycystin-2 (pkd2) are in cilia on a subgroup of sensory neurons that also express the polaris homologue (osm-5) (7, 21, 41). In contrast to osm-5 mutants, cilia appear normal in lov-1 and pkd-2 mutant worms. However, lov-1 and pkd-2 mutant worms do exhibit sensory defects associated with worm mating behavior that are similar to those in the osm-5 mutants, suggesting that male lov-1 and pkd-2 mutants have lost their cilia-mediated sensory function (7).
This potential connection between cilia and PKD is intriguing in light of our limited knowledge of the role of the primary cilium found on epithelial cells lining the nephron and collecting duct. Elucidating the function of primary cilia and their association with PKD will require detailed characterization of proteins involved in both ciliogenesis and cilia function, plus the development of reagents and assays to test their role in renal physiology. In this regard, we describe the generation of an SV40 conditionally immortalized cortical collecting duct cell line from orpk mice. Although these cells develop overtly normal basal bodies as the initiating step in ciliogenesis, they subsequently fail to assemble the cilia axoneme. This ciliary defect can be corrected by reexpressing wild-type polaris. We also describe basic biochemical properties of polaris during cilia assembly and analyze the effect of microtubule destabilization on polaris localization. Further functional characterization of these cells will be important in dissecting the signaling events mediated through renal cilia and how disruption of this organelle can result in PKD phenotype in mice.
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MATERIALS AND METHODS |
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Mice. The generation and genotyping of the Tg737°rpk mutant lines have been described previously (33, 57). Heterozygous orpk female mice were bred with male mice homozygous for the ImmortoMouse transgene (H-2Kb-tsA58; Charles River Laboratories, Wilmington, MA), and the resulting offspring were genotyped from tail biopsies. Mice that were orpk/ImmortoMouse compound heterozygotes were obtained and crossed to generate orpk homozygous mutants that carry the H-2Kb-tsA58 transgene (ImmortoMouse). All mice were maintained at the University of Alabama School of Medicine in accordance with National Institutes of Health guidelines.
Madin-Darby canine kidney cell culture, transfection of Madin-Darby canine kidney T23 cells, and inducible polaris expression. Type II Madin-Darby canine kidney (MDCK) or transfected MDCK-T23 cells (tetracycline-off cell line; provided by K. Mostov and Y. Altschuler) were used between passages 3 and 15. Cells were cultured in MEM containing Earl's balanced salt solution supplemented with 5% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C and 5% CO2. MDCK-T23 cells were cotransfected with a hygromycin selection maker, and the T7-epitope-tagged polaris was cloned into the pBI-G vector (Clontech, Palo Alto, CA) or empty pBI-G vector using the calcium phosphate precipitation method. Drug-resistant clones were selected, and clonally derived lines were established in the presence of 300 mg/ml hygromycin. Expression from the pBI-G vector was repressed by addition of 20 ng/ml doxycycline (Sigma, St. Louis, MO) to the culture medium.
Antibodies.
The generation of antisera against polaris (GN593 and GN594) and
confirmation of their specificity were described previously (51). Other antibodies used include -
-tubulin
(Biogenex, Mu178-UC),
-E-cadherin (Transduction Laboratories,
Lexington, KY),
-ZO (zonal occludin)-1 antibody (obtained from B. Stevenson, Univ. of Alberta) (48),
-SV40 large T
antigen (Oncogene Sciences, Uniondale, NY),
-T7 (Novagen, Madison,
WI),
-pan-cytokeratin (AE1/AE3; Chemicon International, Temecula,
CA), and
-vimentin (3B4; DAKO, Carpinteria, CA). The secondary
antibodies used include goat anti-mouse IgG-Oregon green (Molecular
Probes, Eugene, OR), goat anti-mouse IgG-FITC (Jackson ImmunoResearch
Laboratories, West Grove, PA), goat anti-rabbit-Texas red (TxR; Jackson
ImmunoResearch Laboratories), goat anti-rat-TxR (Jackson
ImmunoResearch Laboratories), donkey anti-rabbit
IgG-tetramethylrhodamine isothiocyanate (Jackson ImmunoResearch
Laboratories), goat anti-rabbit horseradish peroxidase (HRP; Bio-Rad,
Hercules, CA), and goat anti-mouse HRP (Bio-Rad). In all cases,
antibody controls were evaluated to ensure that the results obtained
were not a consequence of the secondary antibodies used.
Time course of polaris solubility and localization to MDCK cilia. Type II MDCK cells were plated at confluence and grown on 6- and 24-mm Transwell filters (Costar, Cambridge, MA) for 6, 24, 48, 72, and 96 h. For immunofluorescent localization of polaris, 6-mm filters were used. For cell lysate preparation, cells on 24-mm filters were rinsed twice with Dulbecco's phosphate-buffered saline containing Mg2+ and Ca2+ (PBS+) at 4°C. Cells were solubilized in 500 µl of 50 mM NaCl, 10 mM PIPES, pH 6.8, 3 mM MgCl2, 0.5% Triton X-100 (Triton-X), and 300 mM sucrose (CSK buffer) containing protease inhibitors [2 mM phenylmethanesulfonyl fluoride (PMSF), 50 µg/ml pepstatin, 50 µg/ml chymostatin and 10 µg/ml antipain] for 20 min at 4°C. The cells were scraped from the filter and sedimented at 4°C in a microcentrifuge. The soluble and pellet fractions were collected. The pellet was resuspended in 100 µl of 15 mM Tris (pH 7.5), 5 mM EDTA, 2.5 mM EGTA, and 1% SDS, heated at 95°C for 20 min, diluted to 0.5 ml with CSK buffer, and centrifuged for 10 min at 4°C at maximum speed. The resulting supernatant was considered the Triton-X-insoluble fraction. Protein concentration of the fractions was determined using a BCA kit (Pierce, Rockford, IL).
Fluorescent labeling of proteins in MDCK cells.
Cells on Transwell filters were fixed in 4% paraformaldehyde (PFA) in
PBS for 20 min. After filters were washed three times with PBS+, the
cells were quenched with 75 mM NH4Cl and 20 mM glycine, pH
8.0, with KOH (quench solution) for 10 min at room temperature. Filters
were washed once with PBS+ and permeabilized with PBS+, 0.7% fish skin
gelatin, and 0.025% saponin (PFS) for 15 min at 37°C. Filters were
then probed with antibodies against polaris (rabbit polyclonal
antibody), ZO-1 (rat monoclonal antibody), or -tubulin (mouse
monoclonal antibody) diluted in PFS at 1:250, 1:2, or 1:50,
respectively, for 1 h at 37°C. Filters were washed with PFS at
room temperature and then probed with FITC (polaris)- or TxR (ZO-1 and
-tubulin)-conjugated secondary antibodies (Jackson ImmunoResearch
Laboratories) diluted 1:100 in PFS for 1 h at 37°C. Filters were
washed four times for 5 min each with PFS, once with PBS+, twice with
PBS+ containing 0.1% TX-100, and once with PBS+. Cells were postfixed
in 4% PFA for 15 min at room temperature, cut from the support with a
scalpel, and mounted in Vectashield mounting medium (Burlingame, CA).
Conventional and confocal laser scanning microscopy of fluorescently labeled MDCK cells. Fluorescently labeled MDCK cell samples were analyzed using either a Leica fluorescence microscope equipped with a Hamamatsu C5810 digital camera or a Leica confocal laser scanning microscope system configured with both an argon ion (5 mW, 488 nm) and a krypton ion (10 mW, 568 nm) laser. The captured photomicrographs were labeled using Adobe Photoshop.
Nocodazole treatment of cells.
Stocks of nocodazole (10 mg/ml) were prepared in dimethylsulfoxide and
stored in single-use aliquots at 20°C. Just before use, the
nocodazole was diluted into the appropriate medium at a final
concentration of 10 µg/ml (33 µM). Cells were incubated in medium
supplemented with 33 µM nocodazole for 4 h at 4°C as described
elsewhere (18). No effect of exposing the cells to 4°C
treatment in the absence of nocodazole was evident in control samples.
Generation of orpk mutant cortical collecting duct cells.
Cortical collecting duct segments were isolated from
collagenase-treated slices of renal cortex of 21-day-old
orpk mutant mice that were heterozygous for the ImmortoMouse
transgene (H-2kb-tsA58) using a procedure as
described previously (44). Briefly, the capsule of the
kidney was removed and 1-mm sections were cut using a Stadie-Riggs
handheld microtome. The sections were examined to ensure they
contained no trace of medullary tissues. The slice was incubated in
DMEM containing 0.1 g/dl collagenase, 5 mM glycine, 50 U/ml DNase, and
50 µg/ml soybean trypsin inhibitor. Cortical collecting duct tubule
segments were selected and transferred to individual wells in 24-well
culture dishes. To establish the lines, cells were grown from the
tubules under permissive conditions for SV40 large-T antigen expression
(33°C, 10 U/ml interferon-) in CD media (DMEM/F-12, 10% FBS, 1.3 µg/l sodium selenite, 1.3 µg/l 3,3'5-triiodo-thyronine, 5 mg/l
insulin, 5 mg/l transferrin, 2.5 mM glutamine, 5 µM dexamethasone,
100 U/ml penicillin, 100 mg/ml streptomycin, 5%
CO2). Once the cortical collecting duct cells were
established, clonal lines were derived from individual cells. The data
presented here were obtained with the 94D series, and similar results
were obtained with other lines derived from these mice. To
promote differentiation and SV40 large-T antigen inactivation, cells
were cultured at nonpermissive conditions (39°C in the absence
of interferon-
for 3 days before the analysis).
Transfection of orpk mutant cortical collecting duct cells. The construction of the Tg737 expression construct (Tg737Bap) and transfection of the 94D cells were accomplished using Lipofectamine Plus according to the manufacturer's protocol (Life Technologies GIBCO BRL, Carlsbad, CA). Stable cell lines were generated by drug selection using 400 µg/ml G418, and several clonal derived lines were obtained. Expression from the Tg737Bap construct was evaluated on Western blots for each line. Two lines, referred to as 94D-Tg737Bap-1 and 94D-Tg737Bap-2, were selected due to their low and endogenous levels of Tg737 expression, respectively. The control in these studies was 94D cells stably transfected with an empty pCDNA3.1 (Invitrogen Life Technologies, Carlsbad, CA) vector alone to ensure that selection in G418 did not alter the properties of the cells.
Fluorescent labeling of proteins in 94D cells.
For immunofluorescence analysis in 94D cells, cells were fixed in 4%
PFA, 0.1% Triton-X in PBS for 10 min and incubated in blocking buffer
(1% BSA in PBS) for 30 min at room temperature. Blocked cells were
probed with rabbit anti-polaris (1:200), rat monoclonal anti-ZO-1
(1:2), or mouse monoclonal anti -tubulin (1:200) diluted in blocking
buffer for 1 h at room temperature. After being washed three times
with PBS, cells were then immunoprobed with fluorophore-conjugated
secondary antibodies. The secondary antibodies used in the analysis of
94D cells included goat anti-rabbit-TxR, goat anti-rat-TxR, and donkey
anti-rabbit IgG-tetramethylrhodamine isothiocyanate (all 3 obtained
from Jackson ImmunoResearch Laboratories). Secondary antibodies were
diluted 1:200 in blocking buffer. Cells were then washed three times in
PBS and mounted onto slides, and the images were captured on an
inverted fluorescence microscope. Nuclei were stained for 5 min using
Hoechst 33528 (Sigma) diluted 1:1,000 in PBS.
Electrophoresis and Western blot analysis. Protein was isolated from cells or tissue in RIPA buffer, and the protein concentration was quantified using a DC protein assay kit (Bio-Rad, Hercules, CA) as described by the manufacturer. Equal amounts of protein were resolved by electrophoresis on SDS-PAGE gels, and the proteins were transferred to nitrocellulose (NitroBind, MSI) or Immobilon P filters (Millipore, Bedford, MA). Filters were blocked in 5% dry milk containing 0.1% Tween 20 in PBS and immunoprobed with mouse monoclonal antibody against E-cadherin (Transduction Laboratories) diluted 1:1,000, rabbit polyclonal against polaris (GN593) diluted 1:250, or mouse monoclonal anti-T7 antibody (Novagen) diluted 1:5,000 in blocking buffer. The filters were washed with PBS containing 0.1% Tween 20 and probed with goat anti-mouse IgG HRP (Bio-Rad) diluted 1:5,000 (for E-cadherin) or goat-anti-rabbit IgG HRP diluted 1:5,000 (for polaris) in block solution for 1 h. Filters were washed five times with PBS containing 0.1% Tween 20. The HRP signal was detected using a Supersignal West Femto chemiluminescence kit (Pierce) or an enhanced chemiluminescence kit (ECL; Amersham).
Northern blot analysis.
Total RNA was isolated from nonciliated, nonconfluent, proliferating,
or ciliated, confluent, nonproliferating MDCK cells using TriZol as
described by the manufacturer (GIBCO BRL) and enriched for
polyadenylated RNA by passage using oligo-dT columns. One microgram of
PolyA+ RNA from MDCK cells or 2 µg of poly A+
RNA from mouse kidney were resolved by denaturing agarose gel electrophoresis, transferred to charged nitrocellulose membranes, and
hybridized with mouse Tg737 cDNA or chick tubulin labeled with [-32P]deoxycytidine 5'-triphosphate (dCTP)
generated by the random hexamer method.
Fixation and scanning electron mirocscopic analysis. For analysis of cilia on 94D cells using scanning electron microscopy (SEM), cells were fixed in SEM grade 2.5% glutaraldehyde (Electron Microscopy Sciences, Ft. Washington, PA) in 0.1 M cacodylate buffer (pH 7.4) for 90 min, washed in cacodylate buffer, postfixed in 1% OsO4 in cacodylate, and washed twice with 0.1 M cacodylate buffer. Fixed cells were then dehydrated through a series of ethanol washes (30, 50, 70, 80, 90, 95, and 100% ethanol-cacodylate buffer) for 5 min each and then through hexamethyldisilasane (25, 33, 50, 66, 75, and 100%) diluted in ethanol for 5 min each. Samples were desiccated under vacuum overnight, sputter coated (50-s gold deposition), and examined using an Hitachi 7000 SEM at ×20,000.
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RESULTS |
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Expression of polaris in MDCK cells during ciliogenesis.
In mice, Tg737 encodes a 3.2-kb transcript that is expressed
at low levels with a wide tissue distribution (33, 58).
Data from mice and humans suggest that Tg737 is a complex
gene that also encodes several low-level, alternatively spliced
transcripts and protein products. The prominent 95-kDa protein encoded
by the 3.2-kb Tg737 mRNA in mice is called polaris
(51). In addition to this 95-kDa protein, several other
protein bands are often detected. Importantly, expression of both
proteins is abolished in the
Tg7372-3
Gal null mice, suggesting
they are polaris isoforms and confirming the specificity of the
affinity-purified antibodies.
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Localization and biochemical properties of polaris.
To begin analyzing polaris properties during ciliogenesis, we plated
cells at confluence on filter inserts. Triton-X-extractable and
-nonextractable proteins were isolated at different time points through
the formation of cilia. The partitioning of polaris into either the
Triton-X-soluble or -insoluble pools were analyzed on Western blots
using anti-polaris antiserum similar to studies conducted for
E-cadherin during adherens junction formation (1). The
results indicated that polaris was completely Triton-X extractable for
the first 24 h after plating (Fig.
2A). However, at 48 h a small fraction of polaris protein enters the Triton-X-nonextractable pool. The amount of insoluble polaris increased through the 72-h time
point. The only polaris isoform detected in the insoluble pool was the
95-kDa form of the protein. In contrast to polaris, E-cadherin was
detected in the insoluble fraction within 6 h of plating (Fig.
2A). The change in E-cadherin solubility is a consequence of
its association with the cytoskeleton as an adherens junction form
(1, 22, 39). Similarly, we predict that the change in
polaris solubility is reflective of either cytoskeletal attachment during cilia formation or its incorporation into large insoluble structures such as the IFT raft.
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Inducible ectopic overexpression of polaris.
To further explore the role of polaris in ciliogenesis, we utilized a
doxycyline-repressible expression system in T23 MDCK cells (Tet-off
line, Clontech) to test whether high levels of polaris expression can
saturate the IFT process and block ciliogenesis. We anticipated that
these cells could then serve as an inducible model system for analyzing
cilia function on the surface of a well-characterized renal epithelia
cell. These cells were stably transfected with a doxycyline-responsive
Tg737 construct (Tg737pBI-G, Clontech). This construct
encodes a T7 epitope-tagged polaris protein that allows us to
distinguish between endogenous and exogenous expression. Clonal cell
lines were generated, and inducible polaris expression was analyzed on
Western blots using anti-polaris and anti-T7 antibodies under repressed
and nonrepressed conditions. A line was identified that induced high
levels of polaris expression on removal of doxycyline but remained
repressed in its presence (Fig.
3A). Both T7 and anti-polaris
antiserum recognize the exogenously expressed protein; however, unlike
endogenous canine polaris, only a single band at slightly larger than
95 kDa was detected with the T7 antibody. The slight increase in size
is due to the presence of the T7 epitope tag. Thus the exogenously
expressed protein of murine origin likely correlates with the lower
95-kDa form of polaris that is detected in the insoluble fraction
during basal body formation. The fact that we see a single band with exogenously expressed protein suggests that the other polaris isoforms
likely arise due to alternative splicing events that do not occur with
the murine Tg737 cDNA cloned into the expression vector and
not by posttranslational modification.
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The effect of nocodazole treatment on polaris localization and
cilia.
IFT is a process originally identified in Chlamydomonas that
describes the movement of large-protein rafts within the cilia axoneme
(25). Microtubule-based motor proteins direct the movement of the IFT rafts along microtubule filaments in both anterograde and
retrograde directions (24, 38, 47). Thus the microtubular network is likely to play a critical role in cilia axoneme extension, motility, and stabilization. To begin assessing the importance of
intact microtubules in cilia maintenance and polaris localization, we
treated polarized MDCK cells with nocodazole for up to 4 h to
disrupt the microtubule filaments. After nocodazole treatment, the
localization and Triton-X solubility of polaris were determined in
three independent samples by immunofluorescence and Western blot
analysis (Fig. 4A). Nocodazole
treatment resulted in a substantial reduction in the expression of the
highest molecular mass (~118 kDa) polaris isoform; however, the level
of the two smaller isoforms (95 and 100 kDa) remained nearly
constant. In addition, the 95-kDa isoform was still detected in
the Triton-X-insoluble pool. Because nocodazole did not alter the
solubility of the 95-kDa isoform, we analyzed whether it had any effect
on polaris localization. By immunofluorescence analysis, polaris was
still detected in the ciliary axoneme and basal bodies of the
nocodazole-treated cells (Fig. 4B). In contrast, -tubulin
was absent (Fig. 4B).
-Tubulin staining in cilia was not
detected even after extended exposure during image capture. In
non-nocodazole-treated cells,
-tubulin staining and cilia were
evident on almost all cells in the monolayer.
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Generation and characterization of orpk cortical collecting duct
cell lines.
Because we were unable to inhibit or induce excess ciliogenesis by
overexpression of polaris in MDCK cells to study renal cilia function,
we generated several conditionally immortalized cortical collecting
duct cell lines from orpk mutants using the ImmortoMouse (see MATERIALS AND METHODS)
(23). Clonal cell lines were established from single
cells. Results similar to those described here for the 94D line were
obtained for other cell lines derived from these mice. The 94D cells
exhibit a cobblestone appearance typical of epithelial cells under both
permissive and nonpermissive conditions (Fig.
5A). Western blot analysis
indicates that these cells express epithelial markers such as
cytokeratins, whereas fibroblast markers such as vimentin were barely
detectable (Fig. 5B). These cells form adherens and tight
junctions as determined by E-cadherin and ZO-1 expression detected by
Western blot analysis and immunofluorescence (Fig. 5, C and
D, and data not shown). Bioelectrical measurements indicate
that both the mutant and the rescued cell lines form tight monolayers
with transepithelial resistance measurements of 12-18
k/cm2 after 3 days postconfluence on Transwell filters.
The temporal loss of the SV40 large T antigen under nonpermissive
conditions was also evaluated by Western blot analysis. The results
indicate that expression of the SV40 large T antigen was reduced within 3-4 days after the shift to the nonpermissive condition as shown for other renal cell lines derived using the ImmortoMouse (data not
shown) (49).
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Rescue of ciliary defects in 94D cells.
In vivo studies have demonstrated that cilia are malformed on ependymal
cells in the brain and on the renal collecting ducts in orpk
mutant mice (37, 51). To assess whether a similar defect
was occurring in vitro in our cell culture model, we plated rescued
(94D-Tg737Bap-1 and 94D-Tg737Bap-2) and mutant 94D-pcDNA cells at
confluence on permeable filters under nonpermissive conditions for 3 days. The effect of reexpressing wild-type polaris on cilia formation
and morphology was analyzed by immunofluorescence using anti--tubulin antisera and by scanning electron microscopy (Fig. 7, A-C). Using
either of these assays, it was evident that cilia formation was
dramatically inhibited in cells lacking polaris, whereas most cells in
the rescued cultures exhibited overtly normal cilia. Measurements of
cilia length indicate that the cilia on rescued cells are normally
~3-4 µm long (Fig. 7B). Similar to what is seen in
vivo in orpk mice, small rudimentary cilia or small
extensions from the basal body structures were detected on the mutant
cells in these cultures (Fig. 7, A-C)
(37). The cilia on mutant cells were normally <1 µm in
length, and they often exhibited bulging of the axonemal membrane (Fig.
7, B and C). Nearly identical results were
obtained with either 94D-Tg737Bap-1 or 94D-Tg737Bap-2 cells; however,
in general more cilia were found in the 94D-Tg737Bap-2 cultures (data
not shown). This may reflect the higher levels of polaris expression in
the 94D-Tg737Bap-2 line relative to the 94D-Tg737Bap-1 line.
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DISCUSSION |
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In mice, mutations in Tg737 result in a complex series
of phenotypes. The hypomorphic allele in the
Tg737°rpk mutants results in cystic kidney
disease, pancreatic and bile duct hyperplasia, hydrocephalus, and
skeletal patterning defects (33, 42, 58). In contrast, the
complete loss of Tg737 in the
Tg7372-3
Gal null mutants results in
midgestation lethality (~embryonic day 9.5). Null mutants
exhibit random patterning of the left-right body axis, neural tube
defects, pericardial sac expansion, and enlarged limb buds
(34). Polaris, the protein encoded by Tg737, is
highly conserved across ciliated eukaryotes (21). Recent analysis of this protein has revealed that its function is required for
normal cilia and flagella assembly. Ciliary defects have now been
observed in both the hypomorphic and the null Tg737 mutant mice and in lower eukaryotes with mutations in the Tg737
homologues (21, 34, 37, 41, 51).
To begin characterizing the possible association between the cilia and
the cystic kidney disease phenotype in
Tg737°rpk mice, we initiated a study to
analyze polaris expression and localization during ciliogenesis in
renal epithelium using MDCK cells as a model system. The levels of
Tg737 mRNA and polaris expression remained constant in
nonciliated and ciliated MDCK cells, indicating that polaris expression
is not altered by induction of cilia formation. Similar to what was
seen in protein extracts from mice, canine polaris was detected as
multiple bands on Western blots (51). It is unlikely that
these additional proteins are a consequence of cross-reactivity of the
affinity-purified antisera becase these proteins are not detected on
Western blots of protein isolated from Tg737 null mice
(Tg7372-3
Gal) (34, 51). They
may represent differentially modified polaris isoforms; however, the
fact that only a single product is seen in cells exogenously expressing
murine polaris argues against this possibility. It seems more likely
that these protein species are derived from alternatively spliced
Tg737 transcripts. In mice, as many as five Tg737
mRNAs, ranging in size from 2.6 to >7 kb, have been observed
(33, 51, 58). Similarly, longer exposure of the Northern
blot of RNA from MDCK cells also reveals the presence of several less
abundant transcripts; whether these transcripts are derived from the
Tg737 gene remains to be determined.
We further characterized polaris by analyzing changes in its Triton-X
extractability during cilia formation. The results indicate that all
three of the polaris isoforms are restricted to the soluble fraction
for at least 24 h after cells are plated at confluence. However,
after 48 h in culture, a fraction of the 95-kDa form of polaris
was detected in the insoluble pool. Comparing the solubility results
with immunofluorescence data indicates that this shift in solubility is
coincident with basal body formation. Similar solubility results have
been obtained with -catenin,
-catenin, and E-cadherin as the
adherens junction complex forms (1, 2, 39). The
partitioning of the adherens junction proteins into the insoluble
fraction is a result of their attachment to the actin-based
cytoskeleton. Rather than cytoskeletal attachment, we predict that
polaris insolubility is a consequence of its incorporation into the IFT
rafts that assemble near the basal bodies early in ciliogenesis
(9).
To evaluate whether maintenance of polaris insolubility and
localization could involve microtubule filaments such as found in the
cilia axoneme, we treated MDCK cells with nocodazole. Although nocodazole reduced the level of the 118-kDa-molecular-mass polaris isoform, the level of the 95- and 100-kDa isoforms remained constant. In addition, the 95-kDa protein was still detected in the insoluble fraction. Immunofluorescence results indicate that microtubule depolymerization did not alter polaris localization in the ciliary axoneme or basal bodies of treated cells. In contrast, -tubulin could not be detected in the cilia of the nocodazole-treated cells. Thus microtubule filaments appear not to be required for maintenance of
polaris in cilia once they have formed. Whether they are needed for the
initial shift of polaris into the insoluble pool has not yet been
evaluated. The toxicity associated with long-term culture in the
presence of nocodazole to test this possibility has made this analysis
problematic. However, the major role of microtubule-based motor
proteins, such as kinesin and dynein, in the IFT process makes this possible.
The nearly ubiquitous nature of primary immotile cilia on many diverse cell types, including neurons, epithelia, endothelium, and fibroblasts, where they have no obvious function, has led some to speculate that they are vestigial organelles of limited utility or specialization (http://www.wadsworth.org/BMS/SCBlinks/cilia1.html) (13, 29, 53). However, data from the orpk mutants and the localization of other PKD-related proteins in mice and in lower eukaryotes have raised the possibility that ciliary function may be important for normal renal function, development, or differentiation and that disruption of these organelles may result in cystic disease (7, 21, 41, 51). It is also interesting that, in addition to kidney lesions, the pkd-1, pkd-2, cpk, and orpk mutant mice exhibit extrarenal pathologies, often involving the liver and pancreas (17, 20, 27, 33, 55). In most of these cases, the pathology is associated with epithelial cells that have a primary cilium, such as those in bile and pancreatic ductule epithelium (3, 32). Thus alterations in cilia function may also be responsible for the spectrum of extrarenal phenotypes associated with cystic kidney disease.
In the kidney, cilia are present throughout much of the nephron and collecting duct (15, 53). Electron microscopic analysis in rat nephron has detected the presence of a primary cilium on cells of the parietal wall of the renal corpuscle, on the proximal tubules, the thin limb of Henle's loop, the distal tubule, and the collecting duct. Cilia project from the apical surface of the epithelia into the tubule lumen, where they are optimally positioned to function in a sensory capacity. Recent data have suggested that deflection of the primary cilium on MDCK cells can serve as a mechanism to evaluate fluid flow rates (40). The pathway activated in response to the cilium deflection in MDCK cells involves a stretch-activated channel located in the cilium and calcium entry through the axoneme membrane. In addition to mechanosensation, it is possible that renal cilia may function in a chemosensory capacity, such as with cilia found on olfactory neurons of mammals and in C. elegans (26, 45, 46, 52). If renal cilia do function as sensors within the tubule lumen, the cilium length may be a critical factor in signaling efficacy. Thus in orpk mutants, where cilia are severely runted, this mechanism of regulating renal function would be aberrant.
To begin elucidating the biological function of primary cilia and to
determine their possible relationship with cystic kidney disease, we
generated a conditionally immortalized cortical collecting duct cell
line from the orpk mutants. These cells grow as tight monolayers, develop high transepithelial resistance, express markers typical of epithelia, and appear morphologically normal with the exception of their ciliary defects. The cilia that form on these mutant
cells are extremely short and display bulbous extensions of the
axonemal membrane. The continued presence of aberrantly formed cilia
rather than complete loss of cilia on mutant cells most likely reflects
the hypomorphic nature of the Tg737°rpk allele
(34, 51). Similarly malformed cilia are seen in vivo in
orpk mutant kidney and brain, whereas cilia are completely absent in the midgestation embryonic lethal phenotype seen in Tg737 null mutant (Tg7372-3
Gal)
(34, 37, 51). To evaluate the importance of polaris in renal cilia formation, we reexpressed polaris using the rescue construct that corrected the kidney defects in vivo in orpk
mice (56, 57). In contrast to the mutant line, cilia on
almost all of the rescued cells were morphologically normal. These data argue that polaris plays a primary role in the formation of renal cilia
and that loss of cilia is a contributing factor to the renal pathology
in orpk mice.
The primary cilium found on diverse cells ranging from fibroblasts to epithelial cells is a relatively unexplored organelle. The major obstacle to analyzing primary cilia is that they are near the resolution limits of conventional light microscopy, and there have not been cell lines available where cilia formation can be regulated without significantly altering the cellular differentiation state. Because the ciliary defects can be corrected in the 94D cells by reexpressing polaris, we can utilize the rescued and mutant cell lines to explore the possible function of primary renal cilia as mechano- or chemosensors in hopes that it will give insight into the possible mechanism by which cilia loss may result in cystic kidney disease.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Drs. Lisa Guay-Woodford and Susan Sell for critically reading the manuscript. We also thank Shawn Williams of the Univ. of Alabama at Birmingham High Resolution Imaging Facility for assistance in image generation.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 1-RO1-DK-55007-01 (to B. K. Yoder). Additional support was provided by the Polycystic Kidney Research Foundation (99028) and the Medical Research Service of the Department of Veterans Affairs (to D. F. Balkovetz). D. F. Balkovetz is a recipient of a Veterans Affairs Career Development Award.
Address for reprint requests and other correspondence: B. K. Yoder, Dept. of Cell Biology, 1530 3rd Ave. South, MCLM652, Univ. of Alabama at Birmingham, Birmingham, AL 35294-0005 (E-mail: Byoder{at}uab.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00273.2001
Received 4 September 2001; accepted in final form 29 October 2001.
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