The Invs Gene Encodes a Microtubule-Associated Protein
Jens Nürnberger*,
Andreas Kribben*,
Anabelle Opazo Saez*,
Gerd Heusch,
Thomas Philipp* and
Carrie L. Phillips
*Department of Nephrology and Hypertension, and Department of Pathophysiology, University of Essen, Essen, Germany; and Indiana University School of Medicine, Division of Nephrology, Department of Medicine, Indianapolis, Indiana.
Corresponding to Dr. Jens Nürnberger, Department of Nephrology and Hypertension, University of Essen, Hufelandstrasse 55, 45122 Essen, Germany. Phone: 49-201-723-3396; Fax: 49-201-723-3855; E-mail: jens.nuernberger{at}uni-essen.de
ABSTRACT. Microtubule networks are important for many vitalprocesses such as mitosis, cell polarity, and differentiation.Ciliary architecture and function closely depend on the microtubulecytoskeleton, and recent studies suggest a role of apical ciliaof renal epithelia in the pathogenesis of polycystic kidneydisease. This study evaluates the localization, potential interactingpartners, and functional aspects of the Invs gene product inversin.Only recently, INVS has been identified as the gene that ismutated in nephronophthisis type 2, an autosomal recessive polycystickidney disease. Using immunoprecipitation and co-pelleting assays,we show that the Invs gene product inversin forms a stable complexwith tubulin in cultured renal epithelial cells. Inversin localizesto several components of the cytoskeleton including ciliary,random, and polarized microtubule pools. During cell divison,inversin is recruited to mitotic spindle fibers. After microtubuledepolymerization using colcemid inversin and tubulin stainingis no longer characterized by a network pattern but by homogeneous,diffuse distribution. Inversin does not coprecipitate with tubulinafter addition of colcemid. After removal of colcemid, inversinimmunofluorescence reappears together with tubulin in centrioles.Treatment with the microtubule stabilizing agent paclitaxelleads to severe alteration of the microtubule cytoskeleton withbundling and formation of long spindles of tubulin and inversin.In conclusion, inversin is closely associated with the microtubulecytoskeleton, and its spatial distribution is dependent on tubulinpolymerization. Hence, altered inversin-tubulin interactionmay impair ciliary function and thereby contribute to cyst developmentin nephronophthisis.
The primary apical cilium in vertebrate epithelia may be involvedin nephrogenesis (1). This idea is supported by the observationthat several proteins implicated in the pathogenesis of polycystickidney disease (PKD) have been localized to nonmotile ciliain cultured renal epithelial cells (2–4). Mutations ingenes encoding cilia-associated proteins result in a severemalformation of the renal tubular system with epithelial linedcysts in both human genes, such as PKD1 (3,5), PKD2 (6,7), PKHD1(8,9), INVS (4,10), and murine genes (11), including Invs (4,6,12),tgN737Rpw (13,14), and Cys1 (2,15). Mutations in the Invs genehave been directly linked to an autosomal recessive form ofPKD in the inv mouse (6,12) and recently in families with nephronophthisistype 2 (10). The Invs gene product inversin has been localizedto cilia (4,16), nuclear compartments, and cell-cell contacts,where it forms a complex with N-cadherin and the catenins (17).However, inversin’s precise cellular function has notyet been elucidated.
Cilia have been implicated in the pathogenesis of polycystickidney disease (1). Cilia consist of various structural molecules,including microtubules as well as microtubule-associated proteins.The role of these molecules in the pathogenesis of PKD has notbeen well studied. However, abnormalities in microtubule-associatedproteins may lead to the development of cystic kidney. Recently,Lin et al. demonstrated that inactivation of the KIF3A subunitof kinesin-II, a microtubule-dependent motor protein, resultedin polycystic kidney disease in mice. Microtubules are importantcytoskeletal molecules in eukaryotic cells and are involvedin many cellular processes, including morphogenesis, division,and signaling (18–20). The microtubule network integritydepends on its dynamic instability, a process that is characterizedby continuous, rapid polymerization and depolymerization (21).Microtubule assembly is controlled by posttranslational acetylationand detyrosination of tubulin, the major component of the microtubulecytoskeleton (22). In addition, several microtubule-associatedproteins modulate microtubule assembly and stability (23). Classically,these proteins have been described as having two domains, onlyone of which binds to the microtubule network; the other onecan link microtubules to other cell components or may presentbinding sites for regulatory molecules such as the Ca++/calmodulincomplex (23–25).
In the present study, we evaluated the localization, potentialinteracting partners and functional aspects of the Invs geneproduct inversin in polarized renal epithelial cells. We showthat inversin forms a stable complex with the cytoskeletal moleculetubulin. Inversin localizes to various microtubule pools inpolarized epithelial cells and mitotic spindles in dividingcells. Studies with the antimitotic drugs colcemid and paclitaxelsuggest that tubulin polymerization is critical for the spatialdistribution of inversin. Altered microtubule cytoskeleton functionmay contribute to impaired ciliary function in inv mice andpotentially result in cyst development.
Reagents and Chemicals
Mouse monoclonal antibodies to -tubulin, pan-cadherin, and Bcl-2were obtained from Sigma (Sigma-Aldrich Laborchemikalien GmbH,Seelze, Germany). Mouse monoclonal antibody to -catenin waspurchased from Zymed Laboratories Inc. (San Francisco, CA.).All secondary antibodies were obtained from Jackson ImmunoResearchLaboratories, Inc. (West Grove, PA). DAPI Nucleic Acid Stainand rhodamine-phalloidin were purchased from Molecular Probes(Eugene, OR). All other chemicals and reagents were suppliedby Sigma.
A new inversin polyclonal antibody was raised in rabbits usinga recombinant mini-inversin protein containing 153 aminoacidsof the C-terminal end of the full length inversin sequence,using methods that have been previously described (17). Briefly,a 17-kD recombinant mini-inversin protein fragment was expressedfrom a restriction digest fragment of an EST clone that was100% identical to the published Invs sequence (12) and was usedfor immunization. The antibody was purified by immunoaffinitychromatography using the inversin mini-protein coupled to activatedbeads.
Protein Extract Preparation
Confluent Madin-Darby canine kidney (MDCK) cells were washedtwice with cold PBS, scraped from the dish with a rubber policeman,and homogenized in extraction buffer (150 mM NaCl; 50 mM TrisCl,pH 8.0; 4 mM EDTA; 1 mM phenylmethyl sulfonyl-flouride [PMSF];Protease-Inhibitor-Cocktail [Sigma-Aldrich LaborchemikalienGmbH]; Triton X-100 at vol/vol of 1.0%) (17). Cell lysates werecentrifuged at 10,000 x g for 10 min at 4°C, and supernatantswere mixed with Laemmli sample buffer (2% SDS; 100 mM Tris-Cl,pH 6.8; 25% [vol/vol] glycerol; 10 mM DTT; 001% [wt/vol] BromophenolBlue) and boiled for 10 min and loaded on 7.5% SDS-PAGE gels(26).
Immunoprecipitation and Co-Pelleting Assays
Confluent MDCK cells were washed with PBS and incubated on icefor 30 min in HEN-buffer (25 mM NaCl; 50 mM hepes, pH 7.4; 10mM EDTA; 1% [vol/vol] Triton X-100; 1 mM PMSF; Protease-Inhibitor-Cocktail1:100) (27). Cells were scraped from dishes, and insoluble materialwas removed by centrifugation at 800 x g for 5 min at 4°C.The collected supernatant was pre-cleared with protein A-sepharose(Amersham plc, Buckinghamshire, UK). Cell extracts were incubatedwith inversin polyclonal antibody for 2 h. Immune complexeswere recovered by incubation with protein A-sepharose for 2h. Protein A-sepharose beads were washed three times in HEN-bufferbefore protein complexes were released by boiling in Laemmlibuffer for 10 min. Precipitated proteins were separated by SDS-PAGEfollowed by immunoblotting. Co-pelleting assays were performedas described previously (28). Briefly, extracts from MDCK cellstreated with colcemid or paclitaxel were centrifuged at 100.000x g for 20 min (Rotor RP55-S; M120 Microultracentrifuge, Sorvall,Kendro Laboratory Products GmbH, Langenselbold, Germany). Underthese circumstances, microtubules are completely pelleted whileless sedimentable oligomers lead to the appearance of tubulinin the supernatant fractions (29). The supernatant and pelletwere separated, and analyzed by SDS-PAGE and immunoblotting.
Immunoblot Analysis
Proteins were separated on SDS-PAGE gels and transferred tonitrocellulose membranes (Schleicher & Schuell BioScienceGmbH, Dassel, Germany). Membranes were blocked with 3% newborncalf serum (NCS) dissolved in Tris-buffered saline containing0.1% vol/vol Tween-20 (TBS-T) and incubated for 60 min withprimary antibodies diluted in 3% NCS in TBS. Membranes werewashed in TBS-T, incubated with HRP-conjugated secondary antibodiesin 3% NCS in TBS for 60 min and then washed as above. Chemiluminescencewas used for detection (Pierce, Rockford, IL).
Cell Culture, Treatment with Colcemid and Paclitaxel, and Treatment with siRNA
MDCK cells were grown on cover slips in DMEM media supplementedwith 10% fetal calf serum, penicillin, and streptomycin. Confluentlygrown MDCK cells were treated with colcemid (0.2 µg/ml)(30) or paclitaxel (2 µg/ml). After 18 h, cells treatedwith colcemid or paclitaxel were washed twice with medium andallowed to recover for 30 min in medium without colcemid orpaclitaxel as described previously (31). A 21-nucleotide duplexsmall interfering RNA (siRNA) including a 2-nucleotide overhangat the 3'-terminus was used to target Invs mRNA, as describedpreviously (32). MDCK cells were treated with anti-Invs siRNA(5'-CUCUgaUcUgUUcaUagUcUU-3') or scrambled control siRNA for24 h using a transfection reagent (MWG-Biotech AG, Ebersberg,Germany).
Immunohistochemistry and Fluorescence Microscopy
Confluently grown MDCK cells were fixed in 4% paraformaldehydein phosphate-buffered saline (PBS) for 10 min, and subsequentlyquenched in 100 mM NH4Cl dissolved in PBS. Samples were incubatedin blocking buffer (1% BSA, 0.1% Triton X-100 in PBS) for 10min before labeling. Specimens were incubated with rabbit polyclonalantibody to inversin, mouse monoclonal antibody to tubulin,and rhodamine-phalloidin. Primary antibodies were detected byCy5-goat anti-rabbit and FITC-goat anti-mouse–conjugatedsecondary antibodies. All antibodies were diluted in blockingbuffer and washed from filters with 1x PBS. Nuclei were labeledwith DAPI (4',6-Diamidino-2-phenyindole) diluted in PBS. Afterpostfixing in 2% PFA in PBS, coverslips were mounted on slidesand examined with a Zeiss LSM 510 laser scanning microscope(Carl Zeiss Jena GmbH, Jena, Germany) equipped with a UV argonlaser, a visible argon laser and two helium-neon lasers. Imageswere collected using sequential scans to eliminate bleedthrough.Images of cells were analyzed for spatial distribution of fluorescenceintensity using a series of line scanning to quantify pixelintensity. Experiments were performed in triplicate and a representativesample is shown in the results section.
Inversin Localizes to Several Microtubule Pools in Polarized Renal Epithelial Cells
The polyclonal antibody to inversin was characterized by immunoblotanalysis and by inhibition of Invs translation by gene specificsiRNA in MDCK cells (Figure 1). In cells treated with anti-InvssiRNA, there was no inversin immunostaining demonstrating specificityof the inversin antibody.
Figure 1. Characterization of inversin antibody. (A) The recombinant mini-inversin protein used for immunization was separated by 15% SDS PAGE, transferred to membranes, and incubated with serum obtained before (A, Preimmune serum, left lane) and after (A, Postimmune serum, right lane) immunization. (B) Total protein from MDCK cells was separated by 10% SDS-PAGE, transferred to membranes, and incubated with affinity-purified inversin antibody. A strong band was detected at 165 kD with the antibody to inversin alone (left lane), but no signal was detected when inversin antibody was preincubated with the antigen used for immunization (middle lane) or the secondary antibody alone (right lane). MDCK cells were incubated with small interfering RNA (siRNA) specific for Invs (C and D) or with scrambled control siRNA (E and F) for 24 h, and double-stained with antibodies to inversin (C and E) and tubulin (D and F), and examined using confocal microscopy. In cells treated with anti-Invs siRNA, there was no specific inversin-immunostaining signal (C). In the same cells, where inversin was depleted, cilia were observed as shown by tubulin immunostaining (D). Treatment with scrambled siRNA did not affect inversin translation, as indicated by the presence of the inversin immunostaining signal (E).
We evaluated the cellular architecture of MDCK cells by stainingthe microtubule network with monoclonal antibody to tubulinand the actin cytoskeleton with rhodamine-phalloidin. Inversinwas closely co-localized with tubulin in several cellular compartments.In the apical compartment, inversin and tubulin co-localizedin a network-resembling structure (Figure 2, F–J). Thisis consistent with previous characterizations of microfilamentsthat form a web of random microtubules in this cell compartment(see microtubules in red in schematic representation in Figure 2)(33,34). No signal was detected when cells were incubatedwith the inversin antibody in the presence of the antigen usedfor immunization or the secondary antibody alone (Figure 2F,insets).
Figure 2. Inversin is localized to ciliary, random, and polarized microtubules. A schematic diagram (left) illustrates the distinct microtubule pools in polarized renal epithelial cells (ciliary microtubules in yellow; random microtubules in red; polarized microtubules in blue). MDCK cells were labeled for inversin (A, F, K; polyclonal antibody to inversin), tubulin (B, G, L; monoclonal antibody to tubulin), F-actin cytoskeleton (C, H, M; rhodamine-phalloidin), and nuclei (D, I, N; DAPI), and subjected to confocal laser fluorescence microscopy. The upper panel (A–E) shows a horizontal section above the apical cell surface as illustrated by the upper dotted line in the schematic diagram. Inversin (A) co-localized (see composite image E) with tubulin (B) at areas above the cell surface. The center panel (F–J) shows a horizontal section through the apical cell compartment as illustrated by the centrally located dotted line in the schematic diagram. Inversin staining (F) resembled a network structure largely overlapping (see merged image J) with tubulin staining (G). Co-staining with DAPI (I) revealed that this random microtubule-containing network structure was located above nuclei (compare with N showing a section through nuclei). The lower panel (K–O) shows a horizontal section through the basal cellular compartment as illustrated by the lower dotted line in the schematic diagram. Inversin (K) and tubulin (L) staining was localized in a ring-like structure overlapping with the actin belt (M). No signal was detected when cells were incubated with the inversin antibody in the presence of the antigen used for immunization or the secondary antibody alone (F, insets).
In the basal cellular compartment, inversin and tubulin stainingwas localized to the cortical cytoskeleton overlapping withthe actin belt (Figure 2, K–O). This is consistent withthe idea that polarized microtubules form cortical filamentsthat are organized along the apical-basal axis of the cytoskeleton(see microtubules in blue in schematic representation in Figure 2)(33,34).
Both inversin and tubulin, the latter a known constituent ofthe cilia axoneme, strongly co-localized above the apical cellsurface in a fashion typical for cilia (Figure 2, A–E),as described previously (4). A three-dimensionally reconstructedstack of images showed that each cell was equipped with a singlecilium at the apical surface as revealed by staining with anti-inversin(Figure 3) and anti-tubulin (data not shown). Because inversinstaining was strongest in cilia compared with other cellularcompartments, reduction of the overall signal allowed to selectivelyvisualize inversin in cilia.
Figure 3. Inversin localizes to the single apical cilium. MDCK cells were labeled for inversin (polyclonal antibody to inversin, green signal) and nuclei (DAPI, red signal) and examined by confocal laser fluorescence microscopy. A three-dimensional reconstruction shows that the majority of the cells exhibited a single cilium at the apical cell surface. Because inversin staining was strongest in cilia compared with inversin staining in other cellular compartments, reduction of the overall signal allowed selective visualizization of inversin in cilia.
Inversin’s association with tubulin was further confirmedby line scanning analysis, immunoprecipitation, and a co-pelletingassay. Cellular co-distribution of inversin and tubulin wasassessed by quantifying pixel intensity of fluorescence usingline scans along a cross-section of the cell monolayer. Thefluorescence intensity from a single representative line scanis shown in Figure 4A. The close overlap of the curve representingfluorescence intensity of inversin and tubulin reflects thehigh degree of co-localization of both molecules. Immunoprecipitationstudies revealed that inversin is in a complex with tubulin(Figure 4B). Immunoblotting of complexes precipitated from MDCKcells with the inversin antibody contained -tubulin, furthersupporting a close association of both molecules. In addition,we performed co-pelleting assays using extracts from MDCK cellstreated with two anti-mitotic drugs, colcemid or paclitaxel,that affect microtubule architecture (Figure 4C). In extractsfrom colcemid-treated cells, inversin did not pellet in theabsence of polymerized microtubules. Both inversin and tubulinremained in the cytosolic fraction indicated by the presenceof Bcl-2, which was used as a cytosolic marker protein and wasnot expected to co-pellet with tubulin. In contrast, after treatmentwith the microtubule stabilisator paclitaxel, tubulin appearedin the pellet fraction. Inversin co-pelleted in this microtubule-containingfraction, supporting the idea of a close association betweeninversin and polymeric tubulin.
Figure 4. Co-distribution, co-precipitation, and co-pelleting of inversin and tubulin. (A) MDCK cells were stained for inversin (left image) and tubulin (middle image). Yellow color indicates overlap of both molecules in the merged image (right). Fluorescence signal intensities of inversin and tubulin were generated from a scanned horizontal line shown in the merged image. Fluorescence intensity profiles are shown in the bottom panel (red inversin; green tubulin). (B) Immunoblotting for tubulin was established by immunoblot analysis of MDCK cell extract. The tubulin antibody detected a single band of 54 kD in MDCK protein extract (left lane). Homogenates from confluent MDCK cells were immunoprecipitated with inversin antibody, and precipitates were analyzed by tubulin immunoblotting. The tubulin antibody detected a single band of 54 kD in inversin immunoprecipates (middle lane). No band was detected when inversin immunoprecipitation was performed in the presence of the recombinant mini-inversin protein used for immunization (right lane). (C) Extracts from MDCK cells treated with colcemid (left panel) or paclitaxel (right panel) were subjected to a microtubule co-pelleting assay. Pellets (P) and supernatants (S) were analyzed by immunoblotting using antibodies to inversin (top row), tubulin (center row), or Bcl-2 (low row). In colcemid-treated cells, inversin and tubulin remained largely in the cytosolic supernatant as indicated by the presence of the cytosolic molecule Bcl-2. In paclitaxel-treated cells, inversin and tubulin co-pelleted.
Inversin Localizes to Mitotic Spindles in Dividing Cells
Tubulin molecules are recruited during cell division to formmitotic spindles. We examined fluorescently stained cells duringcell division to determine whether inversin may also be associatedwith tubulin to form mitotic spindles. We found that inversinwas localized to mitotic spindles during cell division (Figure 5, A–E).Compared with nondividing cells, the cytoplasmiccompartment in dividing cells showed a lower signal intensityfor inversin and tubulin, suggesting that both molecules arerecruited from the cytoplasm for the formation of mitotic spindles.In cytokinesis, inversin was localized to the midbody, a regionwhere microtubules overlap at the end of cell division (Figure 5, F–I).
Figure 5. Inversin localizes to mitotic spindles in dividing cells. MDCK cells were labeled for inversin (A, E, F; polyclonal antibody to inversin), tubulin (B, E, G; monoclonal antibody to tubulin), F-actin cytoskeleton (C; rhodamine-phalloidin), and nuclei (D, H; DAPI), and examined by confocal laser fluorescence microscopy. During cell division, inversin co-localized with tubulin to spindle fibers (A–E). At the end of cell division, intense inversin (F) and tubulin (G) fluorescence signals are observed at the terminal cytoplasmic connection (arrow in I), a region of microtubule overlap.
Inversin’s Spatial Distribution Depends on Microtubule Assembly
To evaluate whether inversin’s subcellular distributiondepends on tubulin polymerization, we again treated MDCK cellswith colcemid and paclitaxel. In one set of experiments, MDCKcells were incubated with the depolymerizing agent colcemid,which reversibly disassembles polymeric microtubules and mitoticspindles (35). MDCK cells treated with colcemid for 18 h werestained for inversin, tubulin, and DNA and examined by laserconfocal microscopy. Colcemid treatment led to disassembly oftubulin as demonstrated by the diffuse uniform cellular distributionof tubulin (Figure 6F) (36). Similarly, inversin staining incolcemid-treated cells was characterized by a diffuse, homogeneouspattern (Figure 6E) that was strikingly different from the network-likearchitecture observed in control cells (Figure 6, A–D).These results suggest that inversin is associated with polarizedmicrotubules. To further evaluate this association between inversinand polaric tubulin, we immunoprecipiated inversin complexesfrom MDCK cells that were treated with colcemid. We found thattubulin was not present in inversin precipitates obtained fromcolcemid-treated MDCK cells, suggesting that the associationbetween inversin and tubulin was disrupted by colcemid treatment(Figure 6Q).
Figure 6. Effect of microtubule depolymerization with colcemid on the distribution of inversin. (Left) MDCK cells were treated with the microtubule destabilizing agent colcemid (0.2 µg/ml) for 18 h (E–H) and were allowed to recover for 30 min in medium free from colcemid (I–P). Treated cells (E–P) and controls (A–D) were stained for inversin (A, E, I, M), tubulin (B, F, J, N), or DNA (C, G, K, O) and examined by laser confocal microscopy. After treatment with colcemid, inversin (E) and tubulin (F) staining appeared as a diffuse, homogeneous immunofluorescence signal in contrast to the network resembling pattern in controls (A–D). In cells that were allowed to recover (I–P), two overlapping signals of inversin and tubulin immunofluorescence appeared in each cell (arrows in L). Magnification (white rectangle in L) revealed that these inversin (M) and tubulin (N) signals were localized to the perinuclear compartment (O). (Right) Homogenates from MDCK cells treated with colcemid (0.2 µl/ml for 18 h) and controls were immunoprecipitated with inversin antibody, and precipitates were analyzed by tubulin immunoblotting. The tubulin antibody detected a single band of 54 kD in inversin immunoprecipates from MDCK cells that were not treated with colcemid (left lane). In contrast, no band was detected in inversin precipitates from MDCK cells treated with colcemid (right lane).
Disruption of microtubule architecture by colcemid is reversibleand recovers after removal of colcemid cells by assembling microtubulesin the two centrioles in the centrosome (37). MDCK cells treatedwith colcemid (for 18 h) were allowed to recover for 30 minin medium free from colcemid, as described previously (31).In addition to a weak ubiquitous cytoplasmic distribution, inversinand tubulin staining revealed two overlapping small areas inthe perinuclear compartment of each cell (Figure 6, I–P),consistent with the two centrioles where microtubule assemblybegins.
In contrast to colcemid, paclitaxel acts as a microtubule-stabilizingagent, favoring microtubule nonreversible polymerization (38).Microtubule mass increases and bundling occurs with severe spatialdisruption and the creation of multiple spindles. Centrosomesand kinetochores largely lose their capacity to organize microtubuleassembly after treatment with paclitaxel. We evaluated the effectof paclitaxel on the distribution of inversin in MDCK cells.We found that MDCK cells treated with paclitaxel over 18 h exhibitedthese characteristic changes of the microtubule cytoskeleton.Similiarly, inversin distribution was severely altered, withvarious spindle-like structures and bundling partly co-localizingwith tubulin (Figure 7, G–H). Removal of paclitaxel didnot significantly alter the changes in tubulin and inversininduced by paclitaxel treatment (data not shown).
Figure 7. Effect of the microtubule-stabilizing agent paclitaxel on the distribution of inversin. MDCK cells were treated with the microtubule stabilizing agent paclitaxel (2 µg/ml) for 18 h. Treated cells (E–H) and controls (A–D) were stained for inversin (A and E), tubulin (B and F), and DNA (C and G) and examined by laser confocal microscopy. In paclitaxel-treated cells, inversin (G) and tubulin (F) staining was severely altered with bundling and formation of long spindles in contrast to the network-resembling pattern in controls (A–D).
To compare the inversin antibody used in the present study withthe inversin antibody previously published (17), we analyzedprotein complexes immunoprecipitated from MDCK cells (Figure 8).Using immunoblot analysis, we found that the inversin antibodyused in this manuscript precipitated only tubulin. Precipitatesobtained by the previously published antibody contained tubulin,cadherin, and -catenin.
Figure 8. Comparison of the inversin antibody in the present manuscript with the inversin antibody previously published by our group (17). Protein complexes were immunoprecipitated from MDCK cells using the inversin antibody characterized in this manuscript (top row) and the inversin antibody previously published (bottom row). Precipitates were analyzed by immunoblotting using antibodies to tubulin, pan-cadherin, and -catenin. The inversin antibody used in this manuscript precipitated only tubulin. Precipitates obtained by the previously published antibody contained tubulin, cadherin, and -catenin.
Using immunofluoresence confocal microscopy, we found in polarizedrenal epithelial cells that inversin was closely associatedwith tubulin at all sites of polarized microtubules, includingciliary, random, and polarized microtubule pools, and mitoticspindles. We confirmed this close association between inversinand tubulin using immunoprecipitation and co-pelleting assaysindicating that both molecules may be part of a stable complex.Using immunofluorescence and immunoprecipitation, we showedthat the association between inversin and tubulin was lost whenmicrotubules were depolymerized by the anti-mitotic agent colcemid.These data suggest that inversin may be associated with polymerictubulin-forming microtubules but not with free nonpolymerictubulin. We evaluated inversin’s subcellular distributionafter treatment with the microtubule-polymerizing agent paclitaxelto further substantiate the association between inversin andpolaric tubulin. Severe disruption of the microtubule cytoskeletoninduced by paclitaxel led to a concomitant alteration in thesubcellular distribution of inversin, with the formation ofbundles and multiple spindles. These parallel changes in tubulinand inversin after paclitaxel treatment support the close associationbetween inversin and polaric tubulin. In cells recovering frommicrotubule disruption with colcemid, we observed that inversinis present in the centrioles, a region of the centrosome wheremicrotubule assembly begins. This suggests that inversin maybe involved during early microtubule assembly in microtubule-organizingcenters.
The immunofluorescence signal of anti-tubulin and anti-inversinlabeled cilia seemed to disappear in cells treated with paclitaxel.This may be due to the dramatic structural changes of the microtubulecytoskeleton in these cells. Jensen et al. (35) previously reportedthat paclitaxel did not affect the structure of primary ciliaformed before the treatment with this drug as evaluated by electronmicroscopy.
To date, inversin has been localized to cilia (4,16), mitoticspindles, midbodies, and centrioles (4). In addition to observinginversin in these cellular organelles, we also found that inversinis associated with the entire microtubule cytoskeleton, includingrandom and cortical microtubules. This association was maintainedeven when microtubule function was severely disturbed with paclitaxel.These data suggest that inversin may have a role in maintainingthe integrity and normal architecture of renal polarized cells.
In a previous study, we reported inversin isoforms of 140, 125,and 90 kD (17). In that report, these isoforms were confirmedby mass spectrometry. We propose that alternative splicing andposttranslation modifications may account for several inversinisoforms. Morgan et al. (12) described alternative splicingin exon 13 of the full-length Invs sequence predicting threeisoforms of 99, 104, and 118 kD. The existence of splicing isoformsis further supported by the study of Schön et al. (39),who described two bands (4.2 and 3.5 kb) on Northern blots fromseveral tissues. The inversin protein may also undergo severalposttranslational modifications, including phosphorylation (50serine, 11 threonine, and 6 tyrosine) as well as type O-glycosylation(11 serine and 2 threonine) (17). We found that the Invs geneproduct inversin is expressed in different cellular compartmentssuch as cell junctions and cilia (17). This phenomenom of geneexpression in different cellular compartments has also beendescribed for various other PKD-associated gene products, includingpolycystin-1, polycystin-2, and nephrocystin (3,40–42).
We found that the antibody used in the present study did notprecipitate catenin and cadherin but only tubulin, suggestingthat a 165-kD inversin isoform may not interact with cell adhesionmolecules. In contrast, our previously published anti-inversinantibody precipitated catenins, cadherin, and tubulin. Thatantibody, in addition to identifying 140-, 125-, and 90-kD bandson immunoblots, also immunoprecipitated a 165-kD inversin isoformthat was not detected on immunoblots. It is possible that thepreviously published inversin antibody precipitated tubulinas a result of precipitating the 165-kD inversin. Because theantibody reported in the present manuscript did not precipitatecell adhesion molecules, we believe that different inversinisoforms may belong to various cellular protein pools with differentfunctions.
Both antibodies were generated to the same recombinant protein,but their immunostaining as well as their immunoblot patterndiffered. The recombinant protein used for immunization wasa relatively large antigen (17 kD, 153 amino acids). Longerproteins increase the number of available epitopes; therefore,the fusion protein used for antibody generation may have elicitedtwo different antibody responses in the two different rabbitsimmunized. We believe that such different immunological responsesmay account for the varying detection patterns of the two antibodies.
Structural Properties of Inversin Suggest a Potential Role as a Microtubule-Associated Molecule
Microtubule assembly and stability is closely controlled bynumerous microtubule-associated proteins (23) including tau(43), EMAP (44), STOP (45), and several MAPs (23). The activityof these microtubule-associated proteins is frequently modulatedby other regulatory molecules. One such common regulatory moleculeis the Ca++/calmodulin complex that controls microtubule-associatedproteins directly (23,24) or indirectly via dependent proteinkinases (25). The inversin protein has two calmodulin bindingsites of the IQ type (46), and Yasuhiko et al. have shown inin vitro experiments that calmodulin binds to inversin (47).These data suggest that inversin may be regulated by the Ca++/calmodulincomplex.
Inversin’s N-terminal end contains 15 highly conservedtandem repeats of the ankyrin motif (6). Ankyrin repeats area common motif and are known to function as protein-proteininteraction domains in numerous molecules, including cytoskeletalproteins. Tubulin has been described as a molecule that caninteract with ankyrin repeats (48). According to Davis et al.(49,50), two members of the ankyrin family exhibit binding sitesfor tubulin. Hence the ankyrin motif in the Invs gene sequencemay be involved in the interaction between inversin and tubulin.Inversin and tubulin are well-conserved molecules in variousspecies. The Invs gene has been sequenced in mice (6,12) andhumans (39), and Invs transcripts have been identified in otherspecies, including chicken, zebrafish, C. elegans, and frog.Sequence analysis reveals that the ankyrin repeat motif of theInvs sequence is highly conserved (46), similar to that of tubulin.
Potential Cellular Function of Inversin
Mutations in the INVS gene lead to defects of left-right axisdevelopment in humans (10) and mice (6,12). Mice homozygousfor a mutation in the Invs gene develop a situs inversus andpolycystic kidneys (6,12). In the inv mouse, a reduced leftwardflow of the extraembryonic fluid has been observed and has beenimplicated in the pathogenesis of the situs inversus (51,52).These defects suggest that inversin has a potential role inleft-right morphogenesis and renal development. Both phenotypeshave been pathogenetically linked to ciliary function. Ciliafunction closely depends on its structural integrity. In fact,knocking out the KIF3A subunit of kinesin-II, a microtubule-dependentmotor protein, results in polycystic kidney disease and perturbationsof the left-right asymmetry in mice (53). Hence our study supportsthe idea that microtubule-associated proteins may be involvedin left-right morphogenesis and renal development by maintainingproper ciliary function. It is unknown whether inversin is involvedin stabilizing microtubules and whether loss of microtubulestability may impair ciliary function, ultimately leading toperturbation of left-right morphogenesis and tubulogenesis inthe inv mouse.
We recently reported that other inversin isoforms localize toseveral subcellular compartments, including nuclei, cytoplasm,and cell-cell junctions in cultured renal epithelial cells (17).At cell-cell junctions, a 125-kD inversin isoform interactswith adhesion molecules, including the catenins and one memberof the cadherin family (17). It is possible that these shorterisoforms may be involved in linking the microtubule cytoskeletonto other cellular structures such as adherens junctions.
In summary, we have shown that inversin is closely associatedwith the microtubule cytoskeleton. Tubulin polymerization appearsto be critical for the spatial distribution of inversin. Impairedfunction of the microtubule cytoskeleton may contribute to impairedciliary function, which in turn may lead to situs inversus andpolycystic kidneys in the inv mouse.
Acknowledgments
JN acknowledges funding from the Deutsche Forschungsgemeinschaft(Nu 118/1–1, Nu 118/3–1). AK acknowledges fundingfrom the Deutsche Forschungsgemeinschaft (Kr 1108/2–2).CLP acknowledges support from the National Institutes of Health(NIH K08 DK02785), George Schreiner MD Young Investigator Grantof the National Kidney Foundation, Polycystic Kidney DiseaseResearch Foundation (99023), Clarian Health Values Fund (VFR21),and the Ralph W. and Grace M. Showalter Research Trust Fund.
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Received for publication December 3, 2003.
Accepted for publication March 24, 2004.
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Abstract
Introduction
-tubulin, pan-cadherin, and Bcl-2 were obtained from Sigma (Sigma-Aldrich Laborchemikalien GmbH, Seelze, Germany). Mouse monoclonal antibody to 
