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Published as doi: 10.1096/fj.06-7786com.
 (The FASEB Journal. 2008;22:168-182.)
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(The FASEB Journal. 2008;22:168-182.)
© 2008 FASEB

β-tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure

Tamar Shultz, Miriam Shmuel, Tehila Hyman and Yoram Altschuler1

Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Israel

1 Correspondence: Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Israel. E-mail: yoram11{at}md.huji.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In epithelial cells, the apical junctional complex (AJC), composed of tight junctions (TJs) and adherens junctions (AJs), maintains cell-surface polarity by forming a fence that prevents lateral movement and diffusion of proteins and lipids between the apical and basolateral PM and holds the epithelial monolayer intact through cell–cell contacts. Disassembly of this complex is a prime event in development and cell transformation. Maintenance of the AJC has been shown to involve mainly the actin cytoskeleton. Recent findings also point to the involvement of the microtubule (MT) system. Here we show the first evidence that in polarized epithelial MDCK cells, ARF-like protein 2 (ARL2) and β-tubulin cofactor D, known to be involved in MT dynamics, have a role in disassembly of the AJC followed by cell dissociation from the epithelial monolayer, which is not dependent on MT depolymerization. In addition, we show that β-tubulin cofactor D is partially localized to the lateral PM through its 15 C-terminal amino acids and intact MTs. ARL2 inhibited β-tubulin cofactor D-dependent cell dissociation from the monolayer and AJC disassembly. To our knowledge, this is the first evidence that β-tubulin cofactor D plays a role in cells independent of its presumed role in folding tubulin heterodimers. We conclude that ARL2 and β-tubulin cofactor D participate in AJC disassembly and epithelial depolarization.—Shultz, T., Shmuel, M., Hyman, T., and Altschuler, Y. β-tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure.


Key Words: MDCK • tight junction • adherens junctions


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN EPITHELIAL CELLS, THE APICAL junctional complex (AJC), composed of tight junctions (TJs) and adherens junctions (AJs), maintains cell-surface polarity by forming a fence that prevents lateral movement of proteins and lipids between the apical and basolateral PM and maintains epithelial monolayer cell–cell contacts (1 , 2) . Disassembly of this complex is a prime event in development and cell transformation. Maintenance of the AJC has been shown to involve mainly the actin cytoskeleton (3) . Recent findings also point to the involvement of the microtubule (MT) system (4) .

In polarized epithelial cells, MTs anchor along the lateral PM with their minus end toward the apical domain and plus end toward the basal membrane, thereby creating a polarized orientation. In addition, columnar epithelial cells reveal a network of nonpolarized MTs under and parallel to the apical PM (5 6 7 8 9) . The minus end, which anchors and stabilizes the MTs, has previously been shown to depend on E-cadherin expression in fibroblasts and the generation of cell–cell contacts (10 , 11) . MT polymerization and depolymerization are regulated in part by the availability of {alpha}-β-tubulin heterodimers. These heterodimers are generated through a complex multi-step process involving several chaperones, one of which is β-tubulin cofactor D. This latter protein participates in the final folding step that generates the {alpha}-β-heterodimer, and it sequesters β-tubulin from the native tubulin dimer, resulting in MT catastrophe (12 , 13) . ARF-like protein 2 (ARL2) was found to associate with β-tubulin cofactor D in nonpolarized cells and to inhibit β-tubulin cofactor D-dependent MT destruction as well as β-tubulin sequestration from the native tubulin dimer. Later, it was realized that the ARL2-β-tubulin cofactor D complex contains the heterotrimeric protein phosphatase 2A (PP2A), producing a 300 kDa complex (14 , 15) . Additionally, PP2A was shown to be involved in disassembly of TJs (16) .

The TJ complex consists of transmembrane proteins (occludin, claudin, and JAM) and membrane-associated proteins (ZO1, -2, and -3; PAR3 and -6; and aPKC) (17 , 18) . The membrane-associated proteins serve as a link between the TJ and the cytoskeleton and cellular-signaling machinery. The association of the TJ with the actin cytoskeleton is well documented and is vital for TJ assembly and integrity. An indication of the association of the MT system with the TJ emerged from work by Ivanov and co-workers that showed partial colocalization of tubulin with actin at the TJ and kinesin 1 colocalization with occludin. In addition, application of nocodazole, a MT-depolymerizing drug, resulted in stabilization of the TJ. Moreover, nocodazole inhibited TJ disassembly during a calcium-dependent TJ disassembly experiment. These results indicate that MTs actively participate in regulating the disassembly of TJs and their loss inhibits TJ disassembly (4 , 19) .

In this study, we provide the first line of evidence that in polarized MDCK epithelial cells, the ARL2-β-tubulin cofactor D complex has a dual function. In addition to its known role in regulating MT dynamics, it also regulates the AJC assembly–disassembly process. Expression of β-tubulin cofactor D causes disassembly of the TJ and AJ followed by cell dissociation from the epithelial monolayer, while coexpression with the small GTPase ARL2 in its GDP bound form inhibits this process. The activity and localization of β-tubulin cofactor D are dependent on its 15 C-terminal amino acids and intact MTs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals were purchased from Sigma-Aldrich (Rehovot, Israel) unless otherwise indicated. Growth media were from Biological Industries (Beit Haemek, Israel). All fluorescent secondary antibodies were from Molecular Probes (Eugene, OR, USA). Flag antibody and anti-{alpha}- and anti-β-tubulin were from Sigma (St. Louis, MO, USA). 12CA5 was from Covance (Berkeley, CA, USA). Rat monoclonal antibody against ZO1 was obtained from Chemicon International (Temecula, CA, USA). Rabbit anti ARL2 was kindly provided by JC Zabala (University of Cantabria, Spain), and mouse monoclonal anti E-cadherin was kindly provided by Barry Gumbiner (University of Virginia, Charlottesville, VA, USA). All fluorescent secondary antibodies were from Molecular Probes. Secondary antibodies conjugated to HRP were from Jackson Immunoresearch (West Grove, PA, USA). All images were compiled using Adobe Photoshop and/or Canvas software (ACD Systems International Inc., Saanichton, British Columbia, Canada) and are representative of the original data. Western-blot quantitation was performed with NIH image software (Bethesda, MD, USA).

Construction of recombinant adenovirus
All recombinant DNA steps were performed using standard techniques. ARL2 was obtained from the UMR cDNA Resource Center (www.cdna.org) and cloned into adenoviral vector pADtet 7 (20) . ARL2 was mutated to generate dominant-negative and active mutants (T30N and Q70L, respectively) using the QuickChange II site-directed mutagenesis kit from Stratagene (La Jolla, CA, USA) as described previously (14) . β-tubulin cofactor D cDNA was amplified from bovine brain cDNA using the primers based on bovine β-tubulin cofactor D accession number U61233 (5' primer containing EcoRI site, flag tag followed by three glycines and the β-tubulin cofactor D sequence: cct cgg gaa ttc acc atg gat tat aag gac gat gac gat aag gat ggt ggt ggt ggc gtg cct aga ccg cag ctg gtt ccc cct cgg gaa ttc acc atg gat tat aag gac gat gac gat aag gat ggt ggt ggt ggc gtg cct aga ccg cag ctg gtt ccc and 3' primer containing NotI and XbaI sites: aag gaa aaa gcg gcc gct cta gat cag cgg aca gca ggc ttg gga acc agc) followed by digestion with EcoRI and XbaI, and cloning into pADtet 7 to obtain N-terminal flag-tagged β-tubulin cofactor D. To obtain β-tubulin cofactor D lacking the 15 C-terminal amino acids, the 3' primer was replaced with the following primer that amplifies the cDNA lacking the 45 3' nucleotides (3' {Delta} 15 primer containing NotI site: aag gaa aaa gcg gcc gct cta gat cag cgg aca gca ggc ttg gga ac). The mutant cDNA was amplified using the 5' and 3' {Delta}15 primer to generate a 3.5 kbp DNA fragment. The fragment was digested with AgeI and NotI and a 700 bp fragment was excised and used to replace the 3' fragment in the wild-type (WT) β-tubulin cofactor D cDNA to generate a mutant lacking the 15 C-terminal amino acids.

Cell culture and adenoviral production
MDCK cells were grown as described previously (20) . Production of adenovirus in HEK293-cre recombinase-expressing cells and use of adenovirus in MDCK cells were as described previously (20 , 21) . Protein levels were regulated by the concentration of doxycycline (Dx), the amount of virus, and the length of time following infection and/or Dx removal. Cells infected by recombinant adenovirus were incubated for the indicated times, ranging between 3 and 19 h, to express the recombinant proteins (ARL2, β-tubulin cofactor D, and mutants). We verified all heterologous expression by means of immunoblot assay to ensure that coinfection of ARL2 and β-tubulin cofactor D does not cause reduction of any one of the proteins over the other or compared to their expression alone. Controls in all experiments included cells that were i) not infected; ii) infected, but the expression of ARL2 or β-tubulin cofactor D was fully repressed by 60 ng/ml Dx; or iii) infected with a control virus encoding β-galactosidase. These caused complete loss of the ARL2- or β-tubulin cofactor D-specific signal in immunofluorescence and biochemical studies. In experiments containing nocodazole or taxol (33 µM and 10 µM, respectively), cells were incubated with the drug 3.5 h after infection. Expression of β-tubulin cofactor D was not affected by either drug treatment.

Viability assay
Cell viability was quantified by the colorimetric MTT (Biological Industries) assay, which measures mitochondrial activity in viable cells (22) . This method is based on the conversion of the MTT to MTT-formazan crystal by mitochondrial enzyme. In brief, MDCK tet off cells were grown in a 96-well plate for 3 days and subsequently infected to express β-tubulin cofactor D for 18 h or infected with β-tubulin cofactor D and expression suppressed by addition of 60 ng/ml dox (Dx) (control). Infection was performed with 0.07, 0.2, 0.66, 2, and 6X the amount of β-tubulin cofactor D adenovirus (1X the amount of virus equals 60 to 90 PFU/cell). MTT assay was performed according to manufacturer’s instructions.

Immunofluorescence of MDCK cells
These procedures were performed as described (20) . Images were taken using a NIKON TE-2000S (Melville, NY, USA) inverted fluorescence microscope with a plan Apo 60X objective lens (Nikon), equipped with a Z stepper and a Hammamatzo CCD ORCAII camera (Hammamatzo, Tucson, AZ, USA). Images were all deconvolved with SimplePCI software (Improvision, Coventry, UK). All images were compiled using Adobe Photoshop and/or Canvas software (ACD Systems International Inc.), and are representative of the original data.

MDCK cell dissociation from epithelial monolayers
MDCK cells were grown as confluent monolayers for 3 days and were then infected with the indicated adenovirus and incubated for the indicated time points to enable expression of either ARL2 or β-tubulin cofactor D or both. At the indicated time points, the media were collected and attached cells were trypsinized. Cells from both fractions were counted in triplicate using a Z1 Beckman Coulter counter (Fullerton, CA, USA). Cell numbers in both attached and dissociated fractions were used to calculate the percentage of detached cells.

Immunoprecipitation
A standard protocol was used for immunoprecipitation of ARL2 and β-tubulin cofactor D. In brief, cells were lysed in immunoprecipitation RIPA buffer containing 50 mM Tris (pH 7.4), 135 mM NaCl, 1% (v/v) Triton X-100, and 60 mM octylglucoside and supplemented with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 mM diisopropyl fluorophosphate, 5 µg/ml pepstatin, and 1 mM EDTA). Lysates were cleared by centrifugation at 12,000 g for 30 min at 4°C. Supernatants were incubated with 12CA5 anti HA (ARL2) tag antibodies (1 µg) and protein A-Sepharose beads (20 µl of packed beads) at 4°C for 1 h. At the end of incubation, beads were washed 5 times with lysis buffer. The resulting immunoprecipitated immunocomplexes were solubilized in 40 µl of sample buffer, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. The protein complex was detected with anti flag antibody (β-tubulin cofactor D) in Western blot analysis and developed by ECL (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Calcium switch of MDCK cell epithelial monolayers
The protocol was according to (23) . In brief, cells expressing a reduced level of β-tubulin cofactor D for 19 h were transferred to growth media lacking calcium and containing 4 mM EGTA for 60 min at 37°C in a 5% CO2-containing environment. Cells were washed extensively with PBS for 30 min to remove EGTA and either fixed or incubated with complete growth media (recovery) for the indicated time points. Subsequently, cells were fixed and processed for immunofluorescence.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The high complexity of epithelial MTs contributes to epithelial polarity and to the maintenance of protein complexes such as TJs, AJs, and focal adhesions, which are the principal components of the epithelial structure. To study the physiological effect of MT assembly and disassembly, we expressed two important proteins in this process: β-tubulin cofactor D and the small GTPase ARL2. The 130 kDa β-tubulin cofactor D has been previously shown to serve as a chaperone in the assembly of β-tubulin with {alpha}-tubulin to form a dimer, which then polymerizes to generate MTs. In addition, β-tubulin cofactor D has been shown to play a role in the regulation of MT dynamics in all eukaryotes tested to date (24 25 26 27) . In nonpolarized cells, ARL2 associates with β-tubulin cofactor D and inhibits its effects on MT instability and {alpha}-tubulin degradation. We wanted to further elucidate the role of β-tubulin cofactor D in polarized MDCK epithelial cells. Using the published sequences and PCR, we cloned β-tubulin cofactor D from bovine brain cDNA. For expression in MDCK cells, we subcloned the N-terminally flag-tagged cDNA coding for β-tubulin cofactor D into an adenovirus vector and generated a recombinant adenovirus that harbors the wild-type (WT) β-tubulin cofactor D, as well as one harboring a mutant lacking the 15 amino acids expected to associate with tubulin. Infection of MDCK cells with both recombinant adenoviruses followed by Western blotting revealed a 130 kDa protein that fit the expected published size of β-tubulin cofactor D (Fig. 1 A). To establish the role of β-tubulin cofactor D in the epithelium, we assayed its ability to degrade {alpha}-tubulin while β-tubulin expression remains unaffected (28) . As shown in Fig. 1B , expression of the WT form of β-tubulin cofactor D resulted in a drastic reduction of {alpha}-tubulin in the cell. We did not manage to generate specific antibodies to β-tubulin cofactor D and, therefore, we could not estimate the level of overexpression of the recombinant protein compared to its endogenous counterpart. To avoid toxic effects as well as unnecessarily high overexpression of β-tubulin cofactor D, we performed all of the experiments in a dose-dependent manner, with the highest level of expression being that which causes {alpha}-tubulin degradation. This fit our overexpression to previously published levels (14 , 15 , 29) . Moreover, expression of increasing amounts of β-tubulin cofactor D by infection with increasing amounts of adenovirus resulted in increased amounts of β-tubulin cofactor D expression correlated with increased degradation of {alpha}-tubulin (Fig. 1C, D ). In Fig. 1E , we show an immunofluorescence image of MTs in polarized MDCK cells. Control cells show the typical epithelial MT structure, while the β-tubulin cofactor D-infected cells illustrate total loss of MT structure with β-tubulin staining remaining as small puncta and total absence of {alpha}-tubulin (Fig. 1E , cells marked by asterisk). Some cells showed an intermediate stage of MT depolymerization in which MT filaments are not directed from the apical domain toward the lateral PM (Fig. 1E , marked by two asterisks). A small fraction of the cells revealed a MT-degradation pattern, which involved shortening of the MT toward the MT organization center (Fig. 1E , bottom panel, cell marked with two asterisks). Therefore, as in the nonpolarized HeLa cells, in polarized MDCK cells, β-tubulin cofactor D expression also results in {alpha}-tubulin degradation while not affecting β-tubulin expression levels (29) . Moreover, this observation supports our contention that the β-tubulin cofactor D adenoviral expression system operates similarly to previously described β-tubulin cofactor D expression systems (14 , 15 , 29) .


Figure 1
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  		Figure 1. Expression of β-tubulin cofactor D results in {alpha}-tubulin degradation and β-tubulin protection. MDCK tet off cells were grown as a confluent monolayer for 3 days. Subsequently, control cells (noninfected or infected and kept with doxycycline to suppress expression) and cells infected with adenoviruses to express β-tubulin cofactor D or its deletion mutant were processed for immunoblotting or immunofluorescence. A) Immunoblot reacted with antiflag to show expression of wild-type β-tubulin cofactor D and its deletion mutant. Western blot reveals the expected 130 kDa protein, the expression of which is suppressed by doxycycline. B) Immunoblot reacted with either anti-{alpha}- or anti-β-tubulin shows the degradation of {alpha}-tubulin and protection of β-tubulin. C) Dose-dependent expression of β-tubulin cofactor D results in dose-dependent degradation of {alpha}-tubulin. The increased expression of β-tubulin cofactor D was achieved by increasing the concentration (indicated) of applied adenovirus. D) Immunoblots from (C) were scanned and densitometry performed to correlate the level of β-tubulin cofactor D expression (black bar) to {alpha}-tubulin degradation. E) Microtubules (MTs) traced by anti-{alpha}- and anti-β-tubulin antibodies (indicated) in cells expressing β-tubulin cofactor D. Control cells show typical MT morphology in polarized MDCK cells. Cells that reveal partial MT degradation (indicated by two asterisks) show disorganized filamentous structure of the MTs. Complete degradation of the MTs (indicated by one asterisk) is seen as small puncta in cells traced with anti-β-tubulin and no staining in cells stained with {alpha}-tubulin. The experiment was repeated five times. Scale bar = 2 µM.

β-tubulin cofactor D promotes cell detachment from the substratum and neighboring cells
We noticed that expression of β-tubulin cofactor D causes substantial detachment of MDCK cells grown on plates, glass coverslips, or transwells. This phenomenon was significantly more pronounced than expected from infection by other recombinant adenoviruses. We thus performed experiments to establish the role of β-tubulin cofactor D in cell detachment. In Fig. 2 A, we show a phase-contrast image of MDCK cells generating a standard epithelial monolayer (control). Within 3 h of β-tubulin cofactor D expression, cells begin to round up and detach from the epithelial monolayer (Fig. 2A , 3 to 18 h, arrows). With time, the number of rounded (Fig. 2A , arrows) and detached cells increases. Within 9 h of expression, the ability to clearly view the cell border diminishes (Fig. 2A , bottom right panel, enlarged view of cell borders). Within 18 h of expression, most cells have detached, and the remaining attached cells reveal a fibroblastic morphology (Fig. 2A , 18 h, arrowheads). To quantitate this phenomenon, we counted the cells in the media as well as the attached cells at the indicated time points and calculated the fraction of detached cells. As show in Fig. 2B , the number of detached cells in the medium increased with time and within 24 h of expression, 90% of them had detached from the substratum. To exclude the possibility that we are observing a side effect of either excess protein expression or detachment related to the amount of adenovirus, we silenced the expression of β-tubulin cofactor D by adding Dx, which inhibits its expression and reveals adenoviral side effects: as a consequence, cell detachment decreased to control levels (Fig. 2C , control). We also performed a β-tubulin cofactor D dose-response experiment in which we observed a correlation between the amount of β-tubulin cofactor D expression and the amount of cells detached from the substratum (Fig. 2C ). Additionally, to exclude the possibility of cell detachment being due to, and provoked by, cell death, we performed an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and found that at no level of expression do the attached cells show any sign of cell death (Supplemental Fig. 1). The viability levels of cells expressing β-tubulin cofactor D were similar to those of uninfected control cells and of cells infected with the β-tubulin cofactor D adenovirus whose expression was inhibited by the addition of Dx as determined by MTT assay (Supplamental Fig. 1). Together, these results indicated that cell detachment is due to the expression of β-tubulin cofactor D rather than reflecting an adenoviral effect or an overexpression artifact. To correlate the cell detachment to the establishment of adherence and epithelial cell polarity, we expressed a reduced amount of β-tubulin cofactor D in cells that had been allowed to adhere and establish polarity for different periods of time. Cells that established adherence and polarity for 1 to 2 days showed significant cell detachment, whereas those that were allowed to adhere and polarize for 3 days or more showed less detachment (Fig. 2D ). This result indicated that a factor critical to cell adherence is absent, present in a reduced amount, or not yet properly localized within the first 2 days of adherence.


Figure 2
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  		Figure 2. Expression of β-tubulin cofactor D results in the dissociation of cells from polarized epithelial MDCK monolayer. MDCK tet off cells were grown as a confluent monolayer for 3 days. Subsequently, control cells (infected with β-tubulin cofactor D and expression suppressed by addition of 60 ng/ml doxycycline) and cells expressing either β-tubulin cofactor D (A–D) or β-tubulin cofactor D lacking the 15 C-terminal amino acids (B–C) were incubated for the indicated times. A) Microscopic images of epithelial MDCK monolayers expressing β-tubulin cofactor D for the indicated periods reveal increased dissociation of cells from the epithelial monolayer with time of expression. In parallel, the cell junction disappears (compare CoD [β-tubulin cofactor D] to control enlargement at bottom right). Arrows point to cells prior to their dissociation. Arrowheads point to cells with fibroblastic extensions. B) Epithelial monolayers expressing either β-tubulin cofactor D or its mutant lacking the 15 C-terminal amino acids were harvested, and attached cells were trypsinized and counted at the indicated time points. The percentage of cells dissociated from the monolayer is shown. β-tubulin cofactor D is seen to cause substantial dissociation of cells from the epithelial monolayer. C) Epithelial monolayer expressing four different doses of either β-tubulin cofactor D or its mutant lacking the 15 C-terminal amino acids was harvested, and attached cells were trypsinized and counted following 18 h expression. Control cells were infected with the 3x concentration and incubated with 60 ng/ml doxycycline (Dx) in the media to inhibit expression. The number of dissociated cells in these controls was similar to that in cells that were not infected. The percentage of dissociated cells from the monolayer is shown. A dose-response effect was observed for both wild-type and mutant forms of β-tubulin cofactor D. The deletion of 15 C-terminal amino acids reduced the cell dissociation considerably in both the time course experiment (B) and the dose-response experiment (C). D) Epithelial monolayer which was polarized for 1 to 4 days expressed reduced amounts of β-tubulin cofactor D for the last 18 h. Dissociated and attached cells were counted, and the percentage of cells dissociated from the monolayer is shown. β-tubulin cofactor D is shown to cause substantial dissociation of cells during the first days of polarization, indicating that following 3 and 4 days of polarization development, the cells acquire some resistance to cell dissociation. Experiments were repeated five times.

ARL2 antagonizes the effect of β-tubulin cofactor D on cell detachment
Bhamidipati and co-workers found that in nonpolarized HeLa cells, ARL2 in its GDP-bound form binds to β-tubulin cofactor D, in vitro and in vivo, and that coexpression of WT ARL2 or an ARL2 mutant defective in GTP-binding specifically prevents the destruction of tubulin and MTs caused by expression of β-tubulin cofactor D. In addition, a mutant ARL2 that does not bind GDP (carrying a mutation in its putative effector loop, which is necessary for its GDP-bound conformation) failed to bind β-tubulin cofactor D or rescue MTs from destruction (14) . To test whether ARL2 participates together with β-tubulin cofactor D in {alpha}-tubulin degradation and epithelial cell detachment, we generated recombinant adenoviruses containing N-terminally HA-tagged ARL2 in its WT form as well as in its GDP (T30N) or GTP (Q70L) mutant forms and expressed them in polarized MDCK cells (Fig. 3 A). When expressed on their own, neither ARL2-WT nor its two mutants had any effect on {alpha}-tubulin degradation or cell detachment (Fig. 3B-D ). In accordance with the results in HeLa cells (14) , β-tubulin cofactor D-dependent {alpha}-tubulin degradation was reduced when β-tubulin cofactor D was coexpressed with either the WT or the GDP-bound mutant of ARL2. Unlike the work of Bhamidipati and co-workers (2000), however, we found that in polarized MDCK cells, the GTP (Q70L) mutant of ARL2 also inhibits {alpha}-tubulin degradation. Nevertheless, a higher degree of expression of ARL2-Q70L was required to protect {alpha}-tubulin from degradation (Fig. 3A ). We reasoned that cell detachment might be related to MT degradation. We, therefore, coexpressed ARL2 and β-tubulin cofactor D and measured the number of detached cells. In Fig. 3B-D , we show that ARL2 in its WT form (46% inhibition; P=0.00004), and to a greater extent in its GDP-bound form (ARL2-T30N) (55% inhibition; P=0.006), inhibits MDCK cell dissociation from the epithelial monolayer, while the GTP-bound form showed statistically insignificant inhibition (11% inhibition P=0.05). These results are in accordance with the inhibitory effect of ARL2 and its mutants on {alpha}-tubulin degradation. Nevertheless, the marked difference between the WT and GDP-bound form compared to the GTP-bound form on cell detachment is not reflected in the inhibition of {alpha}-tubulin degradation. We, therefore, reason that the two phenomena are in mostly unrelated. We aimed to localize ARL2 and its mutants in polarized MDCK cells by immunofluorescence microscopy. To establish a direct association between ARL2 and β-tubulin cofactor D similar to that shown in HeLa cells (14) , we performed a coimmunoprecipitation experiment. In Fig. 3E , we show that ARL2 in its GDP-bound form (T30N) associates tightly with β-tubulin cofactor D; in contrast, only a very weak association is observed for its WT or GTP forms. These results indicate that inhibition of cell dissociation may be achieved by direct association between ARL2 in its GDP-bound state and β-tubulin cofactor D. In accordance with the inhibitory effect of ARL2-WT on cell detachment (Fig. 3B ), we also expected ARL2-WT and β-tubulin cofactor D to associate (Fig. 3E ). The observed lack of association may be due to the documented exceptionally rapid GTP nucleotide binding to ARL2 (in contrast to other small GTPases) and its intrinsic high GAP activity. These would result in a high on-off rate of ARL2-WT with β-tubulin cofactor D (30) .


Figure 3
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  		Figure 3. ARL2 antagonizes the β-tubulin cofactor D effect on cell detachment. MDCK tet off cells were grown as a confluent monolayer for 3 days. Subsequently, control cells (infected with β-tubulin cofactor D and expression suppressed by addition of 60 ng/ml doxycycline) and cells infected with adenoviruses to express β-tubulin cofactor D, with or without adenovirus to coexpress ARL2 wild-type or mutants, were processed for immunoblotting or quantitation of cell dissociation from the monolayer. A) Immunoblot reacted with antiflag to show expression of β-tubulin cofactor D, with anti-HA (12CA5) to detect ARL2 and with anti-{alpha}-tubulin. Western blot reveals the expression of both β-tubulin cofactor D and ARL2, and shows that ARL2 inhibits β-tubulin cofactor D-dependent {alpha}-tubulin degradation. B–D) Epithelial monolayer cells expressing either β-tubulin cofactor D alone or coexpressing ARL2 wild-type or mutants (as indicated) for 19 h were harvested, and attached cells were trypsinized and counted. The percentage of cells dissociated from the monolayer is shown. E) Cells grown as described on a 10 cm plate, expressing the above constructs, were subjected to immunoprecipitation by 12CA5 anti-HA-ARL2 antibody. Immunoprecipitates were resolved by SDS-PAGE and blotted to nitrocellulose membranes. Membranes were subjected to immunoblot analysis by antiflag (β-tubulin cofactor D). t Test was performed, and the P value is shown for each pair (*P<0.05 and **P<0.01). Experiments revealed that ARL2 inhibits β-tubulin cofactor D-dependent cell dissociation and that the ARL2 GDP-bound form has the highest inhibitory capacity. GDP-bound form of ARL2 is shown to strongly immunoprecipitate with β-tubulin cofactor D, explaining its higher inhibition on cell detachment. The experiments were repeated three times.

β-tubulin cofactor D localizes to cell-cell contacts and promotes TJ and AJ disassembly
The epithelial monolayer structure is maintained by cell–cell contacts composed of AJs and TJs. β-tubulin cofactor D-dependent epithelial cell detachment occurs through the dissociation of single cells, rather than epithelial sheets (Fig. 2A and data not shown). Therefore, we reasoned that the cell dissociation is in part due to the disassembly of the AJC, composed of TJs and AJs. To establish the effect and to understand the role of β-tubulin cofactor D in this process, we performed immunofluorescence microscopy studies of MDCK cells expressing β-tubulin cofactor D. The results, shown in Fig. 4 B, revealed that β-tubulin cofactor D partially localizes to filamentous structures within the cytoplasm, and partially overlaps with the lateral PM (Fig. 4C , C1, arrowheads). Frequently, β-tubulin cofactor D is observed to partially colocalize to the TJ protein ZO1 (Fig. 4D , D1, arrows). This β-tubulin cofactor D-ZO1 colocalization is apparent when the TJs appear as a noncontinuous strand and are in the process of disassembly. In many cases, such as that in Fig. 4D1 , the PM and most likely the TJ localization of β-tubulin cofactor D is apparent in ZO1-positive spots and is lacking when ZO1 is not apparent, which may indicate that ZO1 or other TJ proteins are required for this localization. In these cells, we observe TJ disassembly, evidenced by disruption in the TJ (enlargements in Fig. 4C1 , D1, compared to A), and localization of β-tubulin cofactor D to these sites. A closer look at the images reveals that in all cells that express β-tubulin cofactor D, the TJ is disrupted to some extent and TJ loss is correlated with expression of β-tubulin cofactor D. It was interesting to speculate that the appearance of β-tubulin cofactor D along the lateral PM is dependent on intact MTs. To test this hypothesis, we treated the cells with the MT-disrupting agent nocodazole. As shown in Fig. 4E , nocodazole treatment abolished the PM appearance of β-tubulin cofactor D as well as its overlap with the TJ marker ZO1. This result indicates that β-tubulin cofactor D utilizes the MT system for its localization to the lateral PM. As shown here, the TJ disassembly process is accompanied by ZO1’s release from the TJ region and accumulation in the cytoplasm, which led us to postulate that this is also accompanied by ZO1 degradation. Interestingly, we found that β-tubulin cofactor D-dependent ZO1 release from the TJ does not promote its degradation but rather moderately increases its expression (Fig. 4F ). These results support our observation of increased cytoplasmic ZO1 in MDCK cells expressing β-tubulin cofactor D.


Figure 4
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  		Figure 4. β-tubulin cofactor D causes disassembly of tight junctions (TJs). MDCK tet off cells were grown on Corning transwells for 3 days. Subsequently, control cells (infected with β-tubulin cofactor D and expression suppressed by addition of 60 ng/ml doxycycline) (A) and cells infected with adenovirus to express β-tubulin cofactor D (B–E) were incubated for 18 h for expression and subsequently incubated with (E) or without (A–D1, F) 33 µM nocodazole. Cells were then processed for immunofluorescence (A–E) or Western blotting (F). Cells were stained for the TJ marker ZO1 (A–E, red) and antiflag antibody recognizing β-tubulin cofactor D (A–E, green). A) Control cells display a continuous intact TJ. B) Infected cells show a partially disassembled TJ and internalized ZO1. β-tubulin cofactor D appears overlapping the PM (arrowheads) and cytoplasmic locations. C1) β-tubulin cofactor D highly overlaps the PM and partially disassembled TJs (arrowheads). D, D1) In some cells we observed colocalization of β-tubulin cofactor D and ZO1 (arrows). E) Cells expressing β-tubulin cofactor D for 19 h and incubated with 33 µM nocodazole for the last 15 h show β-tubulin cofactor D in a punctate subcellular localization, having lost its lateral PM localization. Scale bar = 2 µM. F) Immunoblot reacted with anti-ZO1 demonstrates that ZO1 did not degrade; in fact, densitometric analyses show 20 to 30% increased ZO1 expression, indicating that β-tubulin cofactor D causes the release of ZO1 from the TJ but does not promote its degradation.

The AJs are the principal components of epithelial cell–cell contacts. To establish the possible disassembly of this structure, we stained MDCK cells expressing β-tubulin cofactor D with antibodies against E-cadherin and β-catenin, known to be major components of these structures (4) . In control cells (Fig. 5 A, left column), E-cadherin and β-catenin show the continuous and enhanced lateral PM staining common to epithelial AJs. In cells expressing β-tubulin cofactor D (Fig. 5A , right column), β-catenin staining is weak and has diffused into the cytoplasmic region, indicating its dissociation and possible degradation. In addition, E-cadherin localization is significantly reduced from the lateral PM and found in the cytoplasm in punctate staining (Fig. 5 , right column, cells marked by arrowheads).


Figure 5
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  		Figure 5. β-tubulin cofactor D causes disassembly of adherens junctions (AJs). MDCK tet off cells were grown on Corning transwells for 3 days. Subsequently, control cells (infected with β-tubulin cofactor D and expression suppressed by addition of 60 ng/ml doxycycline) (A) and cells infected with adenovirus for expression of β-tubulin cofactor D (A–C) were incubated for 18 h for expression and subsequently processed for immunofluorescence. Cells were stained for the tight-junction (TJ) marker ZO1, AJ markers β-catenin, and E-cadherin (A–C) and rabbit antiflag antibody recognizing β-tubulin cofactor D with antidog E-cadherin (B), as indicated. A) Control cells display continuous intact TJs and AJs (left column); β-tubulin cofactor D-expressing cells (right column) reveal fragmented TJs (gaps indicated by arrows); reduced staining of β-catenin on the lateral PM with increased cytoplasmic staining indicates dissociation of β-catenin from the AJ. Additionally, cells overexpressing β-tubulin cofactor D show fragmented and reduced E-cadherin staining on the PM with an increased amount of endocytosed E-cadherin (A, right column and C, arrowheads). B) β-tubulin cofactor D shows partial overlap with E-cadherin on the lateral PM (arrowheads). C) Image taken at the interface where the TJ associates with the AJ shows punctate cytoplasmic staining of endocytosed E-cadherin (arrowheads), and ZO1 staining reveals intracellular ZO1 and fragmented TJ, indicative of its state of disassembly (arrows). Scale bar = 2 µM.

MDCK cells costained for E-cadherin and β-tubulin cofactor D show clear localization of β-tubulin cofactor D to the lateral PM where E-cadherin is seen in reduced amounts (Fig. 5B , arrowheads). An image of cells expressing β-tubulin cofactor D and stained for ZO1 and E-cadherin, taken at the interface where the TJ associates with the AJ, shows punctate cytoplasmic staining of endocytosed E-cadherin (Fig. 5C , arrowheads); in parallel, ZO1 staining reveals fragmented TJ and intracellular localization, indicative of its state of disassembly (Fig. 5C , arrows). Therefore, these results support the notion that β-tubulin cofactor D-dependent AJC disassembly is promoted by the expression and partial localization of β-tubulin cofactor D to these lateral PM structures.

It was important to determine whether ARL2 participates in the β-tubulin cofactor D-dependent AJC disassembly. We, therefore, used ZO1 staining as an indicator for the state of TJ disassembly. Control cells and cells expressing ARL2-T30N showed normal ZO1 staining (Fig. 6 A, C). Cells expressing β-tubulin cofactor D revealed disrupted and noncontinuous ZO1 staining (Fig. 6B , arrows). In contrast, cells expressing both β-tubulin cofactor D and ARL2-T30N showed continuous ZO1 staining, which indicates that ARL2-T30N markedly inhibits β-tubulin cofactor D-dependent ZO1 dissociation (Fig. 6C ). This result indicates that ARL2 participates in the attenuation of β-tubulin cofactor D-dependent TJ disassembly.


Figure 6
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  		Figure 6. ARL2 inhibits β-tubulin cofactor D-induced TJ disassembly. MDCK tet off cells were grown on Corning transwells for 3 days. Subsequently, control cells (infected with β-tubulin cofactor D and expression suppressed by addition of 60 ng/ml doxycycline) (A) and cells infected with adenoviruses for expression of β-tubulin cofactor D (B), ARL2-T30N (C), or both β-tubulin cofactor D and ARL2-T30N (D), as indicated, were incubated for 18 h of expression. Cells were then processed for immunofluorescence and stained for the tight-junction (TJ) marker ZO1. Control cells and ARL2-T30N cells reveal intact TJs (A, C). β-tubulin cofactor D expression causes disruption of TJs as indicated by the fragmented appearance of the ZO1 staining (B). Expression of ARL2 markedly inhibits β-tubulin cofactor D TJ disruption as observed by the continuous appearance of ZO1 (D), similar to that of control (A), and ARL2-T30N-expressing cells (C). Scale bar = 2 µM.

To understand the mechanism by which ARL2 (WT and GDP-bound form) inhibits β-tubulin cofactor D-dependent cell detachment (Fig. 3B-D ), we performed immunofluorescence experiments (Fig. 7 A–C) and analyzed the different localization distributions of each protein (Fig. 7D) . In Fig. 7A, B , we show that ARL2 in its WT form and the T30N mutant localize to a single puncta within the cell or to a single puncta together with cytoplasmic localization; rarely (WT) or never (T30N) does it localize to the cytoplasm alone or the lateral PM (Fig. 7D ). On the other hand, the GTP-bound mutant, ARL2-Q70L, is localized to this single puncta (Figure 7C1 ) but also localizes to the cytoplasm alone (Figure 7C2, D ). In Fig. 7E, we show coexpression of ARL2-WT and β-tubulin cofactor D, which reveals their complete colocalization to a single puncta and the loss of β-tubulin cofactor D localization from the lateral PM or filamentous intracellular structures. Identical results were obtained for ARL2-T30N and β-tubulin cofactor D (data not shown). The lack of localization of ARL2 in either the WT or T30N form to the lateral membrane (Fig. 7A-D ), together with their complete colocalization to a single intracellular puncta with β-tubulin cofactor D (Fig. 7E ), may indicate that ARL2 exerts its inhibitory effect by titrating β-tubulin cofactor D from the lateral PM. To establish this, we performed immunolocalization of β-tubulin cofactor D in cells expressing both β-tubulin cofactor D and the different ARL2 forms.


Figure 7
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  		Figure 7. ARL2 localizes to a single puncta or to the cytoplasm. MDCK tet off cells were grown on Corning transwells for 3 days. Subsequently, cells infected with adenoviruses for the expression of wild-type or mutant ARL2, as indicated, were incubated for 18 h for expression and subsequently processed for immunofluorescence. Cells were stained with rabbit anti-ARL2 antibody (A–C) or rabbit anti-ARL2 with mouse antiflag recognizing β-tubulin cofactor D (E). D) Images of at least 100 cells expressing each construct were counted and localization was determined and calculated. A, B, D) ARL2 in its wild-type and GDP-bound (T30N) forms localizes mainly to a single puncta within the cell with some cytoplasmic localization. (C1, C2) The GTP-bound form of ARL2 is also localized to the single puncta (C1) with some cells showing only cytoplasmic staining (C2) and a limited number of cells showing cytoplasmic and lateral PM localization (D). E) ARL2-WT (green) and β-tubulin cofactor D (red) show complete colocalization to the single puncta. Scale bar = 2 µM.

While ARL2-WT and ARL2-T30N efficiently relocate β-tubulin cofactor D from its lateral PM and cytoplasmic localization and confine it to a single puncta (Fig. 8 A, B, D), the opposite occurs in cells expressing ARL2-Q70L (GTP-bound form): β-tubulin cofactor D’s intracellular localization is unaffected (Fig. 8C, D ). Taken together, the inhibitory effect of ARL2-WT and its T30N mutant on β-tubulin cofactor D-dependent cell dissociation (Fig. 3B-D ) and the direct association of only ARL2-T30N with β-tubulin cofactor D led us to hypothesize that ARL2 inhibition is effected through titration of β-tubulin cofactor D away from the lateral PM.


Figure 8
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  		Figure 8. ARL2-GDP recruits β-tubulin cofactor D to the single puncta within the cell. MDCK tet off cells were grown on Corning transwells for 3 days. Subsequently, cells coinfected with adenoviruses for the expression of ARL2 in its wild-type or mutant form and β-tubulin cofactor D, as indicated, were incubated for 18 h for expression and subsequently processed for immunofluorescence. Cells were stained with rabbit antiflag antibody recognizing β-tubulin cofactor D (A–C). D) Images of at least 100 cells expressing each construct were counted and localization was determined and calculated. A, B, D) ARL2 in its wild-type and GDP-bound (T30N) forms translocate β-tubulin cofactor D to a single puncta within the cell. C, D) The GTP-bound form of ARL2 has no effect on the localization of β-tubulin cofactor D. Scale bar = 2 µM.

Nocodazole attenuates and Taxol promotes β-tubulin cofactor D cell detachment
Our observation that nocodazole abolishes β-tubulin cofactor D’s subcellular distribution as well as its colocalization with the TJ protein ZO1 indicated that MTs play an essential role in β-tubulin cofactor D localization (Fig. 4E ). Therefore, we delved deeper into the effects of nocodazole, known to promote MT disassembly but to inhibit TJ disassembly (4) , and taxol, known to stabilize and promote MT polymerization. As our observation of TJ disassembly (ZO1 dissociation) correlated with AJ disassembly (E-cadherin and β-catenin internalization and dissociation, respectively), we further monitored AJC disassembly via ZO1 dissociation. Cells treated with nocodazole alone showed no sign of cell detachment; instead, they showed more intense ZO1 localization to the TJ (Fig. 9 C). Cells expressing β-tubulin cofactor D and cotreated with nocodazole showed a reduced degree of cell detachment (Fig. 9A ), indicating that MTs and MT dynamics are required for the cell detachment induced by β-tubulin cofactor D (Fig. 9A ). Additionally, β-tubulin cofactor D requires MTs for its localization to the TJ (Fig. 4E ) and, therefore, MT disassembly reduces β-tubulin cofactor D’s ability to localize to the TJ. In addition to enhancing the expression of β-tubulin cofactor D. Treatment of MDCK cells with the MT-stabilizing drug taxol significantly enhanced the cell-detachment phenomenon. This taxol effect supports the notion that MT assembly and stability are required for cell detachment and enable more β-tubulin cofactor D localization at the TJ and at other subcellular sites that participate in cell detachment (Fig. 9B ). Treatment of MDCK cells with nocodazole followed by immunofluorescence microscopy showed TJs with a twisted morphology, indicating that MTs are required for their shape. Short-term expression of β-tubulin cofactor D in MDCK cells cotreated with nocodazole showed reduced fragmentation of the TJ strand (Fig. 9C ). In comparison, taxol treatment alone had no effect on TJ morphology, but short-term expression of β-tubulin cofactor D together with taxol treatment revealed significant disruption of the TJs, supporting our observation of increased detachment (Fig. 9C ). These results indicated that MTs and MT dynamics play an important role in β-tubulin cofactor D-dependent cell detachment, most likely by transporting β-tubulin cofactor D to the TJ and other sites along the PM that participate in cell detachment.


Figure 9
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  		Figure 9. Nocodazole attenuates and taxol promotes β-tubulin cofactor D cell detachment. MDCK tet off cells were grown on Corning transwells for 3 days. Then control cells (infected with β-tubulin cofactor D and expression suppressed by addition of 60 ng/ml doxycycline) (A) and cells infected with adenovirus for the expression of β-tubulin cofactor D, were incubated for 18 h of expression, with 33 µM nocodazole or 10 µM taxol being added during the last 14.5 h of incubation (3.5 h after infection). Cells were then assayed for cell dissociation from the monolayer (A, B), or processed for immunofluorescence (C). A, B) Epithelial monolayer cells either expressing or not expressing β-tubulin cofactor D (as indicated) were harvested from the media, attached cells were trypsinized, and both fractions were counted. The percentage of cells dissociated from the monolayer is shown. The experiment revealed that nocodazole, known to disassemble microtubules (MTs), inhibits β-tubulin cofactor D-dependent cell dissociation and taxol, known to stabilize MTs, promotes cell dissociation from the epithelial monolayer. C) Control cells or cells expressing β-tubulin cofactor D, not treated or treated with either nocodazole or taxol, were stained for the tight-junction (TJ) marker ZO1. Control cells treated with nocodazole show a distorted TJ, while taxol treatment shows no effect. Cells expressing β-tubulin cofactor D show aberrant ZO1 staining. Nocodazole appears to inhibit β-tubulin cofactor D TJ breakdown while taxol enhances it. t Test was performed, and P value is shown for each pair (*P<0.05 and **P<0.01). Scale bar = 2 µM. The experiment was repeated three times.

β-tubulin cofactor D inhibits assembly of the TJ
To explore the potential role of β-tubulin cofactor D in TJ assembly, we performed calcium-switch experiments to follow the rate at which cells that express β-tubulin cofactor D assemble their TJs. As shown in Fig. 10 , polarized MDCK cells incubated in medium lacking calcium exhibited TJ disassembly. When the medium was replaced with one containing calcium, cells required 30 min to assemble their TJs with most of the ZO1 at the TJ, and within 90 min, the TJs exhibited strong ZO1 staining. Cells that expressed β-tubulin cofactor D were unable to form any TJs within 30 min and at 90 min, showed initial recruitment of ZO1 to the TJs. This result indicates that β-tubulin cofactor D plays a vital role in TJ assembly and that it may be titrating out a factor that is required for TJ assembly.


Figure 10
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  		Figure 10. β-tubulin cofactor D inhibits tight junction (TJ) reassembly following calcium depletion. MDCK tet off cells were grown on Corning transwells for 3 days. Subsequently, cells were infected with adenovirus for the expression of β-tubulin cofactor D for 19 h, or infected with β-tubulin cofactor D and expression suppressed by addition of 60 ng/ml doxycycline (control) (A). Cells were transferred to growth media lacking calcium and containing 4 mM EGTA for 60 min, then washed extensively and either fixed (0 min) or incubated with complete growth media for TJ reassembly for 30 or 90 min (as indicated). Subsequently, cells were fixed and processed for immunofluorescence. Control cells reassembled TJs within 30 min, while cells expressing β-tubulin cofactor D hardly reassembled TJs within 90 min.

The 15 C-terminal amino acids of β-tubulin cofactor D participate in cell detachment activity
Our results indicated that MT disruption by nocodazole attenuates β-tubulin cofactor D-dependent TJ disassembly and that nocodazole alone does not disrupt TJs (Fig. 5A, C , nocodazole, and ref. 4 ). The presence of a putative tubulin-binding site within the C-terminal 15 amino acids of β-tubulin cofactor D suggests that β-tubulin cofactor D may bind tubulin through this domain to exert its AJC-disassembly function. To test this hypothesis, we constructed a deletion mutant that lacks the 15 C-terminal amino acids and generated a recombinant adenovirus containing this construct. Expression of this deletion mutant also resulted in cell detachment (Fig. 2B, C ). Nevertheless, the degree of detachment was significantly lower than with WT β-tubulin cofactor D. Within 24 h, only 70% of the cells had detached (Fig. 2B, C ) compared with 90% following expression of the WT β-tubulin cofactor D (Fig. 2B ). Furthermore, our dose-response assay showed an overall 50% reduction in cell detachment (Fig. 2C ) compared to the WT form of β-tubulin cofactor D. These results indicate an important role for the C-terminal portion of the 130 kDa protein (1184 amino acids). Additionally, we found that in contrast to the WT form, the C-terminal deletion mutant does not provoke {alpha}-tubulin degradation (Fig. 11 A). This result may support a mechanism by which the 15 C-terminal amino acids participate in β-tubulin binding. In Fig. 11B , we observe that the expression of mutant β-tubulin cofactor D does not significantly modify normal MT epithelial ultrastructures while the TJ structure is fragmented (Fig. 11B , arrows). Therefore, expression of a β-tubulin cofactor D mutant that lacks the 15 C-terminal amino acids does not affect the degradation pattern of {alpha}-tubulin observed in cells expressing the WT full-length protein (Fig. 11B ). Staining for localization of the mutant β-tubulin cofactor D revealed a very different subcellular distribution compared to that of the WT full-length protein. The mutant protein localized to the cytoplasm in a punctate pattern compared to the WT form, which extensively overlapped with the lateral PM (Fig. 11C ). Similar to the WT form, we did observe fragmentation of the TJ, but to a lesser extent (Fig. 11A , arrows). The total abolishment of {alpha}-tubulin degradation in cells expressing the β-tubulin cofactor D C-terminal deletion mutant, while still partaking in TJ disassembly, indicates that β-tubulin cofactor D has a dual function consisting of two separate events and that MT disruption is not the sole reason for TJ disassembly and cell dissociation from the monolayer.


Figure 11
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  		Figure 11. The 15 C-terminal amino acids of β-tubulin cofactor D are critical for its localization and govern microtubule (MT) disassembly. MDCK tet off cells were grown on Corning transwells for 3 days. They were then infected with adenoviruses for the expression of β-tubulin cofactor D or its mutant lacking the 15 C-terminal amino acids, as indicated, and incubated 18 h for expression. Cells were processed for either immunofluorescence (A, B) or Western blotting (C). A) Cells were stained for the tight-junction (TJ) marker ZO1 and antiflag antibody recognizing the β-tubulin cofactor D deletion mutant. B) Cells were stained for the TJ marker ZO1 and anti-{alpha}-tubulin antibody. Both β-tubulin cofactor D and its mutant show fragmented ZO1 staining (arrowheads). While expression of β-tubulin cofactor D destroys MTs, expression of its mutant has no effect on epithelial MT ultrastructure. Scale bar = 2 µM. C) Immunoblot reacted with antiflag recognizing the β-tubulin cofactor D deletion mutant and anti-{alpha}-tubulin antibody shows that the 15 C-terminal amino acids are essential for β-tubulin cofactor D-dependent {alpha}-tubulin degradation and for localization to the lateral and TJ PM regions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found that in polarized epithelial cells, β-tubulin cofactor D has a dual function. Similar to its role in nonpolarized cells, it regulates MT dynamics in conjunction with ARL2. Its second function lies in the assembly–disassembly of the AJC, composed of TJs and AJs. Heterologous expression of β-tubulin cofactor D results in excess protein relative to others that are known to associate with it (β-tubulin cofactor C and E, ARL2, PP2A) and in an association with and sequestration of β-tubulin from the {alpha}-β-tubulin dimer, which drives {alpha}-tubulin degradation (29) . Here we show that in polarized epithelial MDCK cells, this monomeric state of β-tubulin cofactor D results in its partial localization to the lateral PM where it causes AJC disassembly, eventually causing the cells to dissociate from the epithelial monolayer and substratum. The cells’ dissociation from the monolayer is explained mainly by the disassembly of the TJs and AJs. TJs and AJs are considered to be two distinct structures: nevertheless, key proteins of both structures interact during assembly, as well as in a steady state. The assembly and disassembly of the AJ appears to regulate the fate of the TJ (31 32 33 34 35) . Since polarized MDCK cell monolayers do not demonstrate prominent focal adhesions, the disassembly of focal adhesions most likely does not play a critical role in the detachment of cells in this system, but it most likely does play a role in most epithelial cells. Previous works by several groups have shown that focal-adhesion disassembly is in part regulated by the MTs and is accompanied by tyrosine phosphatases and inhibited by the MT-disrupting agent nocodazole. This, in turn, increases the number of focal adhesions in lung epithelial cells and elimination of nocodazole enables MT regrowth followed by FAK dephosphorylation and focal-adhesion disassembly (36 , 37) . We, therefore, speculate that a common mechanism based on MTs and MT dynamics localizes β-tubulin cofactor D to the TJs, AJs and focal adhesions to regulate their fate.

Kahn and colleagues identified ARL2 in a 300 kDa complex containing β-tubulin cofactor D and a trimer of protein phosphatase 2A (PP2A) in brain extracts. The authors showed that a portion of the total β-tubulin cofactor D and PP2A is associated with ARL2 in its GDP-bound form (15) . In a totally different arena, Sontag and colleagues showed that PP2AB{alpha}, a Ser/Thr protein phosphatase, translocates to the TJ during calcium-induced TJ assembly and that TJ disassembly, provoked by calcium depletion, releases PP2AB{alpha} to the cytoplasm. Elevated expression of PP2AB{alpha} in the TJ region results in occludin dephosphorylation with a concurrent decrease in TER (trans-epithelial resistance) and TJ breakdown. PP2AB{alpha} thus appears to play a direct role in the phosphatase activity on TJ proteins that triggers their disassembly (16) . PP2A is localized to either the TJ or AJ depending on its B subunit, and participates in Ser/Thr phosphorylation of integral proteins of both TJs and AJs and their maintenance (16 , 38) . Our results, together with the published data, suggest that this complex regulates the fate of the AJC through Ser/Thr phosphorylation in a MT-dependent fashion. The regulated disruption of these cell-cell contacts generates a fibroblastic morphology followed by cell detachment from the epithelial monolayer.

The TJ and AJ assembly-disassembly process has been linked with phosphorylation-dephosphorylation cycles of the TJ and AJ membranes and associated proteins. For example, during TJ assembly, occludin is phosphorylated and dephosphorylated during disassembly, followed by endocytosis and localization to the cytoplasm (39 , 40) . These results indicate a general phosphorylation cycle in which kinase activity is correlated to the TJ-assembly process and phosphatase activity is related to the disassembly step. Here we show that the ARL2-β-tubulin cofactor D complex in epithelial cells regulates the dissociation of cell–cell adhesions as observed by cell dissociation and AJC disassembly. We hypothesize that ARL2 is a negative regulator of the cell-detachment process; that PP2AB{alpha} belongs to that complex and controls phosphatase activity, resulting in TJ and AJ disassembly; and that β-tubulin cofactor D may be responsible for the translocation of PP2AB and other proteins that participate in the disassembly process to the TJs and AJs.

β-tubulin cofactor D belongs to a family of proteins containing armadillo repeat domains. This family also includes PP2A, β-catenin, and adenomatous polyposis coli protein and associates (directly or indirectly) with MTs for their intracellular transport and AJ localization (41 , 42) . We hypothesize that the above proteins use a similar MT-dependent mechanism to localize to both the TJs and AJs. Moreover, β-tubulin cofactor D that lacks its 15 C-terminal amino acids appears to have reduced TJ-disassembly properties and shows anomalous localization, similar to β-tubulin cofactor D in cells treated with nocodazole. This may be explained by β-tubulin cofactor D’s ability to associate with MTs via both the 15 C-terminal amino acids and the armadillo repeat (43) .

In this work, we show that β-tubulin cofactor D, in addition to its cytoplasmic localization where it causes MT disruption and {alpha}-tubulin degradation, is partially localized to the lateral PM with some overlap and colocalization to the TJs and AJs, promoting their disassembly. β-tubulin cofactor D localization to the lateral PM is dependent on its 15 C-terminal amino acids and MT integrity. Additionally, β-tubulin cofactor D is localized to intercellular connection sites and its expression causes polarized MDCK cells to dissociate from their neighboring cells and the substratum, followed by complete detachment from the epithelial monolayer. Deletion of the 15 C-terminal amino acids of β-tubulin cofactor D or disassembly of the MTs by nocodazole causes the mis-localization of β-tubulin cofactor D to punctate cytoplasmic regions, as well as the attenuation of its effect on cell dissociation from the epithelial monolayer. However, it does not affect {alpha}-tubulin degradation or MT epithelial structure. A calcium-switch experiment revealed that expression of β-tubulin cofactor D also slows the TJ assembly process dramatically. The small GTPase ARL2, which was found to be in a complex with β-tubulin cofactor D and to regulate MT dynamics, also attenuates β-tubulin cofactor D-dependent cell dissociation from the epithelial monolayer through the translocation of β-tubulin cofactor D to an intracellular site. We conclude that β-tubulin cofactor D actively participates in the assembly-disassembly of the AJC and other intercellular and cellular-substratum binding of epithelial cells.


   ACKNOWLEDGMENTS
 
This research was supported by the Israel Science Foundation (grant no. 1318/04) to Y.A. Y.A. is affiliated with the David R. Bloom Center for Pharmacy at the Hebrew University. We thank Israel Ringel, Alexander Bershadsky, and Karl Matter for critically reading the manuscript.

Received for publication January 22, 2007. Accepted for publication July 19, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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