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Tubulin detyrosination promotes monolayer formationand apical trafficking in epithelial cells

Sabrina Zink, Lena Grosse, Andrea Freikamp, Sebastian Banfer, Frauke Muksch and Ralf Jacob*Department of Cell Biology and Cell Pathology, Philipps-Universitat Marburg, Marburg, Germany

*Author for correspondence (jacob@staff.uni-marburg.de)

Accepted 5 October 2012Journal of Cell Science 125, 5998–6008� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.109470

SummaryThe role of post-translational tubulin modifications in the development and maintenance of a polarized epithelium is not wellunderstood. We studied the balance between detyrosinated (detyr-) and tyrosinated (tyr-) tubulin in the formation of MDCK cell

monolayers. Increased quantities of detyrosinated microtubules were detected during assembly into confluent cell sheets. These tubuleswere composed of alternating stretches of detyr- and tyr-tubulin. Constant induction of tubulin tyrosination, which decreased the levelsof detyr-tubulin by overexpression of tubulin tyrosine ligase (TTL), disrupted monolayer establishment. Detyr-tubulin-depleted cells

assembled into isolated islands and developed a prematurely polarized architecture. Thus, tubulin detyrosination is required for themorphological differentiation from non-polarized cells into an epithelial monolayer. Moreover, membrane trafficking, in particular tothe apical domain, was slowed down in TTL-overexpressing cells. This effect could be reversed by TTL knockdown, which suggests

that detyr-tubulin-enriched microtubules serve as cytoskeletal tracks to guide membrane cargo in polarized MDCK cells.

Key words: Tubulin, Apical transport, Cell polarity, Post-translational modification

IntroductionPolarization of epithelial cells involves the separation of the plasma

membrane into two membrane domains and a completereorganization of the cytoskeletal architecture. Microtubules, as

central elements of the cytoskeleton, play critical roles in thegeneration of an apico-basolateral polarity and specific transport

pathways to the apical or the basolateral cell pole (Musch, 2004).Alterations in microtubule composition thus affect the integrity

of epithelial cell sheets (Yap et al., 1995). Tubulin is subject todistinct post-translational modifications, which include acetylation,

tyrosination, detyrosination, D2 modification, polyglutamylation,palmitoylation and phosphorylation (Hammond et al., 2008).

Tubulin tyrosination was the first tubulin-specific modificationreported (Barra et al., 1973). Pools of tyrosinated (tyr-tubulin) and

detyrosinated (detyr-tubulin) microtubules are generated by a cycle

of removal and subsequent re-ligation of tyrosine to the C-terminusof a-tubulin. Whereas dynamic microtubules are tyrosinated,

detyrosination leads to stabilisation and inhibition of microtubuledisassembly (Peris et al., 2009). This process can be modulated by

the depolymerizing motor mitotic centromere-associated kinesin(MCAK). Tubulin tyrosination is dependent on the synthesis of

tubulin tyrosine ligase (TTL), which acts on ab-tubulinheterodimers and restores tyr-tubulin (Raybin and Flavin, 1975).

A knockout of TTL is lethal in mice within 1 hour after birth due toneuronal disorganization, suggesting that this enzyme is essential

(Erck et al., 2005). The distribution of tyr- and detyr-tubulin hasbeen studied intensively in different cell types and during different

phases of cell division. Dynamic tyrosinated microtubules are

commonly found in the interphase network and the metaphasespindle, whereas stable detyrosinated microtubules have only been

detected in a limited subset of interphase microtubules from TC-7cells (Gundersen et al., 1984). In neuroblastoma cells detyr-tubulin

was restricted to microtubules of elongated cell processes, with aconcentration of tyr-tubulin in the cell bodies (Wehland and Weber,1987). In general, the ratio of detyr- to tyr-tubulin is higher in cells

that have established cell-cell contacts than in isolated cells 1 dayafter seeding (Bre et al., 1991). In polarized neuronal cellstyrosinated tubulin acts as a directional cue to navigate motor

heads of kinesin-1 to axons (Konishi and Setou, 2009). Tubulinmodification thus has the potential to determine cellular polarity bya precise regulation of kinesin-1 transport. This prompted us toexamine the dynamics of tubulin detyrosination to tyrosination

during the formation of polarized epithelial cell sheets. We found anincrease followed by a decrease in the number of detyrosinatedmicrotubules during polarization of MDCK cells. Constant

depletion of detyr-tubulin by overexpression of TTL resulted inprolonged time periods for monolayer formation, and perturbed thesurface delivery of secreted and membrane-bound cargo molecules.

In particular apical transport pathways were affected, whichindicates that detyr-tubulin-containing microtubules are involvedin at least two processes of MDCK cells; the correct assembly of

cells into monolayers and guiding apical protein trafficking in fullypolarized cells.

ResultsDistribution of detyrosinated and tyrosinated tubulin inpolarized epithelial cells

The arrangement of detyrosinated, acetylated and tyrosinated a-

tubulin in polarized epithelial cells was first studied byimmunofluorescence. Line scan analysis of MDCK cell cystsgrown in Matrigel showed an accumulation of all three tubulin

variants close to the apical membrane, followed by a gradualdecline towards the basal cell pole. Detyr- and tyr-tubulin wereequally distributed along the apical–basolateral axis. Steeper

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gradients were occasionally observed for acetylated tubulin

(Fig. 1A; see also supplementary material Fig. S1), which

supports data from Jaulin and Kreitzer who showed an

enrichment of acetylated tubulin at the apical cell pole (Jaulin

and Kreitzer, 2010). In a further study, the quantity of detyr- and

tyr-tubulin at the two cell poles was studied by TIRF microscopy,

which detects fluorescent dyes in close proximity to the apical or

basal plasma membrane of polarized MDCK cells (Schneider

et al., 2010). Most of the membrane-proximal microtubules were

enriched in tyr-tubulin, while a smaller proportion was double

stained by detyr- and tyr-tubulin (supplementary material Fig.

S2A,B). Overall, similar ratios of detyr- and tyr-tubulin were

detected at the apical and basal domain (Fig. 1B), which

indicates that neither tubulin variant preferentially concentrates

at the poles of polarised cells and confirms the line scan data

from MDCK cell cysts.

The distribution of detyr- and tyr-tubulin on double-positive

microtubules was then analysed by high-resolution ground state

depletion with individual molecular return (GSDIM) microscopy

of MDCK cells. The cells were grown for 1 day after seeding on

coverslips and fixed with methanol, then immunostained with anti-

tyr- or anti-detyr-tubulin antibodies. As depicted in Fig. 1, short

detyr-tubulin sections of up to 1 mm long were disrupted by longer

segments of tyr-tubulin, thus leading to a string-of-pearls-like

appearance of the two tubulin variants with a preferential

concentration of tyr-tubulin at the tubulus ends. Similar

arrangements of interspersed detyr- and tyr-tubulin stretches had

been seen for interphase and metaphase microtubules using

immuno-EM (Geuens et al., 1986). However, antibodies directed

against a-tubulin as a control were more or less evenly distributed

along microtubules (supplementary material Fig. S2C).

Fluctuations in detyrosinated tubulin during epithelial

polarisation

Next we addressed whether the balance between the two tubulin

variants varies during the course of epithelial polarisation.

Therefore, MDCK cells were incubated on filters and analysed

after 1–5 days. Immunofluorescence data suggested that detyr-

tubulin decreased during the process of epithelial cell polarisation

(supplementary material Fig. S2D). This was confirmed by

immunoblot analysis of MDCK cell lysates harvested after

different incubation intervals with antibodies directed against a-

tubulin and tyr-, detyr- or acetylated tubulin (supplementary

material Fig. S2E). Densitometric quantification of the

Fig. 1. Tubulin modifications in MDCK cells.

(A) Linescan analysis of tyr- and detyr- or acetylated

tubulin along the apico-basolateral axis (line indicated in

supplementary material Fig. S1A,B). (B) Quantification

of detyr- and tyr-tubulin from TIRF images in

supplementary material Fig. S2A,B using the Volocity

software package (n54). (C) For GSDIM microscopy,

Alexa Fluor 488 and Alexa Fluor 647 were used to label

detyr- and tyr-tubulin, respectively, on cells 1 day after

seeding. Scale bars: 5 mm; enlarged sections, 1 mm.

(D) Quantification of the immunoblot data from MDCK

cell lysed after the indicated time intervals depicted in

supplementary material Fig. S2D. The band intensities

detected with anti-tubulin, anti-tyr-, anti-detyr- and anti-

acetylated tubulin antibodies were normalized to a

GAPDH control. Significance was tested with Student’s

t-test (***P,0.0001), n57.

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immunoblots shows a slow increase in tyr-tubulin as well as

acetylated tubulin during cell polarisation (Fig. 1D), which is in

agreement with the overall level of cellular a-tubulin. The level of

detyr-tubulin, however, increased to a maximum on the second day,

followed by a decrease to less than 10% at day 5. This decrease is

statistically significant and corroborates our immunofluorescence

observations, as well as previously published data by Quinones et al.

(Quinones et al., 2011). Thus, the pattern of tubulin modifications

changes during the course of epithelial polarisation with an initial

increase followed by a decrease in the amount of detyr-tubulin,

resulting in a high prevalence of tyr- and a minor pool of detyr-

tubulin in fully polarised cells.

Alteration of detyr-tubulin by tubulin tyrosine ligase

Tubulin tyrosine ligase (TTL), the enzyme that converts detyr- into

tyr-tubulin, is expressed in the cytosol of polarized MDCK cells as

assessed by immunofluorescence staining with TTL-specific

antibodies (Fig. 2A). To monitor changes in TTL expression

during epithelial polarization, MDCK cells were harvested on

different days after seeding. Equal amounts of protein from cell

lysates were separated by SDS gel electrophoresis and further

processed by western blot analysis with anti-TTL antibodies. A

housekeeping protein, GAPDH was used as a loading control. As

depicted in Fig. 2B,C, TTL expression changed with cellular

differentiation. Just following seeding TTL expression was low,

but dramatically increased during cell polarization from day 2 to 4.

Once the monolayer had been formed at day 5, TTL quantities

once again declined. Hence, the synthesis of TTL is at a maximum

level when MDCK cells are fully polarized. This is in accordance

with the observed decline of detyr-tubulin during cell polarization

(Fig. 1D), since an increase of TTL is expected to enhance the

tubulin tyrosination.

We next artificially decreased detyr-tubulin in MDCK cells by

continuous expression of GFP-tagged TTL (TTL–GFP). TTL–

GFP-expressing MDCK cells (MDCKTTL–GFP) were selected

after transfection in the presence of neomycin and positive clones

stably expressing TTL–GFP were verified by western blot with

anti-TTL and anti-GFP antibodies (supplementary material Fig.S3A). The amount of detyr-tubulin was dramatically decreased in

MDCKTTL–GFP (Fig. 3A,B). Interestingly, polyglutamylatedtubulin was also depleted to about 50% by TTL overexpression.The concentration of tyr- or acetylated tubulin was notsignificantly altered. The reduction in detyr-tubulin in

MDCKTTL–GFP cells was verified by immunohistochemistry withantibodies against tyr- and detyr-tubulin in polarized epithelialcells. A mixture of MDCK and MDCKTTL–GFP cells was analysed

by immunofluorescence microscopy using antibodies directedagainst tyr- or detyr-tubulin 1–4 days after seeding (Fig. 3C;supplementary material Fig. S3B). In direct comparison,

detyrosinated microtubules of non-polarized TTL-overexpressingcells were less frequent compared to control cells. Furthermore,detyr-tubulin was barely detectable in polarized MDCKTTL–GFP

cells, whereas several detyr-tubulin-enriched tubules could be seen

in control MDCK cells. For a quantitative assessment of the effectof TTL overexpression on detyr-tubulin, we used a nocodazoledepolymerisation assay (Khawaja et al., 1988). Nocodazole was

added for various times at a concentration (10 mM) that slowlydepolymerizes stable detyr-tubulin microtubules (supplementarymaterial Fig. S3C). Following a 1 hour treatment with nocodazole,

about 40% of polarized MDCK cells were positive for detyr-tubulin. In contrast, the number of detyr-tubulin-positive polarMDCKTTL–GFP cells was negligible. After nocodazole treatment

for longer periods of time, only a few detyr-tubulin-positiveMDCK cells remained. Thus, polarized MDCK cells harboursignificant amounts of stable detyr-tubulin-enriched microtubules,which slowly depolymerize in the presence of nocodazole. Our

data indicate that this pool of stable microtubules was almostabsent in MDCKTTL–GFP cells at the start of the experiment.

We also addressed whether overexpression of TTL in MDCK

cells affects the ratio of tubulin polymer to soluble dimer. It hadpreviously been shown that TTL binds preferentially to non-assembled tubulin (Arce et al., 1978), so changes in the level of

this enzyme might also influence the total amounts of assembledmicrotubules. We therefore, separated assembled microtubulesfrom soluble tubulin dimers by centrifugation. Antibodiesdirected against cytosolic GAPDH and the microtubule-

associated motor protein KIF5B where used as a control todiscriminate between microtubules in the pellet fractions andsoluble material in the supernatants. Surprisingly, the amount

of assembled versus soluble tubulin was almost equal orsignificantly elevated in MDCKTTL–GFP cells up until 3 daysafter seeding (supplementary material Fig. S4). This changed

following cell polarization, with a decrease in the amount ofpolymerized tubulin. This change in the balance can be explainedby constantly elevated TTL levels in MDCKTTL–GFP cells; the

enzyme would normally decline following cell polarization(Fig. 2B,C). Elevated TTL levels may thus sequester tubulinsubunits and prevent them from being incorporated intomicrotubules, as previously described (Szyk et al., 2011).

Changes in the morphology of TTL-overexpressingMDCK cells

Based on our working hypothesis that changes in the level ofdetyr-tubulin during the process of epithelial polarization arerequired for the correct formation of epithelial cell sheets, we

then studied the formation of polarized MDCKTTL–GFP cellmonolayers in greater detail. Already by 1 day after plating,MDCKTTL–GFP cells showed a much higher tendency to join into

Fig. 2. TTL expression in MDCK cells. Filter grown MDCK cells were

(A) fixed (5 days after seeding) and immunostained with polyclonal antibody

(pAb) anti-TTL/Alexa Fluor 546 for confocal microscopy or (B) lysed after

the indicated number of days in culture for immunoblotting with pAb anti-

TTL. (C) Quantification of the data depicted in B. The band intensities were

normalized to a GAPDH control. Significance was tested with Student’s t-test

(*P,0.05, **P,0.007), n54. Scale bar: 10 mm.

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islands of 10–30 cells than MDCK cells (Fig. 4A). As indicated by

immunostaining of claudin-1 in the upper part of laterally aligned

plasma membranes, tight junctions had already been assembled

12 hours after plating in MDCKTTL–GFP cells (Fig. 4B, upper

panel). In contrast, in MDCKGFP control cells the majority of

claudin-1 was still in intracellular vesicles. Moreover, ZO1 and E-

cadherin were aligned earlier along lateral membranes in

MDCKTTL–GFP than in MDCKGFP cells (supplementary material

Fig. S5A). If cells polarized after 5 days were immunostained, no

differences in the distribution of these polarity markers could be

detected (Fig. 4B, lower panel; supplementary material Fig. S5B).

Comparison of the ground areas of the two cell lines revealed that

12 hours after plating, each MDCKTTL–GFP cell was significantly

smaller than an average MDCKGFP cell (supplementary material

Fig. S5A,C), thus suggesting that the cells become taller during

island formation as indicated by x/z scans (Fig. 4A). TTL

overexpression also correlated with a higher tendency to

assemble into islands of prematurely polarized cells, as

demonstrated by time-lapse analysis (Fig. 4C; supplementary

material Movies 1, 2). MDCKGFP cells formed an almost confluent

cell layer 32 hours after seeding whereas MDCKTTL–GFP cells

seeded at the same density still exhibited extensive lacunae

between cell islands. Another parameter that can be used to

determine monolayer formation is the transepithelial resistance

(TER). This value increased during monolayer formation of

MDCKGFP cells until it reached a plateau when the monolayer

became tight (supplementary material Fig. S5D). TER values for

MDCKTTL–GFP cells increased slightly more slowly compared to

controls, indicating a delay in monolayer formation. In summary,

these data suggest that TTL overexpression in MDCK cells

induces changes in the course of monolayer formation.

TTL expression modulates the apical surface delivery of

gp80 and p75 in MDCK cells

Next, we wished to study whether enhanced levels of TTL also

affect the directed transport of cargo molecules in polarized

epithelial cells. To this end, surface molecules of the apical or

basolateral membrane of MDCKGFP and MDCKTTL–GFP cells

were biotinylated and harvested with streptavidin–Sepharose

beads. Fig. 5A depicts the pattern of proteins isolated from the

two membrane domains in comparison to the corresponding cell

lysates separated by SDS-PAGE analysis and silver stained.

Prominent proteins from the apical and, to a lesser extent, from

the basolateral surface of MDCKGFP cells were dramatically

depleted in MDCKTTL–GFP cells. This can be explained either by

a defect in forward trafficking to the cell surface, or an

accelerated uptake into the cell in detyr-tubulin depleted cells.

To elucidate this, we studied the kinetics of surface delivery of

soluble gp80, the major secretory protein of MDCK cells (Wada

et al., 1994). Secretion of this predominantly apically sorted

glycoprotein into the apical or basolateral medium from

MDCKGFP and MDCKTTL–GFP cells was monitored after

different periods of incubation (Fig. 5B,C). Quantification of

the data revealed a substantial decrease in the transport kinetics

of apical gp80 secretion in TTL-overexpressing cells with a

significance below 0.01 according to Student’s t-test. The effect

on basolateral trafficking was less pronounced and membrane

trafficking of the two basolateral proteins CD29 and E-cadherin

was not significantly affected in TTL-overexpressing cells

(supplementary material Fig. S6A,B). Confocal microscopy

revealed strong intracellular immunostaining of gp80 in

MDCKTTL–GFP cells, whereas only a few tubular gp80-positive

cisternae-like structures were detected in MDCKGFP control cells

Fig. 3. TTL overexpression depletes detyrosinated

microtubules. (A) Lysates of MDCK and MDCKTTL–GFP cells

incubated for 3 days post confluency were analysed by

immunoblot with antibodies directed against a-tubulin and detyr-,

tyr-, acetylated or polyglutamylated (polyglut) a-tubulin.

(B) Quantification of the data depicted in A. The band intensities

were normalized to GAPDH. ***P,0.0001, n53. (C) MDCK and

MDCKTTL–GFP cells (green) were mixed, seeded on coverslips and

fixed after 1 to 4 days. Detyr- or tyr-tubulin were immunostained

with specific antibodies (red, Alexa Fluor 546). Nuclei are

indicated in blue. Scale bars: 10 mm.

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(Fig. 5D), which were possibly endosomal organelles, as

previously described (Apodaca et al., 1994). Together with the

surface biotinylation data, this would suggest that predominantly

apical transport pathways are affected in MDCKTTL–GFP cells and

that a reduced level of detyr-tubulin leads to a dramatic reduction

in apical protein secretion. The opposite effect would be

predicted upon TTL depletion, leading to an increase in levels

of detyr-tubulin.

Therefore, a specific siRNA-mediated knockdown of TTL was

performed with two TTL siRNAs in transiently transfected MDCK

cells and luciferase siRNA transfection as a mock control.

Following siRNA transfection, at 48 hours TTL was depleted to

less than 35% compared to controls (supplementary material Fig.

S7A,B). Fluorescence microscopic analysis of the polarity markers

E-cadherin, claudin and ZO-1 revealed that MDCK cells form

polarized cell monolayers after TTL depletion (supplementary

material Fig. S7C,D). Concurrently, transport kinetics of gp80 to

the apical medium of TTL-depleted cells increased by 25% within

4 hours (Fig. 6A,B). Basolateral delivery of CD29 or E-cadherin

was not affected by TTL knockdown (supplementary material Fig.

S6C–E). In combination with the results from TTL-overexpressing

MDCKTTL–GFP cells, these data indicate that changes in TTL

expression substantially alter the apical transport kinetics of

soluble gp80.

To further assess whether apical trafficking of membrane-

associated glycoproteins is affected in TTL-depleted cells, we

studied the delivery of two polypeptides that use distinct apical

transport platforms and pathways. Lipid-raft-associated sucrase

isomaltase (SI) and non-raft p75–GFP (Delacour et al., 2007) were

biosynthetically labelled and immunoprecipitated from the two

membrane domains of polarized MDCKSI or MDCKp75–GFP cells

after TTL depletion or luciferase siRNA transfection. Fig. 6C

indicates that the proportion of p75–GFP that had reached the

apical plasma membrane was 30% higher in cells with reduced

amounts of TTL than in control cells. Lipid-raft-dependent apical

trafficking, however, was not affected by TTL depletion, as no

significant changes in the quantities of apical SI could be observed

(Fig. 6D). This was unexpected, but could be due to the

involvement of actin microfilaments as a rate-limiting step in

this transport pathway (Jacob et al., 2003). To clarify this

possibility, we analysed surface delivery of endogenously

expressed raft-associated gp135/podocalyxin in MDCKTTL–GFP

cells, which contain radically depleted levels of detyrosinated

microtubules. As assessed by biotinylation, the amount of surface-

resident gp135 was significantly reduced by TTL overexpression

(Fig. 6E). A partial compensation for this effect by simultaneous

knockdown of endogenous TTL and TTL–GFP (Fig. 6F) suggests

that, also for a raft-associated glycoprotein, apical trafficking can

Fig. 4. TTL overexpression promotes the assembly into islands of

prematurely polarised MDCK cells. (A) MDCK and MDCKTTL–GFP

cells were seeded at identical densities on coverslips, incubated for 1 or

5 days and fixed for immunostaining with anti-tubulin antibodies. Scale

bars: 20 mm. (B) 12 hours (upper panel) or 6 days (lower panel) after

seeding, MDCKGFP and MDCKTTL–GFP cells were fixed and

immunostained with pAb anti-Claudin (Alexa Fluor 546). Scale bars:

10 mm. For every time point and strain xy-scans and xz-scans are

depicted. GFP fluorescence is indicated in green. Nuclei are indicated

in blue. (C) Time-lapse recording by DIC microscopy from MDCKGFP

and MDCKTTL-GFP cells started 2 hours after seeding. Images obtained

after 0, 16 and 32 hours are depicted for each cell line (see also

supplementary material Movies 1, 2). Scale bars: 100 mm.

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be modulated by lowering the level of overexpressed TTL.

Moreover, in conjunction with the data observed for SI, apical

delivery of gp135 was not significantly improved if TTL was

knocked down in MDCK wild-type cells. Thus, we conclude that

the kinetics of raft-mediated apical trafficking cannot be further

optimized by TTL knockdown if the level of TTL is not artificially

increased.

Elevation of detyr-tubulin by TTL knockdown in MDCK cells

Finally, the influence of TTL knockdown on the tubulin

composition of MDCKSI or MDCKp75–GFP cells was verified by

immunoblots and immunofluorescence (Fig. 7). At first, antibodies

directed against detyr-, tyr- or acetylated tubulin were used for

immunoblots of control and TTL-depleted cells. Knockdown of

TTL resulted in a substantial increase in detyr-tubulin, whereas the

levels of tyr- or acetylated tubulin were not significantly altered

(Fig. 7A–C). Four days after seeding, levels of detyr- and tyr-tubulin

were monitored in control and TTL-depleted MDCK cells by

immunofluorescence. As seen in Fig. 7D detyr-tubulin staining is

much stronger than in cells expressing normal levels of the ligase

(see also supplementary material Fig. S7E,F). Altogether, these data

clearly demonstrate that TTL depletion raised the level of detyr-

tubulin in microtubules in MDCK cells.

In summary, elevated detyr-tubulin increases the transport rate

of soluble gp80 or non-raft-p75–GFP to the apical membrane. In

contrast, apical transport of gp80 or lipid-raft-associated gp135 is

disrupted by detyr-tubulin depletion, which can be rescued by

TTL knockdown. This suggests that detyrosinated microtubules

Fig. 5. Impaired surface trafficking in TTL-overexpressing MDCK cells.

MDCK (C, control) and MDCKTTL–GFP (TTL) cells were polarized on filters

for 5 days. (A) Proteins were biotinylated on the apical or basolateral surface

of the cells. Following cell lysis biotinylated material was isolated with

neutravidin beads, separated by SDS-PAGE and silver stained. Protein

patterns of the lysates are depicted on the right. Depleted protein candidates

from the surface domains of MDCKTTL–GFP cells are indicated with an

asterisk. (B) Medium from the apical or basolateral domain of the cells was

harvested after 5, 120 or 240 minutes and analysed by immunoblotting with

anti-gp80 antibodies. (C) Quantification of three experiments as shown in B.

Significance was tested with Student’s t-test (**P,0.01), n53. (D) Confocal

images of immunostained gp80 in polarized MDCKGFP and MDCKTTL–GFP

cells. Nuclei in xy- and xz-scans are indicated in blue. Scale bars: 10 mm.

Fig. 6. TTL knockdown enhances transport efficiency to the apical

membrane domain. (A) Medium from the apical or basolateral domain of

filter-grown MDCK cells transfected with TTL siRNA (TTL-kd) or luciferase

siRNA (control) was harvested after 5, 120 or 240 minutes and analysed by

immunoblotting. (B) Quantification of three experiments as in A. MDCKp75

(C) and MDCKSI (D) cells were grown on filters and biosynthetically labelled

with [35S]methionine for 15 minutes (p75) or 2 hours (SI) in the presence of

TTL siRNA or luciferase siRNA as a control. Cell surface

immunoprecipitation of p75 or SI from the apical (a) or basolateral (b) surface

was performed with the corresponding antibodies. The immunoprecipitates

were subjected to SDS-PAGE, followed by phosphoimager analysis and

quantification. Significance was tested with Student’s t-test (P,0.05), n53.

(E) Surface biotinylation followed by western blotting against the apical

marker protein gp135. High amounts of gp135 were transported to the apical

surface in MDCK wild-type cells with and without TTL knockdown. In

MDCKTTL–GFP cells, transport is heavily impaired and could be partially

restored through TTL-kd. (F) Knock down efficiencies of endogenous TTL

and TTL–GFP after siRNA transfection.

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are involved in lipid-raft-dependent and -independent apicalprotein trafficking in MDCK cells.

DiscussionThe role of post-translational modifications in the generationand maintenance of polarized epithelial cells is only poorlyunderstood.

In this study, we analysed differences in the quantities of tyr-and detyr-tubulin and monitored the consequences of animbalance in these two tubulin variants on the cell architecture

and the transport of glycoproteins to both membrane domains ofMDCK cells. A polarized distribution of tyr- or detyr-tubulin wasnot found in these cells, as equal amounts of each population

were detected at the apical and the basolateral cell poles in cellcysts or epithelial monolayers. In contrast, previous studies onthe distribution of post-translationally modified tubulin variants

in nerve cells have described a predominant location of tyr-tubulin in the distal region of the axon and in cell bodies; whereashighly detyrosinated domains of microtubules were found at their

minus ends (Baas and Black, 1990; Brown et al., 1992). InMDCK cells, both the tyrosinated and detyrosinated isoforms

were present along most of the length of double positive

microtubules with alternating sections of the two isoforms.

Segmental labelling of microtubules has been describedpreviously (Geuens et al., 1986; Webster et al., 1987). Detyr-

tubulin stretches can be generated by an individual turnover from

tyr- to detyr-tubulin, which is facilitated on pre-existingmicrotubules (Arce and Barra, 1985). Long stretches of detyr-

tubulin have been found in the Chlamydomonas flagellum along

the outer wall of the B tubules (Johnson, 1998). The authors

speculate that these post-translational modifications of the tubulesurface may enhance dynein motor interactions.

As shown in this study and by Quinones et al., the number ofdetyrosinated microtubules increases as the cells become polarized

(Quinones et al., 2011). In agreement with observations on neuronal

cells (Bisig et al., 2009), these detyr-tubulin-enriched microtubulesare frequently curled. Comparatively rapid accumulation of detyr-

tubulin has been reported for myoblasts cultured in differentiation

Fig. 7. TTL knockdown elevates detyrosinated microtubules in MDCKp75 and MDCKSI cells. (A) Lysates of MDCKp75–GFP or MDCKSI cells transfected

with the indicated siRNAs and incubated for 4 days were analysed by immunoblotting with antibodies against TTL, tubulin, detyr-, tyr- and acetylated tubulin.

The immunoblots from MDCKp75–GFP (B) and MDCKSI (C) cells were quantified, and band intensities were normalized to GAPDH. n53. (D) MDCK cells

transfected with luciferase siRNA or TTL siRNA were immunostained against detyr- and tyr-tubulin. Knockdown efficiency of TTL was assessed by

immunoblotting as indicated in the right panel. E-cadherin was labelled as loading control. Scale bars: 50 mm.

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medium (Gundersen et al., 1984). This morphological differentiationwas blocked by inhibition of microtubule detyrosination (Chang et al.,

2002), which suggests a detyr-tubulin-dependent mechanism formorphological transformations. In agreement with our data, thedevelopment of extensive cell to cell interactions between MDCK orVero cells was found to be accompanied by an increase in

microtubule detyrosination (Bre et al., 1991) in confluent,subconfluent or individual cells within a time interval of 1 day,which is reflected by the initial rise in detyr-tubulin up to 2 days after

seeding detected in this study.

Interestingly, our data suggest that TTL overexpression affectsthe ratio of soluble to polymerized tubulin in MDCK cells. Since

it has been shown that TTL binds efficiently to dimeric tubulin(Arce et al., 1978) and forms an elongated complex with thetubulin dimer it may prevent its incorporation into microtubules(Szyk et al., 2011). This scenario seems to prevail in polarized

MDCKTTL–GFP cells, which maintain constantly high levels ofthe enzyme. However, converse ratios between assembled andsoluble tubulin measured 3 days after seeding cannot be

explained by the sequestration of tubulin dimers. These ratiosare in accordance with the situation in human osteosarcoma cells,where TTL overexpression reduced the average growth rates of

microtubules (Szyk et al., 2011). In addition, microtubuledynamics can be modulated by the presence or absence of cell-specific factors such as CLIP-170 (Peris et al., 2006), which

indicates the complexity of this process. Do variations in thelevel of assembled tubulin within the detected range affectmembrane trafficking in polarized MDCKTTL–GFP cells? Sincemicrotubules appear to be involved in the transport of proteins to

both plasma membranes of MDCK cells (Grindstaff et al., 1998),and since the delivery of two basolateral cargo proteins wasunaffected in MDCKTTL–GFP cells, a general effect on trafficking

does not seem plausible. Moreover, it is unlikely that thedramatic reduction in apical p75 transport or gp80 secretionresults exclusively from the observed 20–30% reduction in

assembled tubulin. However, we cannot exclude the participationof this decrease on apical trafficking in MDCKTTL–GFP cells.

Another query is how TTL overexpression can result in thedepletion of detyrosinated microtubules. TTL acts preferentially

on soluble tubulin, and the detyrosination reaction preferentiallytakes place on microtubules (Arce et al., 1978; Arce and Barra,1985; Deanin et al., 1980). One could argue that a shift in the

equilibrium towards tyr-tubulin elevates the assembly oftyrosinated microtubules and pushes the balance towards arelative decrease in detyrosinated microtubules. Indeed, the

opposite scenario, of low TTL protein expression beingparalleled by increased detyr-tubulin is a common feature ofseveral types of cancer cells (Kato et al., 2004; Mialhe et al.,

2001; Soucek et al., 2006). Increasing the amount of TTL inHEK293T cells accordingly results in a decrease in detyr-tubulin(Kato et al., 2004). It seems that the story is not simple, as we didnot detect elevated levels of tyr-tubulin in MDCKTTL–GFP cells,

possibly suggesting that changes in the equilibrium are beingcompensated by additional cellular mechanisms. Alternatively, itmight well be that alterations in the amount of soluble tyr-tubulin

are covered by the considerably large pool of assembled tyr-tubulin (Gundersen et al., 1984).

Nevertheless, variations in the equilibrium of the two tubulin

modifications have dramatic effects on cellular morphology andfunction. An increase in tubulin detyrosination during epithelial-to-mesenchymal transition (EMT) in immortalized human

mammary epithelial cells promotes morphological alterations,which results in enhanced reattachment to endothelial cells

(Whipple et al., 2010). However, excess tubulin detyrosination inmammalian TTL-null fibroblasts results in defects in both spindlepositioning during mitosis and cell polarization during interphase(Peris et al., 2006). In conclusion, a sudden increase in the level

of detyr-tubulin to a specific degree is required for morphologicalchanges during cellular differentiation in a variety of celltypes. Reciprocally, artificial induction of tubulin-tyrosination

by TTL overexpression perturbs the differentiation into epithelialmonolayers. MDCKTTL–GFP cells are less mobile, assemble intocell islands and acquire a prematurely polarized morphology. The

lack of mobility could be explained by a depletion of detyr-tubulin-enriched stable microtubules in MDCKTTL–GFP cells,which are needed for the developing asymmetry of a cell as itbegins to migrate (Gundersen and Bulinski, 1988). It is thus

likely, that these immobile cells settle down, form cell islandsand develop cell-to-cell contacts with neighbouring partners intheir vicinity.

In polarized three-dimensional monolayers of MDCK cells, thelevel of detyr-tubulin decreases from day 2 to day 5, whereas theconcentration of tyr-tubulin remains constantly high and even

increases slightly in this time interval. A 100% increase in TTLlevels in the same period can be regarded as a reason for thisobservation, since knockdown of TTL reverses the effect andresults in high levels of detyr-tubulin. Similarly, EMT induction

in breast cancer cells is characterized by a decrease in thesynthesis of TTL and an increase in detyr-tubulin (Whipple et al.,2010). This indicates that constant amounts of TTL in epithelial

cell layers maintain the concentration of detyr-tubulin at a basiclevel. In addition, expression of TTL plays a vital role in miceand is essentially required for the polarization of neuronal cells,

since TTL-null neurons display morphogenetic anomalies (Ercket al., 2005).

The question arises about the function of a remaining pool of

detyr-tubulin-positive microtubules in fully polarized epithelialcells. Evidence for detyrosinated microtubules as cytoskeletaltracks for intracellular trafficking came from CHO cells. In thesecells, stable detyrosinated microtubules play a role in the

organization of endocytic recycling compartments and facilitatevesicular transport to the cell surface (Lin et al., 2002). A collapseof the recycling compartment was observed in anti-kinesin

antibody-injected cells, which suggests that kinesin motors areinvolved in endosome export. Particularly the kinesin-1 isoformKIF5b is essential for apical delivery of membrane proteins from

the trans-Golgi network to the cell surface (Jaulin et al., 2007). Wehave previously shown that the kinesin motor Kif5c is alsoinvolved in apical trafficking events following trans-Golginetwork exit (Astanina and Jacob, 2010). This motor

preferentially binds to and traffics along detyrosinatedmicrotubules in live cells (Dunn et al., 2008). In this presentreport we demonstrate that apical trafficking of soluble, raft-

associated or non-associated cargo proteins can be modulated byTTL expression and alterations in intracellular detyr-tubulin,which suggests that stable detyrosinated microtubules serve as

tracks for kinesin-driven post Golgi transport to the apicalmembrane domain of MDCK cells, especially considering thatkinesin motors do not move preferentially along dynamic

microtubules (Cai et al., 2009). Recent data on the function andstability of sensory cilia in C. elegans support the idea of post-translationally modified microtubules that guide molecular motors

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to specific subcellular destinations (O’Hagan et al., 2011). Theauthors propose that these tubulin modifications regulate thevelocity of kinesin-2 motors. This ‘tubulin code’ would thus

provide signposts that regulate the localization or activity of motors(Janke and Kneussel, 2010), which even contributes to theestablishment and maintenance of neuronal polarity in mammaliancells (Konishi and Setou, 2009). Autoinhibition is an alternative

instrument for the regulation of kinesin motors involved inintracellular trafficking (Hammond et al., 2010). However, apartfrom a general subapical concentration of microtubules, we did not

observe that detyrosinated or tyrosinated microtubules werespecifically focused to one of the two cell poles. A predominantlysubapical accumulation could be detected for acetylated tubulin as

previously shown (Jaulin and Kreitzer, 2010). Therefore, wepropose that additional factors assist in intracellular guidance tothe specific membrane domains of these cells.

Further evidence for the role of post-translational modifications ofmicrotubules in intracellular transport comes from cultured neuronalcells, in which acetylation promotes kinesin-1 binding and transport

of cargo to a specific subset of neurites (Reed et al., 2006). Yetanother kinesin motor, kinesin-3 UncA, from Aspergillus nidulans,preferentially binds to detyrosinated microtubules and is required for

fast hyphal extension (Zekert and Fischer, 2009). Kinesin-binding todetyrosinated microtubules is not merely involved in intracellulartransport events because it can, in addition, cross-bridge these

microtubules with specific intermediate filaments (Liao andGundersen, 1998). Cytoskeletal tracks can further be characterizedby the alignment with small GTP-binding septins. Spiliotis et al. have

shown that septin-2 coupling of polyglutamylated microtubules topost-Golgi vesicle transport is required for the morphogenesis ofpolarized MDCK cells (Spiliotis et al., 2008).

Irrespective of the fundamental insight in epithelial biology, agreater understanding of the role of alterations in tubulinmodification in epithelial cells is of wider interest because

pathomechanisms that occur in EMT change the balance oftubulin tyrosination and thus promote cell metastasis. We showhere, for the first time, that changes in the pattern of post-

translational tubulin modifications affect monolayer formationof, and membrane trafficking in MDCK cells. Further studies onall components involved and their interactions should provide an

integrated picture of the function of tubulin modifications in theestablishment and maintenance of epithelial integrity.

Materials and MethodsCell culture, siRNA transfection and surface immunoprecipitation

MDCK cells were cultured at 37 C under 5% CO2 in minimum essential medium(PAA) containing 5% foetal calf serum or in Dulbecco’s modified Eagle’s medium(4.5 g/l glucose) containing 10% FCS supplemented with antibiotics andglutamine. For generation of a cell line stably expressing TTL–GFP thetransfected cells were selected in medium containing neomycin for at least 90%fluorescence. For the generation of polarized monolayers of MDCK orMDCKTTL–GFP cells ,36107 cells per 10 cm culture dish or equivalentquantities per six-well filter were seeded. Cells were counted in a Neubauerchamber. Three-dimensional MDCK cell cysts were obtained by using BDMatrigelTM Basement Membrane Matrix Phenol-Red-Free (BD Biosciences). Inshort, cells were diluted to ,26104 cells/ml PBS and mixed with the same volumeof Matrigel in a cold environment. 30 ml of this mixture were spread out on cold12 mm coverslips lying in cell culture dishes, covered with medium after gellingand incubated for 7–10 days at 37 C, in 5% CO2 with regular medium change.Plasmid and small interfering RNA (siRNA) transfection of MDCK cells wasperformed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’sinstructions. Biosynthetic labelling of MDCKp75 or MDCKSI cells, accumulationof newly synthesized material in the trans-Golgi network at 20 C and surfaceimmunoprecipitation were performed essentially as described before (Cramm-Behrens et al., 2008). Images generated on a phosphoimager were analysed withthe QUANTITY ONE software (Bio-Rad).

DNA constructs

For construction of a TTL-containing plasmid the total RNA was isolated from MDCKcells using the RNeasy Mini Kit (Qiagen) followed by generation of TTL cDNAaccording to the manufacturer’s specifications (Fermentas RevertAidTM M-MuLVreverse transcriptase) using a reverse TTL primer (59-ggatcccccagcttgatgaacgcagc-39).The cDNA was used as template in a PCR (forward TTL primer: 59-gaattcatgtacaccttcgtggtg-39 and reverse TTL primer) and the PCR product was thencloned into pEGFP-N1 vector using the EcoRI and BamHI restriction sites.

RNA interference

For RNA-mediated interference (RNAi) experiments, specific depletion of TTLwas performed with the following two duplexes – (1) 59-UGUUUGGAUU-GCAAAGUCAUU-39/39-UUACAAACCUAACGUUUCAGU-59, (2) 59-CAUU-ACCUUGGAAAGUAGUUU-39/39-UUGUAAUGGAACCUUUCAUCA-59.

As a control, luciferase siRNA was used (59-CGUACGCGGAAUACUUCGA-TT-39/59-UCGAAGUAUUCCGCGUAC+GTT-39).

Antibodies

The following tubulin antibodies were used: monoclonal anti-a-tubulin (Clone DM1A) and anti-acetylated a-tubulin (Sigma-Aldrich), monoclonal anti-tyrosinated a-tubulin (YL1/2, Santa Cruz), polyglutamylated tubulin (GT335, AdipoGen)polyclonal anti-detyrosinated a-tubulin (Millipore) and monoclonal anti-detyrosinated a-tubulin antibody (Synaptic Systems). The polyclonal anti-detyrosinated tubulin antibody (Millipore) not recognizing the polyglutamylatedform of tubulin was used for immunoblots and GSDIM imaging. The followingpolyclonal antibodies were used: anti-TTL (Proteintech Group), anti-Claudin-1(Invitrogen), anti-clusterin-a (gp80; Santa Cruz), anti-b-catenin (Sigma), anti-Kif5B (Abcam) and anti-ZO1 (Zymed). The monoclonal antibodies anti-E-cadherin, anti-CD29 and anti-GAPDH (6C5) were purchased from BD Biosciences(E-cadherin, CD29) and HyTest Ltd (GAPDH). The monoclonal anti-SI,monoclonal anti-p75 (ME20.4) and anti-gp135 antibodies were generouslyprovided by H. P. Hauri (Biozentrum Basel, Switzerland), A. Le Bivic (Facultedes Sciences de Luminy, Marseille, France) and G. Ojakian (State University ofNew York Health Science Center, New York, USA).

Biochemical assays and protein analysis procedures

For preparation of cell lysates the cells were collected in lysis buffer (25 mM Tris-HCl, 50 mM NaCl, 0.5% sodium desoxycholate, 0.5% Triton X-100, pH 8.0) afterthe indicated times, incubated at 4 C on a rotating platform, centrifuged for5 minutes at 12,000 g and the supernatants were separated by SDS-PAGE usingthe Hoefer-Mini-VE system (Amersham Pharmacia Biotech) and transferred tonitrocellulose membranes. The membranes were blocked in 5% skimmed milkpowder in PBS for 1 hours and incubated overnight at 4 C with specific antibodies.Detection was performed with horseradish-peroxidase-conjugated secondaryantibody visualized by ECL (Thermo Fischer Scientific) and an Intas gel imagerCCD camera. The results were quantified using LabImage 1D software.

Microtubule assembly was studied essentially as previously published (Ligonet al., 2003). Cells were harvested 1 (non polar), 3 or 5 days (polar) after seeding,centrifuged and resuspended in pre-warmed medium (37 C) containing 30 mMHEPES, pH 6.1. The cells were washed once in pre-warmed medium, resuspendedand left to recover at 37 C for 15 minutes. After two additional washing steps inpre-warmed PBS, PHEM/2 M glycerol/2% Triton X-100 (50 mM PIPES, 50 mMHEPES, 1 mM EDTA, 2 mM MgCl2, pH 6.9) was added, mixed and incubated for10 minutes at 37 C. Following a centrifugation step (2 minutes, 8000 g) thesoluble tubulin fraction was separated from the polymerized tubulin pellet. Thepolymer to dimer ratio was calculated using the LabImage 1D software. Tubulinsignals were normalized to GAPDH from identical blots. Values of polymerizedtubulin (P) were divided by total tubulin (sum of P and S) for ratio presentations.

For surface biotinylation cells were incubated with the crosslinker Sulfo-NHS-Biotin (Thermo Scientific) to biotinylate surface proteins. After several washingsteps with PBS plus 1 mM CaCl2 and 1 mM MgCl2 (PBS++)/0.1 M glycin andPBS++ alone, the cells were frozen at 220 C and lysed in 20 mM Tris, 150 mMNaCl, 5 mM EDTA, 1% Triton X-100. Together with BSA the cell lysates wereincubated with neutravidin beads (Thermo Scientific) to select the biotinylatedsurface proteins. After renewed washing steps the proteins were eluted with SDS/dithiothreitol-containing buffer, separated by SDS-PAGE and stained using asilver staining procedure. The secretion of gp80 was analysed by replacing theapical and basolateral medium of the relevant cells, then after the indicated timeintervals removing and concentrating this medium. The gp80 content was tested byimmunoblotting. For quantification, cell lysates were separated by SDS-PAGE andsilver stained. The samples were normalized to prominent lysate bands.

Immunofluorescence staining

For immunofluorescence analysis of tubulin modifications, Claudin-1, E-cadherinand ZO-1, the cells were fixed with ice-cold methanol for 5 minutes and blockedin 1% milk powder/PBS++ for 1 hour. For TTL analysis the fixation wasperformed in 4% paraformaldehyde for 10–15 minutes followed by a

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permeabilization step in acetone (5 minutes) and 1 hour blocking in 5% FCS/PBS++. The incubation with primary antibody was performed in blockingreagents/PBS++ for 2 hours and with secondary antibody labelled with theindicated AlexaFluor dye in PBS++ for 1 hour. Nuclei were stained with Hoechst33342. Three-dimensional cysts were stained in a similar manner but with longerincubation times: methanol fixation 20 minutes, blocking 2 hours, primaryantibody 24 hours, secondary antibody 14 hours, Hoechst 33342 30 minutes,washing steps 20 minutes each.

Time-lapse experiments

MDCKGFP or MDCKTTL–GFP cells were seeded on glass-bottomed dishes (WillCoDishH, Willco Wells) and incubated until ,40–50% confluency (2 hours). Afterreplacement of medium with 100 mM HEPES-containing medium the cells wereused for microscopic time-lapse experiments.

Microscopy

Confocal images were acquired on a Leica TCS SP2 AOBS microscope using a406 oil planapochromat objective (Leica Microsystems). The live-cell time-lapseexperiments were recorded on a Leica DMI6000 B using a 206 dry fluotarobjective. The total internal reflection fluorescence microscopy (TIRFM) wasperformed as described before (Schneider et al., 2010) using a Leica DMI6000 Bmicroscope with an HCX PL APO 1006 objective. The high resolution imageswere acquired using a Leica SR GSD super-resolution system (Leica) according tothe manufacturer’s specifications. Data evaluation was performed with the Leicasoftware in combination with the Volocity imaging software package(Improvision).

TER measurement

For the determination of the transepithelial resistance, MDCKGFP andMDCKTTL–GFP cells were seeded onto filters, incubated at 37 C, and starting8 hours after seeding, the medium was changed and TER measurement wasperformed every 24 hours using the Millicell ERS-2 Voltohmmeter (Millipore) induplicate.

AcknowledgementsWe are grateful to M. Dienst and W. Ackermann for technicalassistance. We thank P. Holz and L.A. Ligon for advice and forproviding the tubulin ratio protocol, J.M. Przyborski for carefulreading of the manuscript and B. Lamp for help with the MillicellERS-2 Voltohmmeter (Millipore).

FundingThis work was supported by the Deutsche Forschungsgemeinschaft,Bonn, Germany [grant numbers JA 1033 to R.J., Graduiertenkolleg1216 to R.J., and Sonderforschungsbereich 593 to R.J.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.109470/-/DC1

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