Am J Physiol Cell Physiol 297: C1168–C1177, 2009.
First published September 2, 2009; doi:10.1152/ajpcell.00237.2009.
T-cadherin is located in the nucleus and centrosomes in endothelial cells
Alexandra V. Andreeva,1 Mikhail A. Kutuzov,1 Vsevolod A. Tkachuk,2
and Tatyana A. Voyno-Yasenetskaya1
1
Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois; and 2Cardiology Research Center,
Moscow, Russia
Submitted 1 June 2009; accepted in final form 31 August 2009
cadherin family; cell cycle; cytokinesis; glycosylphosphatidylinositol
anchor; human pulmonary artery endothelial cells
T-CADHERIN IS A GLYCOPROTEIN that belongs to the cadherin cell
adhesion family. In contrast to canonical cadherins, T-cadherin
lacks many amino acids crucial for Ca2⫹-dependent intercellular dimerization (15, 60). As a consequence, it is monomeric
in the absence and in the presence of calcium (15) and is
thought to mainly function as a signaling molecule. Another
unique feature of T-cadherin is the absence of the transmembrane and cytoplasmic domains [although a rare cell linespecific transmembrane form has been reported (54)]. T-cadherin is anchored within lipid rafts through glycosylphosphatidylinositol (GPI) (60, 68) and may possibly use cytoplasmic
domains of several interactive partners (58) to transduce signals inside the cell. T-cadherin may operate through integrinlinked kinase (ILK), which is upstream of the Akt/GSK3␤/␤catenin pathway (41, 42). It has been suggested and confirmed
in an in vivo model that T-cadherin may play an antiapoptotic
and protective role under hypoxic conditions (30, 42).
In confluent endothelial cells (ECs) T-cadherin, unlike vascular endothelial (VE)-cadherin (which is mainly present at the
cell-cell junctions), is located over the entire cell body with
only a slight enrichment at cell borders, whereas in wounded
Address for reprint requests and other correspondence: A. V. Andreeva and
T. A. Voyno-Yasenetskaya, Dept. of Pharmacology (MC 868), Univ. of
Illinois at Chicago, 909 S. Wolcott Ave. COMRB, Chicago, IL 60612 (e-mail:
[email protected] and [email protected]).
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cultures it is polarized to the leading edge of migrating cells
(59). Some recent reports described nuclear localization of
cleaved cytoplasmic domains of canonical cadherins (21, 66)
and their potential role in the modulation of gene transcription (21).
Several lines of evidence point to a potential involvement of
T-cadherin in cell-cycle regulation in different cell lines. Tcadherin expression is decreased or undetectable in some
tumor samples and various cancer cell lines (63, 73). In
contrast, overexpression of T-cadherin was demonstrated in
tumorigenic liver tissue and a hepatocellular carcinoma cell
line (63). The role of T-cadherin in tumor neovascularization
was recently confirmed using in vivo model (30). T-cadherin
expression in T-cadherin-deficient C6 glioma cells results in
G2 phase arrest and aneuploidy, which is dependent on increased expression of p21CIP1 and is eliminated in p21CIP1deficient fibroblasts (35). Ectopic expression of Cdh1, one of
the substrate recognition components (E3 ligase) of the anaphase-promoting complex, stimulates T-cadherin degradation
(5). In human umbilical vein endothelial cells (HUVECs),
overexpression of T-cadherin leads to an increased expression
of cyclin D1, a key regulator of G1 to S-phase progression (24)
and to a rapid entrance into S-phase (38).
Here we report that in ECs a considerable proportion of
T-cadherin is located in the nucleus. It is also present in the
centrosomes and colocalizes with ␥-tubulin. In the telophase,
T-cadherin transiently concentrates in the midbody. Overexpression of T-cadherin results in an increase in the number of
multinuclear cells, whereas its downregulation leads to an
increased number of cells with multiple centrosomes. These
findings indicate that T-cadherin is a nuclear and centrosomal
protein and that its deregulation may interfere with the cell cycle
via disturbance in cytokinesis or centrosomal replication.
MATERIALS AND METHODS
Materials. The antibodies used were the following: Akt1, Bcl2,
calreticulin, GFP, GRP78, LAMP1, T-cadherin (sc-7940; designated
here as SC), ␥-tubulin, VE-cadherin, horseradish persoxidase (HRP)conjugated anti-goat antibody (Santa Cruz Biotechnology); FLAG
(M2; Sigma); histone 1 (AE-4; Millipore), GM130, heat shock protein
90 (Hsp90), PP5, Rab5, Rac1 (BD Transduction Laboratories), and
ZO-1 (Zymed). HRP-conjugated anti-mouse and anti-rabbit secondary
antibodies were from Amersham. Phalloidin, AlexaFluor 488, 594, or
633 anti-mouse, anti-rabbit, or anti-goat antibodies were from Molecular Probes. The affinity-purified polyclonal T-cadherin antibody
designated here as TK was described previously (70). FLAG-tagged
T-cadherin constructs were kindly provided by Drs. Kalpana Ghoshal
and Samson Jacob (Ohio State University) (5). T-cadherin constructs
and specificity of the antibodies used in this work are depicted in Fig. 1A and
Supplementary Fig. 1A.
Cell culture. Human embryonic kidney (HEK) 293A cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM), supple-
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Andreeva AV, Kutuzov MA, Tkachuk VA, Voyno-Yasenetskaya
TA. T-cadherin is located in the nucleus and centrosomes in endothelial
cells. Am J Physiol Cell Physiol 297: C1168 –C1177, 2009. First published September 2, 2009; doi:10.1152/ajpcell.00237.2009.—T-cadherin
(H-cadherin, cadherin 13) is upregulated in vascular proliferative disorders and in tumor-associated neovascularization and is deregulated in
many cancers. Unlike canonical cadherins, it lacks transmembrane and
intracellular domains and is attached to the plasma membrane via a
glycosylphosphatidylinositol anchor. T-cadherin is thought to function in
signaling rather than as an adhesion molecule. Some interactive partners
of T-cadherin at the plasma membrane have recently been identified. We
examined T-cadherin location in human endothelial cells using confocal
microscopy and subcellular fractionation. We found that a considerable
proportion of T-cadherin is located in the nucleus and in the centrosomes.
T-cadherin colocalized with a centrosomal marker ␥-tubulin uniformly
throughout the cell cycle at least in human umbilical vein endothelial
cells. In the telophase, T-cadherin transiently concentrated in the midbody and was apparently degraded. Its overexpression resulted in an
increase in the number of multinuclear cells, whereas its downregulation
by small interfering RNA led to an increase in the number of cells with
multiple centrosomes. These findings indicate that deregulation of Tcadherin in endothelial cells may lead to disturbances in cytokinesis or
centrosomal replication.
NUCLEAR AND CENTROSOMAL LOCATION OF T-CADHERIN
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mented with 10% fetal bovine serum. Human endothelial cells were
obtained at passage 3 from Lonza and cultured as described previously (4). Transient transfection of HEK 293A and ECs was performed using Lipofectamine 2000 (Invitrogen) and SuperFect (Qiagen), respectively, according to manufacturers’ instructions.
Confocal microscopy. Cells cultured on gelatin- or fibronectincoated coverslips were fixed with 3.7% paraformaldehyde or with
50% methanol (for preservation of centrosomes), followed by permeabilization in 0.5% Triton X-100. Cells were incubated with primary
antibodies followed by incubation with appropriate secondary antibodies, using Tris-buffered saline containing bovine serum albumin as
a blocking buffer.
siRNA-mediated depletion. Inhibition of T-cadherin expression was
performed using small interfering RNA (siRNA) designed by Dharmacon. Cells were transfected using siRNA transfection reagent
(Santa Cruz Biotechnology) or Lipofectamine 2000 (Invitrogen).
Control siRNA was purchased from Santa Cruz Biotechnology.
Nuclear fractionation. Cells from a 10-cm dish were washed twice
with PBS, recovered by scraping, and resuspended in 300 ␮l of lysis
buffer containing 20 mM Tris 䡠 HCl, pH 7.4, 100 mM NaCl, 10 mM
EGTA, 5 mM MgCl2, 0.5% Na deoxycholate, 0.5% Nonidet P-40, and
protease inhibitor cocktail (1:200 dilution, Sigma). Cells were left on
ice for 15 min and centrifuged at 1,000 g for 5 min. Completeness of
cell lysis was verified microscopically. The supernatant (“nonnuclear”
fraction) was removed, and the pelleted nuclei were washed twice in
lysis buffer.
Isolation of centrosomes. Centrosome isolation was performed
essentially as previously described (77). Briefly, confluent HUVECs
on four 10-cm plates were treated with cytochalasin D (1 ␮g/ml) and
0.2 ␮M nocodazol for 1 h at 37°C. Cells were collected in 8 ml
hypotonic lysis buffer [1 mM HEPES, pH 7.2, 0.5% Nonidet-40, 0.5
mM MgCl2, 1 mM DTT, protease and phosphatase inhibitors (1:200
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dilution, Sigma)]. Nuclei were pelleted by centrifugation at 2,500 g
for 10 min. The supernatant was underlaid with a 60% sucrose
cushion (0.8 ml), and centrosomes were sedimented at 25,000 g for 30
min. The cushion and two volumes of the buffer above it (2.4 ml total)
were collected, mixed, and laid on the top of a discontinuous sucrose
gradient (1 ml 70%, 0.6 ml 50%, and 0.6 ml 40% sucrose) and
centrifuged at 100,000 g for 1 h. Fractions of 0.45 ml were collected
and diluted to reduce sucrose concentration to ⬍20%. Centrosomes
were pelleted at 25,000 g for 30 min and dissolved in the SDS sample
buffer for electrophoresis.
Immunoprecipitation and Western blotting. Cells were lysed in 50
mM HEPES, pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1% Triton
X-100. Proteins were immunoprecipitated with appropriate antibodies
(as specified in figure legends) and protein A/G agarose (Santa Cruz
Biotechnology) for 4 h at 4°C. Immunoprecipitates were washed three
times with the lysis buffer. Proteins were separated on 5–20% gradient
SDS gels and transferred onto polyvinyldifluoride (Osmonics) or
nitrocellulose (Protran, Shchleicher & Schull) membranes. Membranes were probed with appropriate antibodies and developed using
Dura reagents (Pierce). Densitometry of protein bands was performed
on scanned images using NIH Image 1.63 software.
RESULTS
T-cadherin is located in the nucleus in ECs. In accordance
with a report that T-cadherin is only slightly enriched at the cell
borders (59), we only occasionally observed cell surface staining in the primary cultures of ECs (Fig. 1B, indicated with
arrowheads at the top). In contrast, a robust staining of the
nuclei was observed using two different polyclonal T-cadherin
antibodies, designated here as TK [raised against synthetic
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Fig. 1. Endogenous T-cadherin is present in the nuclei
in endothelial cells (EC). A: T-cadherin domain architecture and the rabbit polyclonal T-cadherin antibodies used
in this work. CD, cadherin domain; CRS, COOHterminal recognition sequence; SP, signal peptide. Antibodies (italicized) and positions of their epitopes are
shown. The TK antibody (70) recognizes human but not
mouse T-cadherin. The antibody designated as SC is
from Santa Cruz Biotechnology (cat. number sc-7940).
B: endogenous T-cadherin is present in the nuclei in three
lines of human ECs, derived from pulmonary artery
(HPAEC), umbilical vein (HUVEC), and microvasculature (HMVEC). Immunostaining was performed with
T-cadherin antibodies (TK or SC as indicated), and
mouse monoclonal VE-cadherin antibody, followed by
AlexaFluor 488- and AlexaFluor 594-conjugated secondary antibodies, respectively. Nuclei were visualized
with DAPI (blue). Arrowheads and arrows indicate the
presence of T-cadherin at the cell surface in some cells
and in a pair of punctate structures in a mitotic cell,
respectively. Bottom: primary antibodies were omitted.
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NUCLEAR AND CENTROSOMAL LOCATION OF T-CADHERIN
HUVECs is due to nonspecific contamination by proteins from
postnuclear supernatant.
The predicted molecular mass of the mature protein without
signal peptide is ⬃65 kDa (60). In ECs, there are two major
forms of T-cadherin, mature 105 kDa and a partially processed
precursor 130 kDa, both of them are presumably glycosylated
(70). The results of subcellular fractionation clearly indicate
that both forms of T-cadherin are present in the nucleus. Both
T-cadherin antibodies gave similar nuclear patterns in immunofluorescence experiments (Fig. 1B). The SC antibody has not
been used in fractionation experiments (Fig. 2), because on
Western blots it failed to recognize full-length T-cadherin,
although it recognized truncated T-cadherin mutant ⌬C3-4
with high efficiency (Supplementary Fig. 1, A–C). Because
T-cadherin has several potential glycosylation sites, this might
be due to conformation sensitivity or epitope masking by
glycosyl group(s). Since the presence of T-cadherin in the
nucleus was highly unexpected and of a considerable potential
interest, we also confirmed this finding using polyclonal antibodies against CD1 and CD5 domains of T-cadherin (40),
respectively (Supplementary Fig. 1D).
Analysis of T-cadherin primary structure did not reveal any
canonical nuclear localization sequences, although it is enriched in basic residues and is predicted to be a nuclear protein
by the ESLPred (8) and SubLoc (28) algorithms. We also
found that T-cadherin contains a Leu-rich sequence LRFSLPS
VLLLSLFSLACL within its COOH-terminal recognition sequence (CSR) that perfectly matches a nuclear export signal
consensus Lx2–3Lx2–3LxL (46). Before mature GPI-anchored
proteins are delivered to the cell surface, they undergo processing in the endothelial reticulum (ER). Their CSRs are
removed by proteolytic cleavage in the ER simultaneously with
the attachment of the GPI anchor to the newly exposed COOHterminus (22). We examined the localization of FLAG-tagged
Fig. 2. Distribution of T-cadherin (105 kDa, mature
glycosylated form; 130 kDa, partially processed precursor) between nuclear and nonnuclear fractions of
HPAECs (A) and HUVECs (B) and assessment of the
purity of respective nuclear fractions (C) are shown.
Untransfected cells or cells transfected with FLAG-Tcadherin were fractionated into the nuclear (N, 1,000 g
pellet) and soluble (S, 1,000 g supernatant) fractions.
Proteins were separated on SDS-PAGE, transferred onto
a polyvinyldifluoride membrane, and probed with the TK
or FLAG antibodies. Positions of molecular mass markers (PageRuler, Fermentas) are indicated. In A and B,
quantification of the immunoblotting data for endogenous T-cadherin forms was performed using ImageJ
software. Data shown are means of three separate experiments (error bars ⫾ SD). H1, histone H1. C: fractions
shown in A and B were probed for various markers for
indicated subcellular structures and compartments (AJ;
adherens junctions; ER, endoplasmic reticulum; PM,
plasma membrane; TJ, tight junctions). 1Proteins that
have been reported in the nucleus. 2Proteins that have
been reported as functional partners of T-cadherin (see
main text for references).
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peptides of the first extracellular subdomain (70)] and SC
(raised against a peptide overlapping with CD2 and CD3
domains, Fig. 1A). Nuclear staining was observed in three
different cell lines (Fig. 1B): human pulmonary artery endothelial cells (HPAECs), human umbilical vein endothelial cells
(HUVECs), and human microvascular endothelial cells
(HMVECs). It was not due to the secondary antibody used in
these experiments, since omitting primary antibodies resulted
in the absence of nuclear staining (Fig. 1B, bottom).
To confirm the nuclear location of T-cadherin, we fractionated ECs into a nuclear fraction and a fraction containing other
cellular compartments and cytoplasmic proteins. The fractionation was confirmed using histone H1 as a nuclear marker (Fig. 2,
A and B). Approximately one-half of the total endogenous
T-cadherin was found in the nuclear fractions in HPAECs, as
revealed by Western blot analysis with the TK antibody, which
recognizes 105- and 130-kDa forms (Fig. 2A). A lower proportion (10 –20%) of endogenous T-cadherin was found in the
nuclear fraction of HUVECs (Fig. 2B). The absence of contamination of the nuclear fractions by proteins present in the
postnuclear supernatant was evident from reprobing the membranes for a number of markers for different subcellular compartments: Bcl2 (mitochondria), calreticulin (endoplasmic reticulum, ER), GRP78 [ER/plasma membrane (PM)], LAMP1
(lysosomes), VE-cadherin (adherens junctions), GM130 (Golgi),
Hsp90 (cytoplasm), Rab5 (early endosomes), ZO-1 (tight junctions). It is worth noting that some of the examined proteins
have been reported to either be present in the nucleus [PP5 (9),
ZO-1 (27), Akt1 (56), Rac1 (50, 51)] or to be functional or
interacting partners of T-cadherin [Akt1 (42), Rac1 (57),
GRP78 (58)]. Only trace amounts (except ZO-1 in HPAEC), if
any, of these proteins could be detected in the nuclear fractions
(Fig. 2C). These data effectively rule out a possibility that the
presence of T-cadherin in the nuclear fractions of HPAECs and
NUCLEAR AND CENTROSOMAL LOCATION OF T-CADHERIN
trosomes. Therefore, we examined whether T-cadherin would
colocalize with a centrosomal marker ␥-tubulin. We found a
clear colocalization between the two proteins throughout the
cell cycle (Fig. 3; methanol fixation was used in this experiment to optimize centrosome preservation, which is not optimal for visualization of the nuclei). This indicated that Tcadherin is indeed present in the centrosomes. In the telophase
it was often transiently concentrated in the midbody, with
reduced or undetectable presence in the centrosomes (note that
the brightness of the green channel is increased in the telophase
panel compared with other panels in Fig. 3). In HUVECs, these
T-cadherin-positive centrosomes could be observed throughout
the cell cycle, including mitosis. In HPAECs, a qualitatively
similar pattern was observed; however, centrosomal T-cadherin staining was most obvious in metaphase and anaphase.
Centrosome-associated T-cadherin could also be detected
using the TK antibody (Supplementary Fig. 2B), although less
efficiently than with the SC antibody (while both antibodies
stain the nuclei similarly, Fig. 1B). Lower efficiency of the TK
antibody might be due to partial unavailability of its epitope
(CD1). In PC12 cells, T-cadherin was reported to be ubiquitinated by Cdh1, a component of a major ubiquitination system
that controls the proteasome-dependent destruction of cellcycle regulators (5). Proteasomes are located in both the
nucleus and the centrosomes (see references in 16). Cdh1
localizes to the nucleus during interphase and to the centrosomes during metaphase and anaphase (78). This prompted us
Fig. 3. T-cadherin is located in the centrosomes in HUVECs. Cells were immunostained with the T-cadherin SC antibody and mouse monoclonal ␥-tubulin
antibody, followed by AlexaFluor 488- and AlexaFluor 594-conjugated secondary antibodies, respectively. Nuclei were visualized with DAPI (blue). The cell
cycle phases are A: mitosis: prophase, metaphase, anaphase and telophase; and B: interphase: G1, S, G2. Centrosomes are indicated with arrows. In the telophase
image, the brightness in the green channel has been enhanced to visualize T-cadherin in the midbody (*) and in centrosomes.
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T-cadherin construct, where the epitope is placed after the CSR
for GPI-attachment (see Supplementary Fig. 1A). COOH-terminal epitope tags attached after CSRs are not expected to
impede the proper protein processing and are removed when
the protein is processed in the ER (2, 7, 65), thus only the
molecules that are not (yet) processed are detectable with the
epitope-specific antibody. The nonprocessed FLAG-tagged Tcadherin could be exclusively detected in the nonnuclear fractions (Fig. 2B, Supplementary Fig. 1C), presumably in the ER,
taken into account a characteristic reticular pattern observed
with FLAG-tagged T-cadherin (Supplementary Fig. 1C).
We also examined the effect of leptomycin B (LMB, an
inhibitor of CRM1-dependent nuclear export) on distribution
of endogenous T-cadherin in HUVECs. The proportion of both
endogenous T-cadherin forms increased by 1.5- to 2-fold in the
nucleus upon LMB treatment (Supplemental Fig. 1E), indicating that the presence of T-cadherin in the nucleus is dynamically regulated. Because it is unlikely that mature T-cadherin
possesses this CSR sequence, it is possible that it interacts with
a partner with functional nuclear export signal.
T-cadherin is present in the centrosomes. In dividing
HPAECs, T-cadherin was associated with two punctate structures located on the opposite sides of chromatin and thus
resembling centrosomes (see Fig. 1B, lower HPAEC panel).
Z-sectioning of individual cells confirmed that each mitotic cell
contained exactly two T-cadherin-positive structures (Supplementary Fig. 2A), consistent with their identification as cen-
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NUCLEAR AND CENTROSOMAL LOCATION OF T-CADHERIN
Fig. 4. T-cadherin is degraded in a proteasome-dependent manner in HUVECs
(A), and cofractionates (B), and physically associates (C) with ␥-tubulin. A: confluent
cells were incubated in the presence of inhibitors [20 ␮M: inhibitor 1 (Inh.1), inhibitor
2 (Inh. 2), or MG132] or DMSO (control) as indicated, and T-cadherin levels were
analyzed by Western blot analysis with the TK antibody. Right: quantification of the
results (means of four replicates; error bars ⫾ SD). B: centrosomes were isolated from
untransfected HUVECs as described in MATERIALS AND METHODS. Sucrose gradient
fractions were analyzed for the presence of ␥-tubulin (centrosomal marker) and
T-cadherin. TL, total lysate. C: HUVECs were transfected with the ⌬C3-4 construct or
empty vector. Cells from two 10-cm plates were subjected to immunoprecipitation
with ␥-tubulin or GFP (negative control) antibodies as described in MATERIALS AND
METHODS. Immunoprecipitated material (IP) was probed with the T-cadherin (SC)
antibody. TL, total lysates.
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the 130-kDa form, of T-cadherin is associated with a subfraction of the centrosomes. Our data are also compatible with a
truncated form or nonglycosylated T-cadherin present in a
distinct centrosomal subfraction.
To examine whether T-cadherin and ␥-tubulin might physically associate, we performed immunoprecipitation from
HUVECs. We first attempted to assess whether endogenous
T-cadherin might associate with ␥-tubulin. However, endogenous T-cadherin could not be detected in ␥-tubulin immunoprecipitates, whereas reciprocal immunoprecipitation proved
inconclusive due to the nearly identical molecular weight of
␥-tubulin and the heavy immunoglobulin chain (data not
shown). Taking into account that only a small proportion of
endogenous T-cadherin is located in the centrosomes compared
with the nucleus in ECs, we next attempted to use overexpressed T-cadherin to assess the possibility of its interaction
with ␥-tubulin. Since the SC antibody recognizes the ⌬C3-4
T-cadherin construct, but not full-length constructs, on Western blots (as discussed above and shown in Supplementary Fig.
1B), HUVECs were transfected with the latter construct or
empty vector. Immunoprecipitation was performed using ␥-tubulin- and GFP-specific antibodies. The presence of a band of
the expected molecular size (see Supplementary Fig. 1B)
recognized by the SC antibody could be detected in the
material precipitated from T-cadherin transfected cells with the
␥-tubulin antibody but not in the negative controls (Fig. 4C).
No corresponding signal could be detected in the immunoprecipitates using FLAG antibody, consistent with a form that has
been COOH terminally processed in the ER.
To assess whether the centrosomal and nuclear location of
T-cadherin is specific for ECs, we examined localization of
endogenous T-cadherin in HEK 293A cells. Immunoblotting
analysis showed that HEK 293A (as well as COS-7) cells
express considerably lower levels of T-cadherin relative to
total protein than ECs (data not shown). We found that in HEK
293A cells, most of endogeneous T-cadherin colocalized with
␥-tubulin and thus was associated with the centrosomes [which
are unusually large structures in this cell line (55, 76)], with
only very faint nuclear and cell surface staining (Supplementary Fig. 3A). In an attempt to detect association of endogenous
T-cadherin with ␥-tubulin, we performed immunoprecipitation
experiments from HEK 293A cells. A ⬃60-kDa band could be
detected in ␥-tubulin immunoprecipitates but not in GFP antibody immunoprecipitates used as a negative control (Supplementary Fig. 3B). The molecular mass of this band is close to
that expected for nonglycosylated T-cadherin without prepeptide (65 kDa).
Interference with T-cadherin expression leads to defects in
cell division. As described above, T-cadherin was often transiently concentrated in the midbody, which plays a key role in
cytokinesis (6). This observation suggested that T-cadherin
might be involved in cytokinesis. Cytokinesis failure leads to
the appearance of cells containing more than one nucleus. To
assess whether overexpression or downregulation of T-cadherin might affect the probability of failed cytokinesis, we
examined the proportion of bi- or multinuclear cells among
HUVECs transfected with either T-cadherin cDNA or Tcadherin siRNA. A small proportion of ECs is naturally bi- or
multinuclear, and an increase in the proportion of such cells
has been associated with some physiological or pathological
conditions (19, 36, 69). In line with these reports, we observed
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to test whether the levels of T-cadherin in HUVECs would be
affected by proteasome inhibition. After 4-h incubation with
proteasome inhibitors I1 and MG132, the levels of T-cadherin
were significantly increased, whereas incubation with I2 (calpain inhibitor that does not affect proteasome) resulted in only
marginal, if any, increase in T-cadherin levels (Fig. 4A). These
data indicate that T-cadherin levels in HUVECs are under
control of proteasome-dependent degradation.
To confirm the centrosomal location of T-cadherin, we
isolated centrosomal fractions from HUVECs by differential
centrifugation using a 20 –70% discontinuous sucrose gradient.
The fractionation was confirmed by using ␥-tubulin as a
centrosomal marker. Immunoblotting showed a biphasic distribution of ␥-tubulin (Fig. 4B). The lower molecular mass
form, but not the 130-kDa form, of T-cadherin could be
detected in the centrosomal fractions using the TK antibody
and was mainly associated with the first of the two ␥-tubulin
peaks (Fig. 4B). Reprobing the same blot with the SC antibody
[which does not recognize full-length glycosylated T-cadherin
on Western blots but may efficiently recognize truncated
form(s), see above] revealed the presence of a ⬃60-kDa band,
associated with the second ␥-tubulin peak, and also found in a
denser fraction (Fig. 4B). Thus the results of centrosome
isolation indicate that only the mature processed form, but not
NUCLEAR AND CENTROSOMAL LOCATION OF T-CADHERIN
F), which suggests a specific role of T-cadherin in the control
of centrosomal organization.
DISCUSSION
We report here that a considerable proportion of T-cadherin
is located in the nucleus, as evidenced by immunofluorescence
and by immunoblotting upon cell fractionation using antibodies raised against different epitopes. Nuclear localization of
cleaved intracellular domains of some other members of the
cadherin family, such as protocadherins ␣ (18), ␥ (29), and
Fat1 (33, 49), E-cadherin (21) was reported. However, Tcadherin has no intracellular domain and is present in the
nucleus as a full-length molecule; therefore trafficking pathways suggested for other cadherins may not be relevant in the
case of T-cadherin. Full-length cell surface receptors, both G
protein-coupled receptors and tyrosine kinase receptors, were
reported to translocate to the nucleus (17, 20, 25, 53, 62, 72),
including LPA1, for which caveolin-mediated endocytosis was
suggested (26). Nucleus is rich in lipid environments, which
contain lipid raft components, including caveolin-1 (1). However, several lines of evidence argue against a role of caveolin
in the nuclear trafficking of T-cadherin: 1) Pertussis toxin
inhibits LPA1 trafficking to the nucleus (26) but does not affect
the nuclear localization of T-cadherin (our unpublished observations), which makes a common trafficking mechanism unlikely. 2) Although caveolae-dependent endocytosis was originally considered for GPI proteins (3), later studies suggested a
caveolae-independent pinocytotic pathway (44, 45, 64) or macropinocytotic pathways triggered by a clustering agent or being
ligand independent (14, 74). 3) In ECs, caveolin is not involved
in T-cadherin-mediated signaling (59). However, a recently
described T-cadherin interaction with GRP78 (58), which is
reportedly involved in caveolae-dependent internalization and
nuclear localization of extracellular matrix protein DMP1 (61),
reintroduces the question about a possible role of caveolae.
Fig. 5. T-cadherin overexpression increases
proportion of bi- or multinuclear cells.
HPAECs were transfected with the full-length
human T-cadherin construct. Cells were collected 24 h after transfection, and T-cadherin
expression was analyzed by Western blotting
(A) and by immunofluorescence (B, top and
middle). Bottom in B shows an example of
binuclear cells overexpressing T-cadherin 72 h
after transfection. C: HUVECs transfected with
the T-cadherin construct or glycosylphosphatidylinositol (GFP) were examined for alterations
in morphology 72 h after transfection. Percentage of cells with more than one nucleus is
shown relative to the total cell number counted
(50 – 80 cells per replicate, 4 replicates; error
bars ⫾ SD).
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that primary HUVEC cultures contained 4 –5% cells with more
than one nucleus (of which absolute majority were binuclear
cells).
T-cadherin could be efficiently overexpressed in ECs (Fig. 5A;
Supplementary Fig. 1C), resulting in an overall increase in
T-cadherin content of up to 10- to 20-fold (ECL quantification
data not shown). Immunofluorescence microscopy revealed
that in some cells T-cadherin was present in a reticular pattern
(Fig. 5B, top), probably reflecting its predominant presence in
the ER, whereas in other cells punctate cytoplasmic as well as
nuclear staining was observed (Fig. 5B, middle). The proportion of cells containing more than one nucleus was increased
among the cells overexpressing T-cadherin (Fig. 5, B and C).
Similar results were obtained using overexpression of FLAGtagged T-cadherin in HUVECs (data not shown) and in HEK
293A cells (Supplementary Fig. 3, C–E).
We also depleted endogenous T-cadherin in ECs using the
siRNA approach and examined the cells for any apparent
defects associated with cell division. Both 105- and 130-kDa
forms of T-cadherin could be efficiently downregulated in
HPAECs and in HUVECs (Fig. 6A). It was reported that the
levels of different cadherins may influence each other, for
example, VE-cadherin expression is reduced in N-cadherindeficient endothelium (48). VE-cadherin levels remained unchanged in the cells with 80 –90% T-cadherin depleted (Fig.
6A). In some experiments, 95–98% T-cadherin depletion was
achieved, which was accompanied by a 30 – 40% decrease in
VE-cadherin levels (data not shown). T-cadherin depletion also
resulted in a considerable decrease in T-cadherin-positive
staining in immunofluorescence experiments (Fig. 6, B and C).
T-cadherin depletion led to an only marginal increase in the
proportion of multinucleated cells, which was not statistically
significant (Fig. 6E). However, we noticed that downregulation
of T-cadherin was accompanied by an increased proportion of
mononuclear ECs with multiple centrosomes (Fig. 6, C, D, and
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In some cell lines, T-cadherin may be not GPI modified but
rather expressed as a protein with a transmembrane domain
(54). Thus it cannot be excluded that precise sites of processing
of T-cadherin in ECs might differ from those assumed on the
basis of canonical processing of GPI-anchored proteins.
If T-cadherin is processed as a canonical GPI-anchored
protein, the questions arise whether it is targeted to the nucleus
after its delivery to the PM or directly from the ER. Some
models for the nuclear translocation of cell surface receptors
have been suggested (47), and although these questions are
beyond the scope of this work, T-cadherin would be an excellent model to study this process.
Apart from the question of how T-cadherin is imported into
the nucleus, another question is what functional role the nuclear T-cadherin may play. Cleaved intracellular domain of
E-cadherin may modulate gene transcription (21). Some other
transmembrane receptors may also act as transcriptional factors
or modulators of expression (17, 20, 25, 34, 53, 62, 72). As
T-cadherin upregulates cyclin D1 (41) and may activate the
serum-response element (our unpublished data), a similar function seems conceivable for T-cadherin.
We also found that T-cadherin is located in the centrosomes.
A quantitative difference between venous (HUVECs) and arterial (HPAECs) endothelial cells could be observed, because
T-cadherin was present in the centrosomes uniformly throughout the cell cycle (except telophase) in HUVECs but was most
noticeable during metaphase in HPAECs. The mechanisms of
protein delivery to the centrosomes are poorly understood. For
AJP-Cell Physiol • VOL
some proteins, such as RGS14 (13), nuclear-cytoplasmic shuttling may be important. In this respect, it may be relevant that
the two lines of ECs studied here also differed in the proportion
of nuclear T-cadherin. Since our data suggest that T-cadherin
may play a role in cytokinesis, it is also worth noting that some
proteins required for cytokinesis are sequestered into the nucleus during interphase (6). Centrosomal T-cadherin may have a
longer half-life than T-cadherin in other compartments, since the
centrosomes were the last location where it could be detected
upon siRNA-mediated depletion. This is similar to the observations for centrosomal ␥-tubulin (67). In line with these data,
in HEK 293A cells (where T-cadherin expression is low), the
endogenous T-cadherin could only be observed in the centrosomes and to a minimal extent in the nucleus.
The role of T-cadherin in the centrosomes may be linked to
the control of their duplication, since siRNA-mediated Tcadherin depletion resulted in an increased fraction of cells
with multiple centrosomes. Since the data were collected for
the cells that have an increased number of centrosomes
without nuclear duplication, this reflects a defect in centrosomal duplication per se. Notably, depletion of most centrosomal proteins leads to cell cycle arrest and defects in
centrosome structure (52).
Our data also suggest that T-cadherin may play a role in
cytokinesis, since it transiently concentrates in the midbody
during telophase, and its overexpression leads to an increased
proportion of cells with more than one nucleus. Notably, both
the expression of T-cadherin (39) and the proportion of bi- and
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Fig. 6. Small interfering RNA (siRNA)-mediated depletion of T-cadherin in ECs results in multiple centrioles. A and B: downregulation of endogenous
T-cadherin in HPAECs and HUVECs. Cells were transfected with control (C) or T-cadherin (T) siRNA and analyzed by immunoblotting 24 or 72 h after
transfection (A) or by immunofluorescence 24 h after transfection (B; HPAECs). Vascular endothelial (VE)-cadherin was examined as a specificity control.
C: centrosomes in T cadherin-depleted HUVECs, visualized with ␥-tubulin antibody 72 h after transfection (methanol fixation). D: Z-sections (␥-tubulin staining)
of the cell shown in the bottom of C. In C and D, centrosomes are indicated by arrows. E and F: HUVECs transfected with T-cadherin or control siRNA were
examined for aberrations in the number of nuclei and centrosomes per cell, respectively, 72 h after transfection. E: percentage of cells with more than one nucleus
is shown relative to the total cell number counted (90 –170 cells per replicate, 4 replicates; error bars ⫾ SD). F: percentage of cells with ⬎2 centrosomes is shown
relative to the total cell number counted; interphase binuclear cells were disregarded (500 cells per replicate, 3 replicates; error bars ⫾ SD).
NUCLEAR AND CENTROSOMAL LOCATION OF T-CADHERIN
ACKNOWLEDGMENTS
We thank Drs. Kalpana Ghoshal and Samson Jacob (Ohio State University)
for the FLAG-tagged T-cadherin constructs, Aleksandar Krbanjevic for plasmid purification, and Dr. Jasmina Profirovic for critical reading of the manuscript.
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