Download 1 T-cadherin is located in the nucleus and centrosomes in

Articles in PresS. Am J Physiol Cell Physiol (September 2, 2009). doi:10.1152/ajpcell.00237.2009
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T-cadherin is located in the nucleus and centrosomes in endothelial cells.
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Alexandra V. Andreeva1*, Mikhail A. Kutuzov1, Vsevolod A. Tkachuk2, Tatyana A. Voyno-
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Yasenetskaya1*
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Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA.
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Cardiology Research Center, Moscow, Russia.
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* Corresponding authors. Deparment of Pharmacology (MC 868), University of Illinois at
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Chicago, 909 S. Wolcott Ave. COMRB, Chicago, IL 60612, USA.
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Tel.: +1 312 996 9823; fax: +1 312 996 1225.
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E-mail addresses: [email protected] (A. V. Andreeva), [email protected] (T. A. Voyno-
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Yasenetskaya).
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Running title: Nuclear and centrosomal location of T-cadherin
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Copyright © 2009 by the American Physiological Society.
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ABSTRACT
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T-cadherin (H-cadherin, cadherin 13) is upregulated in vascular proliferative disorders and in
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tumor-associated neovascularization, and is deregulated in many cancers. Unlike canonical
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cadherins, it lacks transmembrane and intracellular domains and is attached to the plasma
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membrane via a glycosylphosphatidylinositol anchor. T-cadherin is thought to function in
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signaling rather than as an adhesion molecule. Some interactive partners of T-cadherin at the
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plasma membrane have recently been identified. We examined T-cadherin location in human
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endothelial cells using confocal microscopy and subcellular fractionation. We found that a
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considerable proportion of T-cadherin is located in the nucleus and in the centrosomes. T-
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cadherin colocalized with a centrosomal marker γ-tubulin uniformly throughout the cell cycle at
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least in human umbilical vein endothelial cells. In the telophase, T-cadherin transiently
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concentrated in the midbody and was apparently degraded. Its overexpression resulted in an
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increase in the number of multinuclear cells, while its downregulation by siRNA led to an
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increase in the number of cells with multiple centrosomes. These findings indicate that
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deregulation of T-cadherin in endothelial cells may lead to disturbances in cytokinesis or
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centrosomal replication.
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Key words: cadherin family, cell cycle, cytokinesis, GPI anchor, HPAEC.
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INTRODUCTION
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T-cadherin is a glycoprotein that belongs to the cadherin cell adhesion family. In contrast to
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canonical cadherins, T-cadherin lacks many amino acids crucial for Ca2+-dependent intercellular
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dimerization (15, 60). As a consequence, it is monomeric in the absence and in the presence of
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calcium (15) and is thought to mainly function as a signaling molecule. Another unique feature
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of T-cadherin is the absence of the transmembrane and cytoplasmic domains (although a rare
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cell line-specific transmembrane form has been reported (54)). T-cadherin is anchored within
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lipid rafts through glycosylphosphatidylinositol (GPI) (60, 68) and may possibly use cytoplasmic
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domains of several interactive partners (58) to transduce signals inside the cell. T-cadherin may
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operate through integrin-linked kinase (ILK), which is upstream of the Akt / GSK3β / β-catenin
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pathway (41, 42). It has been suggested and confirmed in an in vivo model that T-cadherin may
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play an antiapoptotic and protective role under hypoxic conditions (30, 42).
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In confluent endothelial cells (ECs) T-cadherin, unlike VE-cadherin (which is mainly present at
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the cell-cell junctions), is located over the entire cell body with only a slight enrichment at cell
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borders, while in wounded cultures it is polarized to the leading edge of migrating cells (59).
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Some recent reports described nuclear localization of cleaved cytoplasmic domains of canonical
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cadherins (21, 66) and their potential role in the modulation of gene transcription (21).
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Several lines of evidence point to a potential involvement of T-cadherin in cell cycle regulation in
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different cell lines. T-cadherin expression is decreased or undetectable in some tumor samples
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and various cancer cell lines (63, 73). In contrast, overexpression of T-cadherin was
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demonstrated in tumorigenic liver tissue and a hepatocellular carcinoma cell line (63). The role
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of T-cadherin in tumor neovascularization was recently confirmed using in vivo model (30). T-
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cadherin expression in T-cadherin-deficient C6 glioma cells results in G2 phase arrest and
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aneuploidy, which is dependent on increased expression of p21CIP1 and is eliminated in p21CIP1-
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deficient fibroblasts (35). Ectopic expression of Cdh1, one of the substrate recognition
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components (E3 ligase) of the anaphase promoting complex, stimulates T-cadherin degradation
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(5). In human umbilical vein endothelial cells (HUVECs), overexpression of T-cadherin leads to
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an increased expression of cyclin D1, a key regulator of G1 to S-phase progression (24), and to
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a rapid entrance into S-phase (38).
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Here we report that in ECs a considerable proportion of T-cadherin is located in the nucleus. It
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is also present in the centrosomes and co-localizes with γ-tubulin. In the telophase, T-cadherin
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transiently concentrates in the midbody. Overexpression of T-cadherin results in an increase in
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the number of multinuclear cells, while its downregulation leads to an increased number of cells
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with multiple centrosomes. These findings indicate that T-cadherin is a nuclear and centrosomal
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protein and that its deregulation may interfere with the cell cycle via disturbance in cytokinesis
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or centrosomal replication.
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MATERIALS AND METHODS
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Materials. The following antibodies were used: Akt1, Bcl2, calreticulin, GFP, GRP78, LAMP1, T-
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cadherin (sc-7940; designated here as SC), γ-tubulin, VE-cadherin, HRP-conjugated anti-goat
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antibody (Santa Cruz Biotechnology); FLAG (M2; Sigma); histone 1 (AE-4; Millipore), GM130,
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Hsp90, PP5, Rab5, Rac1 (BD Transduction Laboratories), ZO-1 (Zymed). HRP-conjugated anti-
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mouse and anti-rabbit secondary antibodies were from Amersham. Phalloidin, AlexaFluor 488,
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594, or 633 anti-mouse, anti-rabbit, or anti-goat antibodies were from Molecular Probes. The
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affinity-purified polyclonal T-cadherin antibody designated here as TK was described previously
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(70). FLAG-tagged T-cadherin constructs were kindly provided by Drs Kalpana Ghoshal and
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Samson Jacob (Ohio State University) (5). T-cadherin constructs and specificity of the
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antibodies used in this work are depicted in Fig. 1A and Supplementary Fig. 1A.
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Cell culture. HEK 293A cells were cultured in Dulbecco's modified Eagle's medium (DMEM),
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supplemented with 10% fetal bovine serum. Human endothelial cells were obtained at passage
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3 from Lonza and cultured as described previously (4). Transient transfection of HEK 293A and
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ECs was performed using Lipofectamine 2000 (Invitrogen) and SuperFect (Qiagen),
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respectively, according to manufacturers’ instructions.
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Confocal microscopy. Cells cultured on gelatin or fibronectin-coated coverslips were fixed with
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3.7% paraformaldehyde or with 50% methanol (for preservation of centrosomes), followed by
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permeabilization in 0.5% Triton X-100. Cells were incubated with primary antibodies followed by
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incubation with appropriate secondary antibodies, using Tris buffered saline containing bovine
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serum albumin as a blocking buffer.
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siRNA-mediated depletion. Inhibition of T-cadherin expression was performed using siRNA
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designed by Dharmacon. Cells were transfected using siRNA transfection reagent (Santa Cruz
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Biotechnology) or Lipofectamine 2000 (Invitrogen). Control siRNA was purchased from Santa
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Cruz Biotechnology.
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Nuclear fractionation. Cells from a 10 cm dish were washed twice with PBS, recovered by
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scraping and resuspended in 300 μl of lysis buffer, containing 20 mM Tris-HCl, pH 7.4, 100 mM
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NaCl, 10 mM EGTA, 5 mM MgCl2, 0.5% Na deoxycholate, 0.5% Nonidet P-40, protease
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inhibitor cocktail (Sigma, 1:200 dilution). Cells were left on ice for 15 min and centrifuged at
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1000 g for 5 min. Completeness of cell lysis was verified microscopically. The supernatant
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(“non-nuclear” fraction) was removed and the pelleted nuclei were washed twice in lysis buffer.
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Isolation of centrosomes. Centrosomes isolation was performed essentially according to ref.
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(77). Briefly, confluent HUVECs on four 10 cm plates were treated with cytochalasin D (1 μg/ml)
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and 0.2 μM nocodazol for 1 h at 370C. Cells were collected in 8 ml hypotonic lysis buffer (1mM
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HEPES, pH 7.2, 0.5% NP-40, 0.5 mM MgCl2, 1 mM DTT, protease and phosphatase inhibitors
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(Sigma, 1:200 dilution)). Nuclei were pelleted by centrifugation at 2,500 g for 10 min. The
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supernatant was underlaid with a 60% sucrose cushion (0.8 ml), and centrosomes were
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sedimented at 25,000 g for 30 min. The cushion and two volumes of the buffer above it (2.4 ml
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total) were collected, mixed and laid on the top of a discontinuous sucrose gradient (1 ml 70%,
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0.6 ml 50%, 0.6 ml 40% sucrose) and centrifuged at 100,000 g for 1 h. Fractions of 0.45 ml
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were collected and diluted to reduce sucrose concentration to <20%. Centrosomes were
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pelleted at 25,000 g for 30 min and dissolved in the SDS sample buffer for electrophoresis.
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Immunoprecipitaiton and Western blotting. Cells were lysed in 50 mM HEPES, pH 7.5, 50 mM
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NaCl, 5 mM MgCl2, and 1% Triton X-100. Proteins were immunoprecipitated with appropriate
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antibodies (as specified in figure legends) and protein A/G agarose (Santa Cruz Biotechnology)
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for 4 h at 4°C. Immunoprecipitates were washed 3 times with the lysis buffer. Proteins were
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separated on 5–20% gradient SDS gels and transferred onto a PVDF (Osmonics) or
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nitrocellulose (Protran, Shchleicher & Schull) membranes. Membranes were probed with
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appropriate antibodies and developed using Dura reagents (Pierce). Densitometry of protein
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bands was performed on scanned images using NIH Image 1.63 software.
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RESULTS
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T-cadherin is located in the nucleus in ECs. In accordance with a report that T-cadherin is only
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slightly enriched at the cell borders (59), we only occasionally observed cell surface staining in
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the primary cultures of ECs (Fig. 1B, indicated with arrowheads in the upper panel). On the
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contrary, a robust staining of the nuclei was observed using two different polyclonal T-cadherin
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antibodies, designated here as TK (raised against synthetic peptides of the first extracellular
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subdomain (70)) and SC (raised against a peptide overlapping with CD2 and CD3 domains, Fig.
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1A). Nuclear staining was observed in three different cell lines (Fig 1B): human pulmonary
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artery endothelial cells (HPAECs), human umbilical vein endothelial cells (HUVECs) and human
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microvascular endothelial cells (HMVECs). It was not due to the secondary antibody used in
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these experiments, since omitting primary antibodies resulted in the absence of nuclear staining
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(Fig. 1B, bottom panel).
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To confirm the nuclear location of T-cadherin, we fractionated ECs into a nuclear fraction and a
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fraction containing other cellular compartments and cytoplasmic proteins. The fractionation was
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confirmed using histone H1 as a nuclear marker (Fig. 2 A, B). Approximately half of total
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endogenous T-cadherin was found in the nuclear fractions in HPAECs, as revealed by Western
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blotting with the TK antibody, which recognizes 105 and 130 kDa forms (Fig. 2A). A lower
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proportion (10-20%) of endogenous T-cadherin was found in the nuclear fraction of HUVECs
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(Fig. 2B). The absence of contamination of the nuclear fractions by proteins present in the post-
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nuclear supernatant was evident from reprobing the membranes for a number of markers for
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different subcellular compartments: Bcl2 (mitochondria), calreticulin (ER), GRP78 (ER/PM),
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LAMP1 (lysosomes), VE-cadherin (adherens junctions), GM130 (Golgi), Hsp90 (cytoplasm),
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Rab5 (early endosomes), ZO-1 (tight junctions). It is worth noting that some of the examined
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proteins have been reported to either be present in the nucleus (PP5 (9), ZO-1 (27), Akt1 (56),
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Rac1 (50, 51)), or to be functional or interacting partners of T-cadherin (Akt1 (42), Rac1 (57),
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GRP78 (58)). Only trace amounts (except ZO-1 in HPAEC), if any, of these proteins could be
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detected in the nuclear fractions (Fig. 2C). These data effectively rule out a possibility that the
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presence of T-cadherin in the nuclear fractions of HPAECs and HUVECs is due to non-specific
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contamination by proteins from post-nuclear supernatant.
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The predicted molecular mass of the mature protein without signal peptide is approximately 65
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kDa (60). In ECs, there are two major forms of T-cadherin, mature 105 kDa and a partially
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processed precursor 130 kDa, both of them are presumably glycosylated (70). The results of
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subcellular fractionation clearly indicate that both forms of T-cadherin are present in the nucleus.
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Both T-cadherin antibodies gave similar nuclear patterns in immunofluorescence experiments
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(Fig. 1B). The SC antibody has not been used in fractionation experiments (Fig. 2), since on
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Western blots it failed to recognize full length T-cadherin, although it recognized truncated T-
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cadherin mutant ΔC3-4 with high efficiency (Supplementary Fig. 1A-C). Since T-cadherin has
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several potential glycosylation sites, this might be due to conformation sensitivity or epitope
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masking by glycosyl group(s). Since the presence of T-cadherin in the nucleus was highly
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unexpected and of a considerable potential interest, we also confirmed this finding using
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polyclonal antibodies against CD1 and CD5 domains of T-cadherin (40), respectively
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(Supplementary Fig. 1D).
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Analysis of T-cadherin primary structure did not reveal any canonical nuclear localization
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sequences, although it is enriched in basic residues and is predicted to be a nuclear protein by
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the ESLPred (8) and SubLoc (28) algorithms. We also found that T-cadherin contains a Leu-rich
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sequence LRFSLPSVLLLSLFSLACL within its C-terminal recognition sequence (CSR) that
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perfectly matches a nuclear export signal consensus Lx2-3Lx2-3LxL (46). Before mature GPI-
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anchored proteins are delivered to the cell surface, they undergo processing in the ER. Their
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CSRs are removed by proteolytic cleavage in the ER simultaneously with the attachment of the
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GPI-anchor to the newly exposed C-terminus (22). We examined the localization of FLAG-
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tagged T-cadherin construct, where the epitope is placed after the CSR for GPI-attachment (see
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Supplementary Fig. 1A). C-terminal epitope tags attached after CSRs are not expected to
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impede the protein proper processing, and are removed when the protein is processed in the
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ER (2, 7, 65), thus only the molecules that are not (yet) processed are detectable with the
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epitope-specific antibody. The non-processed FLAG-tagged T-cadherin could be exclusively
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detected in the non-nuclear fractions (Fig. 2B, Supplementary Fig. 1C), presumably in the ER,
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taken into account a characteristic reticular pattern observed with FLAG-tagged T-cadherin
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(Supplementary Fig. 1C).
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We also examined the effect of leptomycin B (LMB, an inhibitor of CRM1-dependent nuclear
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export) on distribution of endogenous T-cadherin in HUVECs. The proportion of both
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endogenous T-cadherin forms increased by 1.5-2 fold in the nucleus upon LMB treatment,
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indicating that the presence of T-cadherin in the nucleus is dynamically regulated. As it is
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unlikely that mature T-cadherin possesses this CSR sequence, it is possible that it interacts with
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a partner with functional nuclear export signal.
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T-cadherin is present in the centrosomes. In dividing HPAECs, T-cadherin was associated with
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two punctate structures located on the opposite sides of chromatin and thus resembling
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centrosomes (see Fig. 1B, lower HPAEC panel). Z-sectioning of individual cells confirmed that
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each mitotic cell contained exactly two T-cadherin-positive structures (Supplementary Fig. 2A),
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consistent with their identification as centrosomes. Therefore, we examined whether T-cadherin
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would co-localize with a centrosomal marker γ-tubulin. We found a clear co-localization between
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the two proteins throughout the cell cycle (Fig. 3; methanol fixation was used in this experiment
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to optimize centrosome preservation, which is not optimal for visualization of the nuclei). This
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indicated that T-cadherin is indeed present in the centrosomes. In the telophase it was often
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transiently concentrated in the midbody, with reduced or undetectable presence in the
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centrosomes (note that the brightness of the green channel is increased in the telophase panel
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as compared to other panels in Fig. 3). In HUVECs, these T-cadherin-positive centrosomes
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could be observed throughout the cell cycle, including mitosis. In HPAECs, a qualitatively similar
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pattern was observed, however centrosomal T-cadherin staining was most obvious in
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metaphase and anaphase.
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Centrosome-associated
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(Supplementary Fig. 2B), although less efficiently than with the SC antibody (while both
T-cadherin
could
also
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be
detected
using
the
TK
antibody
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antibodies stain the nuclei similarly, Fig. 1B). Lower efficiency of the TK antibody might be due
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to partial unavailability of its epitope (CD1). In PC12 cells, T-cadherin was reported to be
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ubiquitinated by Cdh1, a component of a major ubiquitination system that controls the
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proteasome-dependent destruction of cell cycle regulators (5). Proteasomes are located both in
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the nucleus and in the centrosomes (see Refs in (16)). Cdh1 localizes to the nucleus during
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interphase, and to the centrosomes during metaphase and anaphase (78). This prompted us to
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test whether the levels of T-cadherin in HUVECs would be affected by proteasome inhibition.
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After 4 h incubation with proteasome inhibitors I1 and MG132, the levels of T-cadherin were
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significantly increased, while incubation with I2 (calpain inhibitor that does not affect proteasome)
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resulted in only marginal if any increase in T-cadherin levels (Fig. 4A). These data indicate that
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T-cadherin levels in HUVECs are under control of proteasome-dependent degradation.
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To confirm the centrosomal location of T-cadherin, we isolated centrosomal fractions from
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HUVECs by differential centrifugation using a 20-70% discontinuous sucrose gradient. The
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fractionation was confirmed using γ-tubulin as a centrosomal marker. Immunoblotting showed a
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biphasic distribution of γ-tubulin (Fig. 4B). The lower molecular weight form, but not the 130 kDa
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form of T-cadherin could be detected in the centrosomal fractions using the TK antibody and
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was mainly associated with the first of the two γ-tubulin peaks (Fig. 4B). Reprobing the same
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blot with the SC antibody (which does not recognize full length glycosylated T-cadherin on
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Western blots, but may efficiently recognize truncated form(s), see above) revealed the
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presence of a ~60 kDa band, associated with the second γ-tubulin peak and also found in a
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denser fraction (Fig. 4B). Thus, the results of cenrosome isolation indicate that only the mature
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processed form but not the 130 kDa form of T-cadherin is associated with a subfraction of the
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centrosomes. Our data are also compatible with a truncated form or non-glycosylated T-
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cadherin present in a distinct centrosomal subfraction.
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To examine whether T-cadherin and γ-tubulin might physically associate, we performed
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immunoprecipitation from HUVECs. We first attempted to assess whether endogenous T-
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cadherin might associate with γ-tubulin. However, endogenous T-cadherin could not be
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detected in γ-tubulin immunoprecipitates, while reciprocal immunoprecipitation proved
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inconclusive due to nearly identical molecular weight of γ-tubulin and the heavy immunoglobulin
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chain (data not shown). Taking into account that only a small proportion of endogenous T-
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cadherin is located in the centrosomes in comparison with the nucleus in ECs, we next
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attempted to use overexpressed T-cadherin to assess the possibility of its interaction with γ-
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tubulin. Since the SC antibody recognizes the ΔC3-4 T-cadherin construct, but not full length
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constructs on Western blots (as discussed above and shown in Supplementary Fig. 1B),
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HUVECs were transfected with the latter construct or empty vector. Immunoprecipitation was
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performed using γ-tubulin- and GFP-specific antibodies. The presence of a band of the
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expected molecular size (see Supplementary Fig. 1B) recognized by the SC antibody could be
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detected in the material precipitated from T-cadherin transfected cells with the γ-tubulin antibody,
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but not in the negative controls (Fig. 4C). No corresponding signal could be detected in the
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immunoprecipitates using FLAG antibody, consistent with a form that has been C-terminally
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processed in the ER.
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To assess whether the centrosomal and nuclear location of T-cadherin is specific for ECs, we
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examined localization of endogenous T-cadherin in HEK 293A cells. Immunoblotting analysis
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showed that HEK 293A (as well as COS-7) cells express considerably lower levels of T-
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cadherin relative to total protein than ECs (data not shown). We found that in HEK 293A cells,
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most of endogeneous T-cadherin co-localized with γ-tubulin and thus was associated with the
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centrosomes (which are unusually large structures in this cell line (55, 76)), with only very faint
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nuclear and cell surface staining (Supplementary Fig. 3A). In an attempt to detect association of
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endogenous T-cadherin with γ-tubulin, we performed immunoprecipitation experiments from
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HEK 293A cells. A ~60 kDa band could be detected in γ-tubulin immunoprecipitates, but not in
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GFP antibody immunoprecipitates used as a negative control (Supplementary Fig 3B). The
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molecular weight of this band is close to that expected for non-glycosylated T-cadherin without
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prepeptide (65 kDa).
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Interference with T-cadherin expression leads to defects in cell division. As described above, T-
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cadherin was often transiently concentrated in the midbody, which plays a key role in
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cytokinesis (6). This observation suggested that T-cadherin might be involved in cytokinesis.
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Cytokinesis failure leads to appearance of cells containing more than one nucleus. To assess
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whether overexpression or downregulation of T-cadherin might affect the probability of failed
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cytokinesis, we examined the proportion of bi- or multinuclear cells among HUVECs transfected
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with either T-cadherin cDNA or T-cadherin siRNA. A small proportion of ECs is naturally bi- or
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multinuclear, and an increase in the proportion of such cells has been associated with some
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physiological or pathological conditions (19, 36, 69). In line with these reports, we observed that
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primary HUVEC cultures contained 4-5 % cells with more than one nucleus (of which absolute
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majority were binuclear cells).
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T-cadherin could be efficiently overexpressed in ECs (Fig. 5A; Supplementary Fig. 1C),
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resulting in an overall increase in T-cadherin content of up to 10-20 fold (ECL quantification data
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not shown). Immunofluorescence microscopy revealed that in some cells T-cadherin was
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present in a reticular pattern (Fig. 5B, upper panel), probably reflecting its predominant
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presence in the endoplasmic reticulum, while in other cells punctate cytoplasmic as well as
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nuclear staining was observed (Fig. 5B, middle panel). The proportion of cells containing more
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than one nucleus was increased among the cells overexpressing T-cadherin (Fig. 5 B, C).
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Similar results were obtained using overexpression of FLAG-tagged T-cadherin in HUVECs
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(data not shown) and in HEK 293A cells (Supplementary Fig. 3 C-E).
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We also depleted endogenous T-cadherin in ECs using siRNA approach, and examined the
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cells for any apparent defects associated with cell division. Both 105 and 130 kDa forms of T-
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cadherin could be efficiently downregulated in HPAECs and in HUVECs (Fig. 6A). It was
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reported that the levels of different cadherins may influence each other, for example, VE-
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cadherin expression is reduced in N-cadherin-deficient endothelium (48)). VE-cadherin levels
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remained unchanged in the cells with 80-90% T-cadherin depleted (Fig. 6A). In some
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experiments, 95-98% T-cadherin depletion was achieved, which was accompanied by a 30-40%
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decrease in VE-cadherin levels (data not shown). T-cadherin depletion also resulted in a
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considerable decrease in T-cadherin positive staining in immunofluorescence experiments (Fig
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6B, C).
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T-cadherin depletion led to an only marginal increase in the proportion of multinucleated cells,
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which was not statistically significant (Fig. 6E). However, we noticed that downregulation of T-
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cadherin was accompanied by an increased proportion of mononuclear ECs with multiple
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centrosomes (Fig. 6 C,D,F), which suggests a specific role of T-cadherin in the control of
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centrosomal organization.
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DISCUSSION
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We report here that a considerable proportion of T-cadherin is located in the nucleus, as
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evidenced by immunofluorescence and by immunoblotting upon cell fractionation using
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antibodies raised against different epitopes. Nuclear localization of cleaved intracellular domains
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of some other members of the cadherin family, such as protocadherins α (18), γ (29), Fat1 (33,
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49), E-cadherin (21) was reported. However, T-cadherin has no intracellular domain and is
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present in the nucleus as a full-length molecule; therefore trafficking pathways suggested for
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other cadherins may not be relevant in the case of T-cadherin. Full length cell surface receptors,
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both G-protein coupled receptors and tyrosine kinase receptors, were reported to translocate to
13
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the nucleus (17, 20, 25, 53, 62, 72), including LPA1, for which caveolin-mediated endocytosis
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was suggested (26). Nucleus is rich in lipid environments, which contain lipid raft components,
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including caveolin-1 (1). However, several lines of evidence argue against a role of caveolin in
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the nuclear trafficking of T-cadherin. (i) Pertussis toxin inhibits LPA1 trafficking to the nucleus
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(26), but does not affect the nuclear localization of T-cadherin (our unpublished observations),
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which makes a common trafficking mechanism unlikely. (ii) Although caveolae-dependent
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endocytosis was originally considered for GPI proteins (3), later studies suggested a caveolae-
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independent pinocytotic pathway (44, 45, 64) or macropinocytotic pathways triggered by a
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clustering agent or being ligand-independent (14, 74). (iii) In ECs, caveolin is not involved in T-
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cadherin-mediated signaling (59). However, a recently described T-cadherin interaction with
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GRP78 (58), which is reportedly involved in caveolae-dependent internalization and nuclear
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localization of extracellular matrix protein DMP1 (61), reintroduces the question about a possible
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role of caveolae.
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In some cell lines, T-cadherin may be not GPI-modified, but rather expressed as a protein with a
320
transmembrane domain (54). Thus, it cannot be excluded that precise sites of processing of T-
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cadherin in ECs might differ from those assumed on the basis of canonical processing of GPI-
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anchored proteins.
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If T-cadherin is processed as a canonical GPI-anchored protein, the questions arise whether it
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is targeted to the nucleus after its delivery to the PM, or directly from the ER. Some models for
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the nuclear translocation of cell surface receptors have been suggested (47), and although
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these questions are beyond the scope of this work, T-cadherin would be an excellent model to
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study this process.
328
Apart from the question of how T-cadherin is imported into the nucleus, another question is what
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functional role the nuclear T-cadherin may play. Cleaved intracellular domain of E-cadherin may
330
modulate gene transcription (21). Some other transmembrane receptors may also act as
14
331
transcriptional factors or modulators of expression (17, 20, 25, 34, 53, 62, 72). As T-cadherin
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upregulates cyclin D1 (41) and may activate the serum response element (our unpublished
333
data), a similar function seems conceivable for T-cadherin.
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We also found that T-cadherin is located in the centrosomes. A quantitative difference between
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venous (HUVECs) and arterial (HPAECs) endothelial cells could be observed, as T-cadherin
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was present in the centrosomes uniformly throughout the cell cycle (except telophase) in
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HUVECs, but was most noticeable during metaphase in HPAECs. The mechanisms of protein
338
delivery to the centrosomes are poorly understood. For some proteins, such as RGS14 (13),
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nuclear-cytoplasmic shuttling may be important. In this respect, it may be relevant that the two
340
lines of ECs studied here also differed in the proportion of nuclear T-cadherin. Since our data
341
suggest that T-cadherin may play a role in cytokinesis, it is also worth noting that some proteins
342
required for cytokinesis are sequestered into the nucleus during interphase (6). Centrosomal T-
343
cadherin may have a longer half life than T-cadherin in other compartments, since the
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centrosomes were the last location where it could be detected upon siRNA-mediated depletion.
345
This is similar to the observations for centrosomal γ−tubulin (67). In line with these data, in HEK
346
293A cells (where T-cadherin expression is low), the endogenous T-cadherin could only be
347
observed in the centrosomes and to a minimal extent in the nucleus.
348
The role of T-cadherin in the centrosomes may be linked to the control of their duplication, since
349
siRNA-mediated T-cadherin depletion resulted in an increased fraction of cells with multiple
350
centrosomes. Since the data were collected for the cells that have an increased number of
351
centrosomes without nuclear duplication, this reflects a defect in centrosomal duplication per se.
352
Notably, depletion of most centrosomal proteins leads to cell cycle arrest and defects in
353
centrosome structure (52).
354
Our data also suggest that T-cadherin may play a role in cytokinesis, since it transiently
355
concentrates in the midbody during telophase, and its overexpression leads to an increased
15
356
proportion of cells with more than one nucleus. Notably, both the expression of T-cadherin (39)
357
and the proportion of bi- and multinucleated ECs (69) are elevated in atherosclerotic lesions.
358
Aneuploidy, which may be caused by different mitotic aberrations, including errors in
359
centrosome duplication or defective cytokinesis, is a characteristic feature of many cancerous
360
cells. At the same time, T-cadherin expression is known to be either down- or upregulated in
361
many cancer cell lines (e.g. (71) and references therein). In the light of our findings, it would be
362
interesting to examine whether T-cadherin expression is affected in tumors of endothelial origin,
363
which are also characterized by centrosome abnormalities (31). We did not detect statistically
364
significant differences in the percentage of multinuclear cells upon T-cadherin depletion.
365
However, since T-cadherin promotes cell cycle progression in vascular cells (38), T-cadherin
366
depletion may “freeze” the culture in the same state it was before siRNA transfection.
367
Overall, our findings reported here point to novel role(s) of T-cadherin in the nucleus and/or in
368
cell division. Although localization of T-cadherin described here is rather unexpected, its links to
369
the cell cycle have been reported previously in endothelial and other cell types: (i) T-cadherin
370
has been identified among cell cycle-related genes upregulated in G1 phase fibroblasts (37). (ii)
371
T-cadherin increases the expression of cyclin D1 in ECs (41). (iii) Ectopic expression of Cdh1
372
(which localizes dynamically to the nucleus during interphase and to the centrosomes during
373
metaphase and anaphase and is involved in the destruction of cell cycle regulators (75)),
374
stimulates T-cadherin degradation in PC12 cells (5). Our data, which indicate that T-cadherin is
375
under control of proteasome-dependent degradation in HUVECs and may be degraded in
376
telophase, are compatible with a role of Cdh1 in T-cadherin degradation in ECs as well. (iv)
377
Enforced expression of T-cadherin induces cell cycle arrest in hepatocellular carcinoma cells
378
(12). (v) Three kinases that act downstream of T-cadherin (41) and supposedly transduce T-
379
cadherin signals from the PM, are also implicated in the control of centrosomes and/or mitotic
380
spindles: (a) GSK-3β regulates localization of the γ-tubulin ring complex to the spindle poles and
16
381
thereby controls the formation of proper mitotic spindles (32). (b) Akt may localize to the
382
centrosomes in a manner dependent on its phosphorylation at specific sites (43), and its
383
reduced levels result in incomplete centrosome migration around the nucleus and in bent
384
misoriented mitotic spindles (10, 11). (c) ILK has recently been found to localize to mitotic
385
centrosomes and to be essential for spindle pole organization and mitosis (23).
386
Since our data suggest that either depletion or overexpression of T-cadherin results in defects in
387
cell division, findings reported here suggest a mechanistic insight into a previously described
388
correlation between either downregulation or upregulation of T-cadherin with cancers. Possible
389
contributions of diverse pathways to the function of T-cadherin in cell division will be the subject
390
of future studies.
391
392
ACKNOWLEGMENTS We would like to thank Drs Kalpana Ghoshal and Samson Jacob (Ohio
393
State University) for the FLAG-tagged T-cadherin constructs, Mr Aleksandar Krbanjevic for
394
plasmid purification, and Dr Jasmina Profirovic for critical reading of the manuscript.
395
396
17
397
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611
FIGURE LEGENDS
612
Fig. 1. Endogenous T-cadherin is present in the nuclei in endothelial cells (EC). A: T-cadherin
613
domain architecture and the rabbit polyclonal T-cadherin antibodies used in this work.
614
Abbreviations: CD, cadherin domain; CRS, C-terminal recognition sequence; SP, signal peptide.
615
Antibodies (italicized) and positions of their epitopes are shown. The TK antibody
616
recognizes human but not mouse T-cadherin. The antibody designated as SC is from Santa
617
Cruz Biotechnology (cat. number sc-7940). B: Endogenous T-cadherin is present in the nuclei in
618
three lines of human ECs, derived from pulmonary artery (HPAEC), umbilical vein (HUVEC) and
619
microvasculature (HMVEC). Immunostaining was performed with T-cadherin antibodies (TK or
620
SC as indicated), and mouse monoclonal VE-cadherin antibody, followed by AlexaFluor 488-
621
and AlexaFluor 594-conjugated secondary antibodies, respectively. Nuclei were visualized with
622
DAPI (blue). Arrowheads and arrows indicate the presence of T-cadherin at the cell surface in
623
some cells and in a pair of punctate structures in a mitotic cell, respectively. Lower panel:
624
primary antibodies were omitted.
(70)
625
626
Fig. 2. Distribution of T-cadherin (105 kDa, mature glycosylated form; 130 kDa, partially
627
processed precursor) between nuclear and non-nuclear fractions of HPAECs. (A) and HUVECs
628
(B) and assessment of the purity of respective nuclear fractions (C). Untransfected cells or cells
629
transfected with FLAG-T-cadherin were fractionated into the nuclear (N, 1000 g pellet) and
630
soluble (S, 1000 g supernatant) fractions. Proteins were separated on SDS-PAGE, transferred
631
onto a PVDF membrane and probed with the TK or FLAG antibodies. Positions of molecular
632
weight markers (PageRuler, Fermentas) are indicated. In A and B, quantification of the
633
immunoblotting data for endogenous T-cadherin forms was performed using ImageJ software.
634
Data shown are means of three separate experiments (error bars, S.D.). H1, histone H1. C,
635
fractions shown in A and B were probed for various markers for indicated subcellular structures
27
636
and compartments (AJ; adherens junctions; ER, endoplasmic reticulum; PM, plasma membrane;
637
TJ, tight junctions). 1 Proteins that have been reported in the nucleus.
638
reported as functional partners of T-cadherin (see main text for references).
2
Proteins that have been
639
640
Fig. 3. T-cadherin is located in the centrosomes in HUVECs. Cells were immunostained with the
641
T-cadherin SC antibody and mouse monoclonal γ-tubulin antibody, followed by AlexaFluor 488-
642
and AlexaFluor 594-conjugated secondary antibodies, respectively. Nuclei were visualized with
643
DAPI (blue). The cell cycle phases are indicated (A: mitosis: prophase, metaphase, anaphase
644
and telophase; B: interphase: G1, S, G2). Centrosomes are indicated with arrows. In the
645
telophase image, the brightness in the green channel has been enhanced to visualize T-
646
cadherin in the midbody (*) and in centrosomes.
647
648
Fig. 4. T-cadherin is degraded in a proteasome-dependent manner in HUVECs (A), and co-
649
fractionates (B) and physically associates (C) with γ-tubulin. A: Confluent cells were incubated in
650
the presence of inhibitors (20 μM) or DMSO (control) as indicated, and T-cadherin levels
651
analyzed by Western blotting with the TK antibody. Right panel shows quantification of the
652
results (means of four replicates; error bars, S.D.). B: Centrosomes were isolated from
653
untransfected HUVECs as described under Materials and Methods. Sucrose gradient fractions
654
were analyzed for the presence of γ-tubulin (centrosomal marker) and T-cadherin. TL, total
655
lysate. C: HUVECs were transfected with the ΔC3-4 construct or empty vector. Cells from two
656
10 cm plates were subjected to immunoprecipitation with γ-tubulin or GFP (negative control)
657
antibodies as described under Materials and Methods. Immunoprecipitated material (IP) was
658
probed with the T-cadherin (SC) antibody. TL, total lysates.
659
28
660
Fig. 5. T-cadherin overexpression increases proportion of bi- or multinuclear cells. HPAECs
661
were transfected with the full length human T-cadherin construct. Cells were collected 24 h after
662
transfection, and T-cadherin expression analyzed by Western blotting (A) and by
663
immunofluorescence (B, upper and middle panels). Lower panel in B shows an example of
664
binuclear cells overexpressing T-cadherin 72 h after transfection. C: HUVECs transfected with
665
the T-cadherin construct or GFP were examined for alterations in morphology 72 h after
666
transfection. Percentage of cells with more than one nucleus is shown relative to the total cell
667
number counted (50-80 cells per replicate, 4 replicates; error bars, S.D.).
668
669
Fig. 6. siRNA-mediated depletion of T-cadherin in ECs results in multiple centrioles. A, B:
670
Downregulation of endogenous T-cadherin in HPAECs and HUVECs. Cells were transfected
671
with control (C) or T-cadherin (T) siRNA and analyzed by immunoblotting 24 or 72 h after
672
transfection (A) or by immunofluorescence 24 h after transfection (B; HPAECs). VE-cadherin
673
was examined as a specificity control. C: Multiple centrosomes in T cadherin-depleted HUVECs,
674
visualized with γ-tubulin antibody 72 h after transfection (methanol fixation). D: Z-sections (γ-
675
tubulin staining) of the cell shown in the bottom panel in (C). In C and D, centrosomes are
676
indicated by arrows. E and F: HUVECs transfected with T-cadherin or control siRNA were
677
examined for aberrations in the number of nuclei and centrosomes per cell, respectively, 72 h
678
after transfection. E: Percentage of cells with more than one nucleus is shown relative to the
679
total cell number counted (90-170 cells per replicate, 4 replicates; error bars, S.D.). F:
680
Percentage of cells with >2 centrosomes is shown relative to the total cell number counted;
681
interphase binuclear cells were disregarded (500 cells per replicate, 3 replicates; error bars,
682
S.D.).
683
29
684
Supplementary Fig. 1. A: FLAG-tagged mouse T-cadherin constructs used in this work
685
(designated according to Ref. (70)). For abbreviations, see Fig. 1. B-D:, Full-length and
686
truncated T-cadherin forms as detected by immunoblotting (B and D) and immunofluorescence
687
(C) using different antibodies. B: HUVECs were transfected with different T-cadherin constructs
688
as indicated, and their expression analyzed using FLAG antibody and two T-cadherin antibodies
689
(TK and SC, see Fig. 1). Note that the TK antibody does not recognize mouse T-cadherin, and
690
the SC antibody only recognizes the ΔC3-4 deletion mutant. In the TK panel, a short ECL
691
exposure is shown that gives signal intensity comparable to two other panels; endogenous T-
692
cadherin becomes visible at longer exposures (see Fig. 5A). Positions of molecular weight
693
markers are indicated. C: Expression of FLAG-tagged T-cadherin constructs in HUVEC,
694
visualized with SC (green) and FLAG (red) antibodies. Cells were collected 24 h after
695
transfection. D: Distribution of endogenous T-cadherin forms between nuclear and non-nuclear
696
fractions of HPAECs. Cells were fractionated as in Fig. 2, and T-cadherin detected using three
697
independently raised antibodies (see Fig. 1 for their epitopes). Positions of molecular weight
698
markers (Fermentas) are indicated. E: Effect of leptomycin B (LMB) treatment on distribution of
699
endogenous T-cadherin in HUVECs. Cells were treated with LMB (10 ng/ml) for 6 h and
700
fractionated into nuclear and non-nuclear fractions as in D. Relative distribution of 130 kDa and
701
105 kDa forms of T-cadherin was calculated separately from the ECL signals. Data shown are
702
means of 3 replicates (± S.D.).
703
704
Supplementary Fig. 2. Co-localization of T-cadherin with centrosomes. A: Z-sections showing
705
localization of T-cadherin (SC antibody) in a metaphase HPAEC from the experiment shown in
706
Fig. 1B. B: Co-localization of T-cadherin with γ-tubulin in HUVECs. The experiment was
707
performed as in Fig. 3, except that TK antibody was used for T-cadherin staining and para-
708
formaldehyde fixation was used in the left panel.
30
709
710
Supplementary Fig. 3. T-cadherin in HEK 293A cells. A: Endogenous T-cadherin is located
711
predominantly in the centrosomes. Untransfected HEK 293A cells were immunostained with T-
712
cadherin (SC) and γ-tubulin antibodies. B: An endogenous T-cadherin-positive 60 kDa protein
713
physically associates with γ-tubulin. HEK 293A cells from four 10 cm plates were subjected to
714
immunoprecipitation with the γ-tubulin or GFP (negative control) antibodies. Immunoprecipitated
715
material (IP) was probed with the T-cadherin (SC) antibody. TL, total lysate. C-E: HEK 293A
716
cells were transfected with the human T-cadherin construct or GFP and examined by Western
717
blotting (C) or immunostaining (D: an example of binuclear T-cadherin-expressing cells; nuclei
718
were visualized with DAPI), and were assessed for alterations in morphology 72 h after
719
transfection. Percentage of cells with more than one nucleus is shown in E relative to the total
720
cell number counted (150-200 cells per replicate, 3 replicates; error bars, S.D.).
721
31
CD1
EC1
SP
TK
CD2
SC
CD3
HUVEC
SC
CD4
CD5
EC5
C
SC
CRS
HMVEC
N
HPAEC
B
T-cadherin VE-cadherin
TK
HPAEC
A
HMVEC
(control)
Figure 1
SC
DAPI
Merge
HPAEC
WB: N
TK
S
kDa
130
95
H1
C
B
T-cadherin in nuclear
fraction (relative to total)
A
HPAEC
N S
TFLAG
WB: N S
TK
250
130
LAMP1
Rab5
Calreticulin
72
GRP782
ZO11
55
VE-cadherin1
35
Bcl2
Hsp901
95
27
17
11
N
S kDa
130
95
130
95
FLAG
H1
WB:
GM130
kDa
HUVEC
Akt11,2
Rac11,2
PP51
HPAEC HUVEC
N S N S
T-cadherin in nuclear
fraction (relative to total)
Figure 2
HUVEC
N S
Golgi
Lysosomes
Endosomes
ER
ER / PM
PM (TJ)
PM (AJ)
Mitochondria
Cytoplasm
kDa
250
130
95
72
55
35
27
17
Figure 3
Telophase Anaphase
Metaphase Prophase
A
T-cadherin J-Tubulin
Merge
B
G1
S
G2
*
T-cadherin J-Tubulin
Merge
Figure 4
A
T-cadherin levels
(relative to control)
2.5
2
1.5
1
0.5
0
Control I1
I2 MG
132
B
kDa
TL
1
2
3
4
5
6
7
55
8
9 10 WB:
J-Tub
130
105
TK
60
SC
C
WB:
SC
Vector T-cadherin
TL
IP:
J-Tub
GFP
Figure 5
A
T-cad
C
Pg: 0.1 0.3 0.5 0.5
WB:
T-cad
(TK)
Longer exposure
VE-cad
C
Cells with >1 nucleus
( % total )
Hsp90
12
*
10
8
6
4
2
0
GFP T-cad
B T-cadherin (TK)
VE-cadherin
Merge
Figure 6
C SC
J-Tubulin
Merge
VE-cad
Hsp90
8
6
4
2
0
siRNA: Control T-cad
VE-cadherin Merge
D
0.00 Pm
Z-sections (JJ-Tubulin)
T-cad siRNA Control siRNA
B
TK
E
Cells with >1 nucleus
( % total )
HUVEC
HPAEC
24 h
72 h
72 h
siRNA: C T
C T
C T
WB:
T-cad
0.66 Pm
0.22 Pm
0.88Pm
Pm
1.10
0.44 Pm
1.10 Pm
F
Mononuclear cells with
>2 centrosomes (% total)
A
2
1.5
1
0.5
0
1
2
siRNA: Control T-cad