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Articles in PresS. Am J Physiol Cell Physiol (September 2, 2009). doi:10.1152/ajpcell.00237.2009 1 T-cadherin is located in the nucleus and centrosomes in endothelial cells. 2 3 Alexandra V. Andreeva1*, Mikhail A. Kutuzov1, Vsevolod A. Tkachuk2, Tatyana A. Voyno- 4 Yasenetskaya1* 5 6 1 Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA. 7 2 Cardiology Research Center, Moscow, Russia. 8 9 * Corresponding authors. Deparment of Pharmacology (MC 868), University of Illinois at 10 Chicago, 909 S. Wolcott Ave. COMRB, Chicago, IL 60612, USA. 11 Tel.: +1 312 996 9823; fax: +1 312 996 1225. 12 E-mail addresses: [email protected] (A. V. Andreeva), [email protected] (T. A. Voyno- 13 Yasenetskaya). 14 15 Running title: Nuclear and centrosomal location of T-cadherin 16 1 Copyright © 2009 by the American Physiological Society. 17 ABSTRACT 18 19 T-cadherin (H-cadherin, cadherin 13) is upregulated in vascular proliferative disorders and in 20 tumor-associated neovascularization, and is deregulated in many cancers. Unlike canonical 21 cadherins, it lacks transmembrane and intracellular domains and is attached to the plasma 22 membrane via a glycosylphosphatidylinositol anchor. T-cadherin is thought to function in 23 signaling rather than as an adhesion molecule. Some interactive partners of T-cadherin at the 24 plasma membrane have recently been identified. We examined T-cadherin location in human 25 endothelial cells using confocal microscopy and subcellular fractionation. We found that a 26 considerable proportion of T-cadherin is located in the nucleus and in the centrosomes. T- 27 cadherin colocalized with a centrosomal marker γ-tubulin uniformly throughout the cell cycle at 28 least in human umbilical vein endothelial cells. In the telophase, T-cadherin transiently 29 concentrated in the midbody and was apparently degraded. Its overexpression resulted in an 30 increase in the number of multinuclear cells, while its downregulation by siRNA led to an 31 increase in the number of cells with multiple centrosomes. These findings indicate that 32 deregulation of T-cadherin in endothelial cells may lead to disturbances in cytokinesis or 33 centrosomal replication. 34 35 Key words: cadherin family, cell cycle, cytokinesis, GPI anchor, HPAEC. 36 37 2 38 INTRODUCTION 39 T-cadherin is a glycoprotein that belongs to the cadherin cell adhesion family. In contrast to 40 canonical cadherins, T-cadherin lacks many amino acids crucial for Ca2+-dependent intercellular 41 dimerization (15, 60). As a consequence, it is monomeric in the absence and in the presence of 42 calcium (15) and is thought to mainly function as a signaling molecule. Another unique feature 43 of T-cadherin is the absence of the transmembrane and cytoplasmic domains (although a rare 44 cell line-specific transmembrane form has been reported (54)). T-cadherin is anchored within 45 lipid rafts through glycosylphosphatidylinositol (GPI) (60, 68) and may possibly use cytoplasmic 46 domains of several interactive partners (58) to transduce signals inside the cell. T-cadherin may 47 operate through integrin-linked kinase (ILK), which is upstream of the Akt / GSK3β / β-catenin 48 pathway (41, 42). It has been suggested and confirmed in an in vivo model that T-cadherin may 49 play an antiapoptotic and protective role under hypoxic conditions (30, 42). 50 In confluent endothelial cells (ECs) T-cadherin, unlike VE-cadherin (which is mainly present at 51 the cell-cell junctions), is located over the entire cell body with only a slight enrichment at cell 52 borders, while in wounded cultures it is polarized to the leading edge of migrating cells (59). 53 Some recent reports described nuclear localization of cleaved cytoplasmic domains of canonical 54 cadherins (21, 66) and their potential role in the modulation of gene transcription (21). 55 Several lines of evidence point to a potential involvement of T-cadherin in cell cycle regulation in 56 different cell lines. T-cadherin expression is decreased or undetectable in some tumor samples 57 and various cancer cell lines (63, 73). In contrast, overexpression of T-cadherin was 58 demonstrated in tumorigenic liver tissue and a hepatocellular carcinoma cell line (63). The role 59 of T-cadherin in tumor neovascularization was recently confirmed using in vivo model (30). T- 60 cadherin expression in T-cadherin-deficient C6 glioma cells results in G2 phase arrest and 61 aneuploidy, which is dependent on increased expression of p21CIP1 and is eliminated in p21CIP1- 62 deficient fibroblasts (35). Ectopic expression of Cdh1, one of the substrate recognition 3 63 components (E3 ligase) of the anaphase promoting complex, stimulates T-cadherin degradation 64 (5). In human umbilical vein endothelial cells (HUVECs), overexpression of T-cadherin leads to 65 an increased expression of cyclin D1, a key regulator of G1 to S-phase progression (24), and to 66 a rapid entrance into S-phase (38). 67 Here we report that in ECs a considerable proportion of T-cadherin is located in the nucleus. It 68 is also present in the centrosomes and co-localizes with γ-tubulin. In the telophase, T-cadherin 69 transiently concentrates in the midbody. Overexpression of T-cadherin results in an increase in 70 the number of multinuclear cells, while its downregulation leads to an increased number of cells 71 with multiple centrosomes. These findings indicate that T-cadherin is a nuclear and centrosomal 72 protein and that its deregulation may interfere with the cell cycle via disturbance in cytokinesis 73 or centrosomal replication. 74 75 MATERIALS AND METHODS 76 Materials. The following antibodies were used: Akt1, Bcl2, calreticulin, GFP, GRP78, LAMP1, T- 77 cadherin (sc-7940; designated here as SC), γ-tubulin, VE-cadherin, HRP-conjugated anti-goat 78 antibody (Santa Cruz Biotechnology); FLAG (M2; Sigma); histone 1 (AE-4; Millipore), GM130, 79 Hsp90, PP5, Rab5, Rac1 (BD Transduction Laboratories), ZO-1 (Zymed). HRP-conjugated anti- 80 mouse and anti-rabbit secondary antibodies were from Amersham. Phalloidin, AlexaFluor 488, 81 594, or 633 anti-mouse, anti-rabbit, or anti-goat antibodies were from Molecular Probes. The 82 affinity-purified polyclonal T-cadherin antibody designated here as TK was described previously 83 (70). FLAG-tagged T-cadherin constructs were kindly provided by Drs Kalpana Ghoshal and 84 Samson Jacob (Ohio State University) (5). T-cadherin constructs and specificity of the 85 antibodies used in this work are depicted in Fig. 1A and Supplementary Fig. 1A. 4 86 Cell culture. HEK 293A cells were cultured in Dulbecco's modified Eagle's medium (DMEM), 87 supplemented with 10% fetal bovine serum. Human endothelial cells were obtained at passage 88 3 from Lonza and cultured as described previously (4). Transient transfection of HEK 293A and 89 ECs was performed using Lipofectamine 2000 (Invitrogen) and SuperFect (Qiagen), 90 respectively, according to manufacturers’ instructions. 91 Confocal microscopy. Cells cultured on gelatin or fibronectin-coated coverslips were fixed with 92 3.7% paraformaldehyde or with 50% methanol (for preservation of centrosomes), followed by 93 permeabilization in 0.5% Triton X-100. Cells were incubated with primary antibodies followed by 94 incubation with appropriate secondary antibodies, using Tris buffered saline containing bovine 95 serum albumin as a blocking buffer. 96 siRNA-mediated depletion. Inhibition of T-cadherin expression was performed using siRNA 97 designed by Dharmacon. Cells were transfected using siRNA transfection reagent (Santa Cruz 98 Biotechnology) or Lipofectamine 2000 (Invitrogen). Control siRNA was purchased from Santa 99 Cruz Biotechnology. 100 Nuclear fractionation. Cells from a 10 cm dish were washed twice with PBS, recovered by 101 scraping and resuspended in 300 μl of lysis buffer, containing 20 mM Tris-HCl, pH 7.4, 100 mM 102 NaCl, 10 mM EGTA, 5 mM MgCl2, 0.5% Na deoxycholate, 0.5% Nonidet P-40, protease 103 inhibitor cocktail (Sigma, 1:200 dilution). Cells were left on ice for 15 min and centrifuged at 104 1000 g for 5 min. Completeness of cell lysis was verified microscopically. The supernatant 105 (“non-nuclear” fraction) was removed and the pelleted nuclei were washed twice in lysis buffer. 106 Isolation of centrosomes. Centrosomes isolation was performed essentially according to ref. 107 (77). Briefly, confluent HUVECs on four 10 cm plates were treated with cytochalasin D (1 μg/ml) 108 and 0.2 μM nocodazol for 1 h at 370C. Cells were collected in 8 ml hypotonic lysis buffer (1mM 109 HEPES, pH 7.2, 0.5% NP-40, 0.5 mM MgCl2, 1 mM DTT, protease and phosphatase inhibitors 5 110 (Sigma, 1:200 dilution)). Nuclei were pelleted by centrifugation at 2,500 g for 10 min. The 111 supernatant was underlaid with a 60% sucrose cushion (0.8 ml), and centrosomes were 112 sedimented at 25,000 g for 30 min. The cushion and two volumes of the buffer above it (2.4 ml 113 total) were collected, mixed and laid on the top of a discontinuous sucrose gradient (1 ml 70%, 114 0.6 ml 50%, 0.6 ml 40% sucrose) and centrifuged at 100,000 g for 1 h. Fractions of 0.45 ml 115 were collected and diluted to reduce sucrose concentration to <20%. Centrosomes were 116 pelleted at 25,000 g for 30 min and dissolved in the SDS sample buffer for electrophoresis. 117 Immunoprecipitaiton and Western blotting. Cells were lysed in 50 mM HEPES, pH 7.5, 50 mM 118 NaCl, 5 mM MgCl2, and 1% Triton X-100. Proteins were immunoprecipitated with appropriate 119 antibodies (as specified in figure legends) and protein A/G agarose (Santa Cruz Biotechnology) 120 for 4 h at 4°C. Immunoprecipitates were washed 3 times with the lysis buffer. Proteins were 121 separated on 5–20% gradient SDS gels and transferred onto a PVDF (Osmonics) or 122 nitrocellulose (Protran, Shchleicher & Schull) membranes. Membranes were probed with 123 appropriate antibodies and developed using Dura reagents (Pierce). Densitometry of protein 124 bands was performed on scanned images using NIH Image 1.63 software. 125 126 127 RESULTS 128 T-cadherin is located in the nucleus in ECs. In accordance with a report that T-cadherin is only 129 slightly enriched at the cell borders (59), we only occasionally observed cell surface staining in 130 the primary cultures of ECs (Fig. 1B, indicated with arrowheads in the upper panel). On the 131 contrary, a robust staining of the nuclei was observed using two different polyclonal T-cadherin 132 antibodies, designated here as TK (raised against synthetic peptides of the first extracellular 133 subdomain (70)) and SC (raised against a peptide overlapping with CD2 and CD3 domains, Fig. 6 134 1A). Nuclear staining was observed in three different cell lines (Fig 1B): human pulmonary 135 artery endothelial cells (HPAECs), human umbilical vein endothelial cells (HUVECs) and human 136 microvascular endothelial cells (HMVECs). It was not due to the secondary antibody used in 137 these experiments, since omitting primary antibodies resulted in the absence of nuclear staining 138 (Fig. 1B, bottom panel). 139 To confirm the nuclear location of T-cadherin, we fractionated ECs into a nuclear fraction and a 140 fraction containing other cellular compartments and cytoplasmic proteins. The fractionation was 141 confirmed using histone H1 as a nuclear marker (Fig. 2 A, B). Approximately half of total 142 endogenous T-cadherin was found in the nuclear fractions in HPAECs, as revealed by Western 143 blotting with the TK antibody, which recognizes 105 and 130 kDa forms (Fig. 2A). A lower 144 proportion (10-20%) of endogenous T-cadherin was found in the nuclear fraction of HUVECs 145 (Fig. 2B). The absence of contamination of the nuclear fractions by proteins present in the post- 146 nuclear supernatant was evident from reprobing the membranes for a number of markers for 147 different subcellular compartments: Bcl2 (mitochondria), calreticulin (ER), GRP78 (ER/PM), 148 LAMP1 (lysosomes), VE-cadherin (adherens junctions), GM130 (Golgi), Hsp90 (cytoplasm), 149 Rab5 (early endosomes), ZO-1 (tight junctions). It is worth noting that some of the examined 150 proteins have been reported to either be present in the nucleus (PP5 (9), ZO-1 (27), Akt1 (56), 151 Rac1 (50, 51)), or to be functional or interacting partners of T-cadherin (Akt1 (42), Rac1 (57), 152 GRP78 (58)). Only trace amounts (except ZO-1 in HPAEC), if any, of these proteins could be 153 detected in the nuclear fractions (Fig. 2C). These data effectively rule out a possibility that the 154 presence of T-cadherin in the nuclear fractions of HPAECs and HUVECs is due to non-specific 155 contamination by proteins from post-nuclear supernatant. 156 The predicted molecular mass of the mature protein without signal peptide is approximately 65 157 kDa (60). In ECs, there are two major forms of T-cadherin, mature 105 kDa and a partially 158 processed precursor 130 kDa, both of them are presumably glycosylated (70). The results of 7 159 subcellular fractionation clearly indicate that both forms of T-cadherin are present in the nucleus. 160 Both T-cadherin antibodies gave similar nuclear patterns in immunofluorescence experiments 161 (Fig. 1B). The SC antibody has not been used in fractionation experiments (Fig. 2), since on 162 Western blots it failed to recognize full length T-cadherin, although it recognized truncated T- 163 cadherin mutant ΔC3-4 with high efficiency (Supplementary Fig. 1A-C). Since T-cadherin has 164 several potential glycosylation sites, this might be due to conformation sensitivity or epitope 165 masking by glycosyl group(s). Since the presence of T-cadherin in the nucleus was highly 166 unexpected and of a considerable potential interest, we also confirmed this finding using 167 polyclonal antibodies against CD1 and CD5 domains of T-cadherin (40), respectively 168 (Supplementary Fig. 1D). 169 Analysis of T-cadherin primary structure did not reveal any canonical nuclear localization 170 sequences, although it is enriched in basic residues and is predicted to be a nuclear protein by 171 the ESLPred (8) and SubLoc (28) algorithms. We also found that T-cadherin contains a Leu-rich 172 sequence LRFSLPSVLLLSLFSLACL within its C-terminal recognition sequence (CSR) that 173 perfectly matches a nuclear export signal consensus Lx2-3Lx2-3LxL (46). Before mature GPI- 174 anchored proteins are delivered to the cell surface, they undergo processing in the ER. Their 175 CSRs are removed by proteolytic cleavage in the ER simultaneously with the attachment of the 176 GPI-anchor to the newly exposed C-terminus (22). We examined the localization of FLAG- 177 tagged T-cadherin construct, where the epitope is placed after the CSR for GPI-attachment (see 178 Supplementary Fig. 1A). C-terminal epitope tags attached after CSRs are not expected to 179 impede the protein proper processing, and are removed when the protein is processed in the 180 ER (2, 7, 65), thus only the molecules that are not (yet) processed are detectable with the 181 epitope-specific antibody. The non-processed FLAG-tagged T-cadherin could be exclusively 182 detected in the non-nuclear fractions (Fig. 2B, Supplementary Fig. 1C), presumably in the ER, 8 183 taken into account a characteristic reticular pattern observed with FLAG-tagged T-cadherin 184 (Supplementary Fig. 1C). 185 We also examined the effect of leptomycin B (LMB, an inhibitor of CRM1-dependent nuclear 186 export) on distribution of endogenous T-cadherin in HUVECs. The proportion of both 187 endogenous T-cadherin forms increased by 1.5-2 fold in the nucleus upon LMB treatment, 188 indicating that the presence of T-cadherin in the nucleus is dynamically regulated. As it is 189 unlikely that mature T-cadherin possesses this CSR sequence, it is possible that it interacts with 190 a partner with functional nuclear export signal. 191 T-cadherin is present in the centrosomes. In dividing HPAECs, T-cadherin was associated with 192 two punctate structures located on the opposite sides of chromatin and thus resembling 193 centrosomes (see Fig. 1B, lower HPAEC panel). Z-sectioning of individual cells confirmed that 194 each mitotic cell contained exactly two T-cadherin-positive structures (Supplementary Fig. 2A), 195 consistent with their identification as centrosomes. Therefore, we examined whether T-cadherin 196 would co-localize with a centrosomal marker γ-tubulin. We found a clear co-localization between 197 the two proteins throughout the cell cycle (Fig. 3; methanol fixation was used in this experiment 198 to optimize centrosome preservation, which is not optimal for visualization of the nuclei). This 199 indicated that T-cadherin is indeed present in the centrosomes. In the telophase it was often 200 transiently concentrated in the midbody, with reduced or undetectable presence in the 201 centrosomes (note that the brightness of the green channel is increased in the telophase panel 202 as compared to other panels in Fig. 3). In HUVECs, these T-cadherin-positive centrosomes 203 could be observed throughout the cell cycle, including mitosis. In HPAECs, a qualitatively similar 204 pattern was observed, however centrosomal T-cadherin staining was most obvious in 205 metaphase and anaphase. 206 Centrosome-associated 207 (Supplementary Fig. 2B), although less efficiently than with the SC antibody (while both T-cadherin could also 9 be detected using the TK antibody 208 antibodies stain the nuclei similarly, Fig. 1B). Lower efficiency of the TK antibody might be due 209 to partial unavailability of its epitope (CD1). In PC12 cells, T-cadherin was reported to be 210 ubiquitinated by Cdh1, a component of a major ubiquitination system that controls the 211 proteasome-dependent destruction of cell cycle regulators (5). Proteasomes are located both in 212 the nucleus and in the centrosomes (see Refs in (16)). Cdh1 localizes to the nucleus during 213 interphase, and to the centrosomes during metaphase and anaphase (78). This prompted us to 214 test whether the levels of T-cadherin in HUVECs would be affected by proteasome inhibition. 215 After 4 h incubation with proteasome inhibitors I1 and MG132, the levels of T-cadherin were 216 significantly increased, while incubation with I2 (calpain inhibitor that does not affect proteasome) 217 resulted in only marginal if any increase in T-cadherin levels (Fig. 4A). These data indicate that 218 T-cadherin levels in HUVECs are under control of proteasome-dependent degradation. 219 To confirm the centrosomal location of T-cadherin, we isolated centrosomal fractions from 220 HUVECs by differential centrifugation using a 20-70% discontinuous sucrose gradient. The 221 fractionation was confirmed using γ-tubulin as a centrosomal marker. Immunoblotting showed a 222 biphasic distribution of γ-tubulin (Fig. 4B). The lower molecular weight form, but not the 130 kDa 223 form of T-cadherin could be detected in the centrosomal fractions using the TK antibody and 224 was mainly associated with the first of the two γ-tubulin peaks (Fig. 4B). Reprobing the same 225 blot with the SC antibody (which does not recognize full length glycosylated T-cadherin on 226 Western blots, but may efficiently recognize truncated form(s), see above) revealed the 227 presence of a ~60 kDa band, associated with the second γ-tubulin peak and also found in a 228 denser fraction (Fig. 4B). Thus, the results of cenrosome isolation indicate that only the mature 229 processed form but not the 130 kDa form of T-cadherin is associated with a subfraction of the 230 centrosomes. Our data are also compatible with a truncated form or non-glycosylated T- 231 cadherin present in a distinct centrosomal subfraction. 10 232 To examine whether T-cadherin and γ-tubulin might physically associate, we performed 233 immunoprecipitation from HUVECs. We first attempted to assess whether endogenous T- 234 cadherin might associate with γ-tubulin. However, endogenous T-cadherin could not be 235 detected in γ-tubulin immunoprecipitates, while reciprocal immunoprecipitation proved 236 inconclusive due to nearly identical molecular weight of γ-tubulin and the heavy immunoglobulin 237 chain (data not shown). Taking into account that only a small proportion of endogenous T- 238 cadherin is located in the centrosomes in comparison with the nucleus in ECs, we next 239 attempted to use overexpressed T-cadherin to assess the possibility of its interaction with γ- 240 tubulin. Since the SC antibody recognizes the ΔC3-4 T-cadherin construct, but not full length 241 constructs on Western blots (as discussed above and shown in Supplementary Fig. 1B), 242 HUVECs were transfected with the latter construct or empty vector. Immunoprecipitation was 243 performed using γ-tubulin- and GFP-specific antibodies. The presence of a band of the 244 expected molecular size (see Supplementary Fig. 1B) recognized by the SC antibody could be 245 detected in the material precipitated from T-cadherin transfected cells with the γ-tubulin antibody, 246 but not in the negative controls (Fig. 4C). No corresponding signal could be detected in the 247 immunoprecipitates using FLAG antibody, consistent with a form that has been C-terminally 248 processed in the ER. 249 To assess whether the centrosomal and nuclear location of T-cadherin is specific for ECs, we 250 examined localization of endogenous T-cadherin in HEK 293A cells. Immunoblotting analysis 251 showed that HEK 293A (as well as COS-7) cells express considerably lower levels of T- 252 cadherin relative to total protein than ECs (data not shown). We found that in HEK 293A cells, 253 most of endogeneous T-cadherin co-localized with γ-tubulin and thus was associated with the 254 centrosomes (which are unusually large structures in this cell line (55, 76)), with only very faint 255 nuclear and cell surface staining (Supplementary Fig. 3A). In an attempt to detect association of 256 endogenous T-cadherin with γ-tubulin, we performed immunoprecipitation experiments from 11 257 HEK 293A cells. A ~60 kDa band could be detected in γ-tubulin immunoprecipitates, but not in 258 GFP antibody immunoprecipitates used as a negative control (Supplementary Fig 3B). The 259 molecular weight of this band is close to that expected for non-glycosylated T-cadherin without 260 prepeptide (65 kDa). 261 Interference with T-cadherin expression leads to defects in cell division. As described above, T- 262 cadherin was often transiently concentrated in the midbody, which plays a key role in 263 cytokinesis (6). This observation suggested that T-cadherin might be involved in cytokinesis. 264 Cytokinesis failure leads to appearance of cells containing more than one nucleus. To assess 265 whether overexpression or downregulation of T-cadherin might affect the probability of failed 266 cytokinesis, we examined the proportion of bi- or multinuclear cells among HUVECs transfected 267 with either T-cadherin cDNA or T-cadherin siRNA. A small proportion of ECs is naturally bi- or 268 multinuclear, and an increase in the proportion of such cells has been associated with some 269 physiological or pathological conditions (19, 36, 69). In line with these reports, we observed that 270 primary HUVEC cultures contained 4-5 % cells with more than one nucleus (of which absolute 271 majority were binuclear cells). 272 T-cadherin could be efficiently overexpressed in ECs (Fig. 5A; Supplementary Fig. 1C), 273 resulting in an overall increase in T-cadherin content of up to 10-20 fold (ECL quantification data 274 not shown). Immunofluorescence microscopy revealed that in some cells T-cadherin was 275 present in a reticular pattern (Fig. 5B, upper panel), probably reflecting its predominant 276 presence in the endoplasmic reticulum, while in other cells punctate cytoplasmic as well as 277 nuclear staining was observed (Fig. 5B, middle panel). The proportion of cells containing more 278 than one nucleus was increased among the cells overexpressing T-cadherin (Fig. 5 B, C). 279 Similar results were obtained using overexpression of FLAG-tagged T-cadherin in HUVECs 280 (data not shown) and in HEK 293A cells (Supplementary Fig. 3 C-E). 12 281 We also depleted endogenous T-cadherin in ECs using siRNA approach, and examined the 282 cells for any apparent defects associated with cell division. Both 105 and 130 kDa forms of T- 283 cadherin could be efficiently downregulated in HPAECs and in HUVECs (Fig. 6A). It was 284 reported that the levels of different cadherins may influence each other, for example, VE- 285 cadherin expression is reduced in N-cadherin-deficient endothelium (48)). VE-cadherin levels 286 remained unchanged in the cells with 80-90% T-cadherin depleted (Fig. 6A). In some 287 experiments, 95-98% T-cadherin depletion was achieved, which was accompanied by a 30-40% 288 decrease in VE-cadherin levels (data not shown). T-cadherin depletion also resulted in a 289 considerable decrease in T-cadherin positive staining in immunofluorescence experiments (Fig 290 6B, C). 291 T-cadherin depletion led to an only marginal increase in the proportion of multinucleated cells, 292 which was not statistically significant (Fig. 6E). However, we noticed that downregulation of T- 293 cadherin was accompanied by an increased proportion of mononuclear ECs with multiple 294 centrosomes (Fig. 6 C,D,F), which suggests a specific role of T-cadherin in the control of 295 centrosomal organization. 296 297 DISCUSSION 298 We report here that a considerable proportion of T-cadherin is located in the nucleus, as 299 evidenced by immunofluorescence and by immunoblotting upon cell fractionation using 300 antibodies raised against different epitopes. Nuclear localization of cleaved intracellular domains 301 of some other members of the cadherin family, such as protocadherins α (18), γ (29), Fat1 (33, 302 49), E-cadherin (21) was reported. However, T-cadherin has no intracellular domain and is 303 present in the nucleus as a full-length molecule; therefore trafficking pathways suggested for 304 other cadherins may not be relevant in the case of T-cadherin. Full length cell surface receptors, 305 both G-protein coupled receptors and tyrosine kinase receptors, were reported to translocate to 13 306 the nucleus (17, 20, 25, 53, 62, 72), including LPA1, for which caveolin-mediated endocytosis 307 was suggested (26). Nucleus is rich in lipid environments, which contain lipid raft components, 308 including caveolin-1 (1). However, several lines of evidence argue against a role of caveolin in 309 the nuclear trafficking of T-cadherin. (i) Pertussis toxin inhibits LPA1 trafficking to the nucleus 310 (26), but does not affect the nuclear localization of T-cadherin (our unpublished observations), 311 which makes a common trafficking mechanism unlikely. (ii) Although caveolae-dependent 312 endocytosis was originally considered for GPI proteins (3), later studies suggested a caveolae- 313 independent pinocytotic pathway (44, 45, 64) or macropinocytotic pathways triggered by a 314 clustering agent or being ligand-independent (14, 74). (iii) In ECs, caveolin is not involved in T- 315 cadherin-mediated signaling (59). However, a recently described T-cadherin interaction with 316 GRP78 (58), which is reportedly involved in caveolae-dependent internalization and nuclear 317 localization of extracellular matrix protein DMP1 (61), reintroduces the question about a possible 318 role of caveolae. 319 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- 321 cadherin in ECs might differ from those assumed on the basis of canonical processing of GPI- 322 anchored proteins. 323 If T-cadherin is processed as a canonical GPI-anchored protein, the questions arise whether it 324 is targeted to the nucleus after its delivery to the PM, or directly from the ER. Some models for 325 the nuclear translocation of cell surface receptors have been suggested (47), and although 326 these questions are beyond the scope of this work, T-cadherin would be an excellent model to 327 study this process. 328 Apart from the question of how T-cadherin is imported into the nucleus, another question is what 329 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 332 upregulates cyclin D1 (41) and may activate the serum response element (our unpublished 333 data), a similar function seems conceivable for T-cadherin. 334 We also found that T-cadherin is located in the centrosomes. A quantitative difference between 335 venous (HUVECs) and arterial (HPAECs) endothelial cells could be observed, as T-cadherin 336 was present in the centrosomes uniformly throughout the cell cycle (except telophase) in 337 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), 339 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 344 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 REFERENCES 398 1. Albi E and Viola Magni MP. The role of intranuclear lipids. Biol Cell 96: 657-667, 2004. 399 2. 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Fibroblast growth factor 2 25 598 75. 599 related (fzr) increases natural killer cell-mediated cell death and suppresses tumor growth. 600 Blood 96: 259-263, 2000. 601 76. 602 Thomas PJ. Dynamic association of proteasomal machinery with the centrosome. J Cell Biol 603 145: 481-490, 1999. 604 77. 605 different microtubule-dependent processes. Mol Biol Cell 17: 2476-2487, 2006. 606 78. 607 regulator CDH1 and its regulation by phosphorylation. J Biol Chem 278: 12530-12536, 2003. Wang CX, Fisk BC, Wadehra M, Su H, and Braun J. Overexpression of murine fizzy- Wigley WC, Fabunmi RP, Lee MG, Marino CR, Muallem S, DeMartino GN, and Zhou C, Cunningham L, Marcus AI, Li Y, and Kahn RA. Arl2 and Arl3 regulate Zhou Y, Ching YP, Chun AC, and Jin DY. Nuclear localization of the cell cycle 608 609 610 26 611 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