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Colocalization of Emerin and Lamins in Interphase Nuclei and Changes during Mitosis
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
249, 643–647 (1998)
RC989209
Colocalization of Emerin and Lamins in Interphase Nuclei and Changes during Mitosis S. Manilal, Nguyen thi Man, and G. E. Morris1 MRIC Biochemistry Group, N.E. Wales Institute, Wrexham LL11 2AW, United Kingdom
Received July 20, 1998
Emerin is a nuclear membrane protein which is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. We have previously suggested that emerin is a member of a family of type II integral membrane proteins which associate with the nuclear lamina and which include lamina-associated proteins and the lamin B receptor. We now show that emerin in COS cells is not restricted to the nuclear rim but is also found at intranuclear sites, where it colocalizes with nuclear lamins B1, B2 and A/C. During mitosis, emerin is dispersed throughout the cell and then participates in the reconstitution of membranes around the daughter nuclei. Although emerin and lamins do not remain colocalized during mitosis, they all show some association with the midbody of the mitotic spindle. q 1998 Academic Press Key Words: Emery-Dreifuss muscular dystrophy; nuclear lamina; nuclear envelope; mitotic spindle; lamin B; lamin A.
Emery-Dreifuss muscular dystrophy (EDMD) is characterized by early contractures of the elbows, Achilles tendons and spine, slowly-progressive muscle wasting and cardiac conduction defects [1]. The Xlinked disorder is caused by mutations in the gene for emerin, a 254 amino acid, serine rich protein which is ubiquitously expressed in most tissues and which contains two regions of homology with thymopoietins [2]. Emerin is absent in most X-linked EDMD patients and is localized at the nuclear rim in muscle and several other tissues [3, 4, 5]. In subcellular fractionation experiments, most of the emerin was found in the nuclear fraction, but a small proportion in the microsomal fraction may reflect transport to the nucleus through the endoplasmic reticulum [4]. It has recently been suggested that cardiac conduction defects in EDMD might 1 Author for correspondence. Fax: 44-1978-290008. E-mail: morrisge@ newi.ac.uk.
be explained by an additional localization of emerin at intercalated disks in the heart [6]. Emerin appears to belong to a family of type II integral membrane proteins which are anchored to the inner nuclear membrane via hydrophobic tails, with the remainder of the molecule projecting into the nucleoplasm [4]. This family includes lamina-associated protein 1 (LAP1) [7], lamina-associated protein 2 (LAP2; now known to be b-thymopoietin) [8, 9] and the lamin B receptor (LBR) [10]. Although all other members of this family are lamin-binding proteins, this has not yet been established experimentally for emerin. At the beginning of mitosis, phosphorylation of B-lamins, LBR and LAP2 is followed by disruption of the nuclear lamina and membrane [11, 12, 13]. A generally-accepted view has been that the nuclear membrane with associated B-lamins is dispersed as membrane vesicles during mitosis. There is evidence that both LAP1 [14] and LBR [12] remain at least partly associated with lamin B in mitotic vesicles, whereas LAP2 dissociates from lamin B after phosphorylation [13]. Recent studies with transfected COS cells, however, have suggested that LBR may also disperse throughout a largely intact ER during mitosis [15] in a reversal of the process by which newly-synthesized type II nuclear membrane proteins are thought to be captured in the nucleus as they migrate through the rough ER. Further studies are clearly needed to establish precisely what happens to the nuclear envelope membranes and to each of the lamina-associated proteins during mitosis. The purpose of the present study was to determine the distribution of emerin during mitosis, especially in relation to the nuclear lamins, using COS cell cultures which contain a high proportion of mitotic cells. MATERIALS AND METHODS COS-7 cells, an African Green monkey kidney cell line, were grown on glass coverslips in Dulbecco’s MEM with 10% horse serum. Pre-confluent cells were fixed briefly by immersion in 50% methanol/ 50% acetone, air-dried and stored at 0707C. For detection of emerin alone, MANEM1 mAb [4] was used with FITC-labelled anti- (mouse Ig) (DAKO) as described previously [16]. For double0006-291X/98 $25.00
643
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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BBRC 9209
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08-05-98 10:48:21
bbrcg
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Vol. 249, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Emerin disperses throughout the cell in late prophase and participates in reforming the nuclear envelope during telophase. (a) Interphase, (b) late prophase, (c) metaphase, (d) early telophase. The primary emerin antibody was MANEM1 [4].
labelling, a rabbit polyclonal antibody was raised against a recombinant protein containing the first 188 amino-acids of emerin [4]. It was used at 1:100 dilution with TRITC-labelled anti-(rabbit IgG) to detect emerin. MAbs against lamin B2 (1:50 dilution; Novocastra Laboratories, Newcastle-upon-Tyne, UK), lamin B1 (1:50; Chemicon, Temecula, CA), lamin A (1:5; Chemicon) and lamin A/C (1:200; Chemicon) were used with FITC-labelled anti- (mouse Ig) (DAKO).
RESULTS Fig. 1 shows the distribution of emerin in COS cells (monkey kidney cell line) at various stages of mitosis, determined using a monoclonal antibody against emerin [4]. Fig. 1a is the interphase distribution, mainly at the nuclear membrane but also in internal spots and fibres. In late prophase, when the nuclear membrane has disassembled, emerin is observed throughout the cytoplasm which surrounds the chromosomal material (Fig. 1b). In metaphase, emerin staining is slightly brighter at the two poles of the mitotic spindle, as seen in the centre of the cell in Fig. 1c. Finally, Fig. 1d shows early telophase cells in which emerin is participating in the re-assembly of the nuclear membrane around the two daughter nuclei. During mitosis, B lamins often take up characteristic distribution patterns in dividing nuclei and cells, as described previously for Ishikawa adenocarcinoma cells and rat kidney cells [14, 17]. Figs. 2 and 3 show that these distribution patterns can be observed in COS cells when double-labelling for emerin and various lamins. A rabbit polyclonal antibody against emerin was required for this double-label study and a control experiment in which COS cells were double-labelled with rabbit polyclonal and mouse monoclonal antibodies against emerin showed that both antibodies gave the same localizations for emerin (results not shown). In Fig. 2a/b, emerin and lamin B2 exhibit an almost identical pattern of discrete internal staining in large interphase nuclei, in addition to nuclear membrane staining, although there are variations in intensity. In mitotic cells, emerin and lamin B2 are dispersed around the chromosomes throughout the cell, but the two proteins no longer co-localize precisely, although they both tend to concentrate near the spindle poles
(Fig. 2c/d). In late telophase, Maison et al. [14] have shown that lamin B and LAP1 both remain partly associated with the mid-body of the mitotic spindle between the separating daughter nuclei. Fig. 2e/f shows this for both lamin B1 and emerin in COS cells and it was observed in most cells at this stage of mitosis. The mAbs against lamins B1 and B2 gave similar results. Internal nuclear staining is much brighter for lamin B1 than emerin at this stage of mitosis (Fig. 2e/f), though whether this implies earlier relocation by lamin B1 after mitosis requires further investigation. Co-localization with emerin in interphase nuclei was also observed with mAbs against lamins A and C (Fig. 3a/b) and lamin A (not shown), but staining by these lamin mAbs was greatly reduced in mitotic cells relative to emerin (Fig. 3c/d). The mid-body of the spindle between daughter cells is recognized by both emerin and lamin A or A/C mAbs, but emerin staining is usually continuous whereas lamin staining is usually broken in the middle (Fig. 3e/f; small arrow). This can also be seen in Fig. 2e/f where lamin B1 is virtually absent from the longer mid-body which still contains emerin (arrow). This suggests that, as daughter cells separate, lamins are retracted into the nucleus earlier than emerin. Fig. 3e/f (large arrow) shows a deep indentation of the nuclear membrane characteristic of early prophase and Georgatos et al [17] have shown that this is caused by growing microtubules which will eventually form the mitotic spindle. This suggests that the daughter nuclei in Fig. 3e/f have begun to re-enter prophase while the late telophase mid-body is still present. Cytoplasmic staining by the rabbit anti-emerin antibody is mainly non-specific since it is seen to a lesser extent with mAbs (Fig. 1a), although there is subcellular fractionation evidence for the presence of some emerin outside the nucleus [4]. DISCUSSION We have shown that the distribution of emerin in interphase cells is very similar to that of lamins B1, B2 and A/C and is consistent with a direct or indirect interaction between emerin and the nuclear lamina. The spotty distribution of emerin and lamins inside interphase nuclei is similar to that observed by Bridger et al [18] for lamins A/C. The ‘‘spots’’ in this study may correspond to the deep and narrow invaginations of the nuclear envelope transecting the nucleus described by Fricker et al [19]. Our studies of lamin B2 distribution during mitosis in COS cells agree very well with recent studies in other cell lines by Georgatos and co-workers [14, 17]. These authors showed that association with spindle microtubules during mitosis was a feature of B lamins and LAP1, and they suggested that a linker protein, such as plectin which binds both lamin B and microtubules, might be responsible. LAP2 has two regions of strong sequence similarity with emerin, but
644
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AP: BBRC
Vol. 249, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Distribution of emerin and B-lamins in interphase COS cell nuclei and during mitosis. (a) Emerin or (b) lamin B2 interphase, (c) emerin or (d) lamin B2 metaphase, (e) emerin or (f) lamin B1 late telophase. Similar results were obtained with lamin B1 and B2 antibodies at all stages. Double labelling with rabbit polyclonal anti-emerin and mouse monoclonal anti-lamin (see Methods). See text for arrows. 645
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Vol. 249, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Distribution of emerin and lamin A/C in interphase COS cell nuclei and during mitosis. (a/b) Interphase, (c/d) early telophase, (e/f) late telophase. Similar results were obtained with lamin A antibody. Double labelling with rabbit polyclonal anti-emerin and mouse monoclonal anti-lamin (see Methods). See text for arrows.
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Vol. 249, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
neither is thought to be involved in lamin binding. One is the C-terminal transmembrane helix and the other is of unknown function near the N-terminus of both proteins. The minimal lamin-binding (298-373) and chromatin-binding (1-88) sequences identified in LAP2 [8, 20] have no significant sequence similarity to emerin. LAP2 is phosphorylated by cdc2 kinase during mitosis and this causes its dissociation from lamin B [13], but emerin has no cdc2 kinase site. Thus, although sequence homologies exist between emerin and LAP2, there is nothing in them to suggest that emerin interacts directly with lamins or chromatin. Co-distribution of emerin and lamins does not necessarily mean that they interact directly, since the association could be mediated by other proteins or by membrane vesicles. We have demonstrated immunoprecipitation of emerin from tissue extracts (10,000g supernatant) by lamin B2 antibody (results not shown) although this is of limited value for showing direct interaction since the emerin is present in very large particles (pelleted at 100,000g [4]) which might contain fragments of lamina and other proteins. The nuclear function of emerin is not known, but it is clearly not essential for the survival of most cell types since the clinical phenotype in emerin-negative EDMD patients is limited to certain tissues and develops only slowly. It is possible that, in the absence of emerin, most of its functions can be carried out by other nuclear proteins.
ACKNOWLEDGMENT We thank the British Heart Foundation for a research grant (PG97142).
REFERENCES 1. Emery, A. E. H. (1989) J. Med. Genet. 26, 637–641. 2. Bione, S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G., and Toniolo, D. (1994) Nature Genet. 8, 323–327. 3. Nagano, A., Koga, R., Ogawa, M., Kurano, Y., Kawada, J., Okada, R., Hayashi, Y. K., Tsukuhara, T., and Arahata, K. (1996) Nature Genet. 12, 254–259. 4. Manilal, S., Nguyen thi Man, Sewry, C. A., and Morris, G. E. (1996) Hum. Mol. Genet. 5, 801–808. 5. Manilal, S., Sewry, C. A., Nguyen thi Man, Muntoni, F., and Morris, G. E. (1997) Neuromusc. Disord. 7, 63–66. 6. Cartegni, L., di Barletta, M. R., Barresi, R., et al. (1997) Hum. Mol. Genet. 6, 2257–2264. 8. Furukawa, K., Pante´, N., Aebi, U., and Gerace, L. (1995) EMBO J. 14, 1626–1636. 9. Harris, C. A., Andryuk, P. J., Cline, S. W., Mathew, S., Siekierka, J. J., and Goldstein, G. (1995) Genomics 28, 198–205. 10. Soullam, B., and Worman, H. J. (1993) J. Cell Biol. 120, 1093– 1100. 11. Nigg, E. A. (1992) Curr. Opin. Cell Biol. 4, 105–109. 12. Courvalin, J-C., Segil, N., Blobel, G., and Worman, H. J. (1992) J. Biol. Chem. 135, 19035–19038. 13. Foisner, R., and Gerace, L. (1993) Cell 73, 1267–1279. 14. Maison, C., Pyrpasopoulou, A., Theodoropoulos, P. A., and Georgatos, S. D. (1997) EMBO J. 16, 4839–4850. 15. Ellenberg, J., Siggia, E. D., Moreira, J. E., Smith, C. L., Presley, J. F., Worman, H. J., and Lippincott-Schwartz, J. (1997) J. Cell Biol. 138, 1193–1206. 16. Nguyen thi Man, Ellis, J. M., Love, D. R., Davies, K. E., Gatter, K. C., Dickson, G., and Morris, G. E. (1991) J. Cell Biol. 115, 1695–1700. 17. Georgatos, S. D., Pyrpasopoulou, A., and Theodoropoulos, P. A. (1997) J. Cell Sci. 110, 2129–2140. 18. Bridger, J. M., Kill, I. R., O’Farrell, M., and Hutchinson, C. J. (1993) J. Cell Sci. 104, 297–306. 19. Fricker, M., Hollinshead, M., White, N., and Vaux, D. (1997) J. Cell Biol. 136, 531–544. 20. Yang, L., Guan, T., and Gerace, L. (1997) J. Cell Biol. 139, 1077– 1087.
647
AID
BBRC 9209
/
695c$$$222
08-05-98 10:48:21
bbrcg
AP: BBRC
249, 643–647 (1998)
RC989209
Colocalization of Emerin and Lamins in Interphase Nuclei and Changes during Mitosis S. Manilal, Nguyen thi Man, and G. E. Morris1 MRIC Biochemistry Group, N.E. Wales Institute, Wrexham LL11 2AW, United Kingdom
Received July 20, 1998
Emerin is a nuclear membrane protein which is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. We have previously suggested that emerin is a member of a family of type II integral membrane proteins which associate with the nuclear lamina and which include lamina-associated proteins and the lamin B receptor. We now show that emerin in COS cells is not restricted to the nuclear rim but is also found at intranuclear sites, where it colocalizes with nuclear lamins B1, B2 and A/C. During mitosis, emerin is dispersed throughout the cell and then participates in the reconstitution of membranes around the daughter nuclei. Although emerin and lamins do not remain colocalized during mitosis, they all show some association with the midbody of the mitotic spindle. q 1998 Academic Press Key Words: Emery-Dreifuss muscular dystrophy; nuclear lamina; nuclear envelope; mitotic spindle; lamin B; lamin A.
Emery-Dreifuss muscular dystrophy (EDMD) is characterized by early contractures of the elbows, Achilles tendons and spine, slowly-progressive muscle wasting and cardiac conduction defects [1]. The Xlinked disorder is caused by mutations in the gene for emerin, a 254 amino acid, serine rich protein which is ubiquitously expressed in most tissues and which contains two regions of homology with thymopoietins [2]. Emerin is absent in most X-linked EDMD patients and is localized at the nuclear rim in muscle and several other tissues [3, 4, 5]. In subcellular fractionation experiments, most of the emerin was found in the nuclear fraction, but a small proportion in the microsomal fraction may reflect transport to the nucleus through the endoplasmic reticulum [4]. It has recently been suggested that cardiac conduction defects in EDMD might 1 Author for correspondence. Fax: 44-1978-290008. E-mail: morrisge@ newi.ac.uk.
be explained by an additional localization of emerin at intercalated disks in the heart [6]. Emerin appears to belong to a family of type II integral membrane proteins which are anchored to the inner nuclear membrane via hydrophobic tails, with the remainder of the molecule projecting into the nucleoplasm [4]. This family includes lamina-associated protein 1 (LAP1) [7], lamina-associated protein 2 (LAP2; now known to be b-thymopoietin) [8, 9] and the lamin B receptor (LBR) [10]. Although all other members of this family are lamin-binding proteins, this has not yet been established experimentally for emerin. At the beginning of mitosis, phosphorylation of B-lamins, LBR and LAP2 is followed by disruption of the nuclear lamina and membrane [11, 12, 13]. A generally-accepted view has been that the nuclear membrane with associated B-lamins is dispersed as membrane vesicles during mitosis. There is evidence that both LAP1 [14] and LBR [12] remain at least partly associated with lamin B in mitotic vesicles, whereas LAP2 dissociates from lamin B after phosphorylation [13]. Recent studies with transfected COS cells, however, have suggested that LBR may also disperse throughout a largely intact ER during mitosis [15] in a reversal of the process by which newly-synthesized type II nuclear membrane proteins are thought to be captured in the nucleus as they migrate through the rough ER. Further studies are clearly needed to establish precisely what happens to the nuclear envelope membranes and to each of the lamina-associated proteins during mitosis. The purpose of the present study was to determine the distribution of emerin during mitosis, especially in relation to the nuclear lamins, using COS cell cultures which contain a high proportion of mitotic cells. MATERIALS AND METHODS COS-7 cells, an African Green monkey kidney cell line, were grown on glass coverslips in Dulbecco’s MEM with 10% horse serum. Pre-confluent cells were fixed briefly by immersion in 50% methanol/ 50% acetone, air-dried and stored at 0707C. For detection of emerin alone, MANEM1 mAb [4] was used with FITC-labelled anti- (mouse Ig) (DAKO) as described previously [16]. For double0006-291X/98 $25.00
643
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 9209
/
695c$$$221
08-05-98 10:48:21
bbrcg
AP: BBRC
Vol. 249, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Emerin disperses throughout the cell in late prophase and participates in reforming the nuclear envelope during telophase. (a) Interphase, (b) late prophase, (c) metaphase, (d) early telophase. The primary emerin antibody was MANEM1 [4].
labelling, a rabbit polyclonal antibody was raised against a recombinant protein containing the first 188 amino-acids of emerin [4]. It was used at 1:100 dilution with TRITC-labelled anti-(rabbit IgG) to detect emerin. MAbs against lamin B2 (1:50 dilution; Novocastra Laboratories, Newcastle-upon-Tyne, UK), lamin B1 (1:50; Chemicon, Temecula, CA), lamin A (1:5; Chemicon) and lamin A/C (1:200; Chemicon) were used with FITC-labelled anti- (mouse Ig) (DAKO).
RESULTS Fig. 1 shows the distribution of emerin in COS cells (monkey kidney cell line) at various stages of mitosis, determined using a monoclonal antibody against emerin [4]. Fig. 1a is the interphase distribution, mainly at the nuclear membrane but also in internal spots and fibres. In late prophase, when the nuclear membrane has disassembled, emerin is observed throughout the cytoplasm which surrounds the chromosomal material (Fig. 1b). In metaphase, emerin staining is slightly brighter at the two poles of the mitotic spindle, as seen in the centre of the cell in Fig. 1c. Finally, Fig. 1d shows early telophase cells in which emerin is participating in the re-assembly of the nuclear membrane around the two daughter nuclei. During mitosis, B lamins often take up characteristic distribution patterns in dividing nuclei and cells, as described previously for Ishikawa adenocarcinoma cells and rat kidney cells [14, 17]. Figs. 2 and 3 show that these distribution patterns can be observed in COS cells when double-labelling for emerin and various lamins. A rabbit polyclonal antibody against emerin was required for this double-label study and a control experiment in which COS cells were double-labelled with rabbit polyclonal and mouse monoclonal antibodies against emerin showed that both antibodies gave the same localizations for emerin (results not shown). In Fig. 2a/b, emerin and lamin B2 exhibit an almost identical pattern of discrete internal staining in large interphase nuclei, in addition to nuclear membrane staining, although there are variations in intensity. In mitotic cells, emerin and lamin B2 are dispersed around the chromosomes throughout the cell, but the two proteins no longer co-localize precisely, although they both tend to concentrate near the spindle poles
(Fig. 2c/d). In late telophase, Maison et al. [14] have shown that lamin B and LAP1 both remain partly associated with the mid-body of the mitotic spindle between the separating daughter nuclei. Fig. 2e/f shows this for both lamin B1 and emerin in COS cells and it was observed in most cells at this stage of mitosis. The mAbs against lamins B1 and B2 gave similar results. Internal nuclear staining is much brighter for lamin B1 than emerin at this stage of mitosis (Fig. 2e/f), though whether this implies earlier relocation by lamin B1 after mitosis requires further investigation. Co-localization with emerin in interphase nuclei was also observed with mAbs against lamins A and C (Fig. 3a/b) and lamin A (not shown), but staining by these lamin mAbs was greatly reduced in mitotic cells relative to emerin (Fig. 3c/d). The mid-body of the spindle between daughter cells is recognized by both emerin and lamin A or A/C mAbs, but emerin staining is usually continuous whereas lamin staining is usually broken in the middle (Fig. 3e/f; small arrow). This can also be seen in Fig. 2e/f where lamin B1 is virtually absent from the longer mid-body which still contains emerin (arrow). This suggests that, as daughter cells separate, lamins are retracted into the nucleus earlier than emerin. Fig. 3e/f (large arrow) shows a deep indentation of the nuclear membrane characteristic of early prophase and Georgatos et al [17] have shown that this is caused by growing microtubules which will eventually form the mitotic spindle. This suggests that the daughter nuclei in Fig. 3e/f have begun to re-enter prophase while the late telophase mid-body is still present. Cytoplasmic staining by the rabbit anti-emerin antibody is mainly non-specific since it is seen to a lesser extent with mAbs (Fig. 1a), although there is subcellular fractionation evidence for the presence of some emerin outside the nucleus [4]. DISCUSSION We have shown that the distribution of emerin in interphase cells is very similar to that of lamins B1, B2 and A/C and is consistent with a direct or indirect interaction between emerin and the nuclear lamina. The spotty distribution of emerin and lamins inside interphase nuclei is similar to that observed by Bridger et al [18] for lamins A/C. The ‘‘spots’’ in this study may correspond to the deep and narrow invaginations of the nuclear envelope transecting the nucleus described by Fricker et al [19]. Our studies of lamin B2 distribution during mitosis in COS cells agree very well with recent studies in other cell lines by Georgatos and co-workers [14, 17]. These authors showed that association with spindle microtubules during mitosis was a feature of B lamins and LAP1, and they suggested that a linker protein, such as plectin which binds both lamin B and microtubules, might be responsible. LAP2 has two regions of strong sequence similarity with emerin, but
644
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BBRC 9209
/
695c$$$222
08-05-98 10:48:21
bbrcg
AP: BBRC
Vol. 249, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Distribution of emerin and B-lamins in interphase COS cell nuclei and during mitosis. (a) Emerin or (b) lamin B2 interphase, (c) emerin or (d) lamin B2 metaphase, (e) emerin or (f) lamin B1 late telophase. Similar results were obtained with lamin B1 and B2 antibodies at all stages. Double labelling with rabbit polyclonal anti-emerin and mouse monoclonal anti-lamin (see Methods). See text for arrows. 645
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Vol. 249, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Distribution of emerin and lamin A/C in interphase COS cell nuclei and during mitosis. (a/b) Interphase, (c/d) early telophase, (e/f) late telophase. Similar results were obtained with lamin A antibody. Double labelling with rabbit polyclonal anti-emerin and mouse monoclonal anti-lamin (see Methods). See text for arrows.
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Vol. 249, No. 3, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
neither is thought to be involved in lamin binding. One is the C-terminal transmembrane helix and the other is of unknown function near the N-terminus of both proteins. The minimal lamin-binding (298-373) and chromatin-binding (1-88) sequences identified in LAP2 [8, 20] have no significant sequence similarity to emerin. LAP2 is phosphorylated by cdc2 kinase during mitosis and this causes its dissociation from lamin B [13], but emerin has no cdc2 kinase site. Thus, although sequence homologies exist between emerin and LAP2, there is nothing in them to suggest that emerin interacts directly with lamins or chromatin. Co-distribution of emerin and lamins does not necessarily mean that they interact directly, since the association could be mediated by other proteins or by membrane vesicles. We have demonstrated immunoprecipitation of emerin from tissue extracts (10,000g supernatant) by lamin B2 antibody (results not shown) although this is of limited value for showing direct interaction since the emerin is present in very large particles (pelleted at 100,000g [4]) which might contain fragments of lamina and other proteins. The nuclear function of emerin is not known, but it is clearly not essential for the survival of most cell types since the clinical phenotype in emerin-negative EDMD patients is limited to certain tissues and develops only slowly. It is possible that, in the absence of emerin, most of its functions can be carried out by other nuclear proteins.
ACKNOWLEDGMENT We thank the British Heart Foundation for a research grant (PG97142).
REFERENCES 1. Emery, A. E. H. (1989) J. Med. Genet. 26, 637–641. 2. Bione, S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G., and Toniolo, D. (1994) Nature Genet. 8, 323–327. 3. Nagano, A., Koga, R., Ogawa, M., Kurano, Y., Kawada, J., Okada, R., Hayashi, Y. K., Tsukuhara, T., and Arahata, K. (1996) Nature Genet. 12, 254–259. 4. Manilal, S., Nguyen thi Man, Sewry, C. A., and Morris, G. E. (1996) Hum. Mol. Genet. 5, 801–808. 5. Manilal, S., Sewry, C. A., Nguyen thi Man, Muntoni, F., and Morris, G. E. (1997) Neuromusc. Disord. 7, 63–66. 6. Cartegni, L., di Barletta, M. R., Barresi, R., et al. (1997) Hum. Mol. Genet. 6, 2257–2264. 8. Furukawa, K., Pante´, N., Aebi, U., and Gerace, L. (1995) EMBO J. 14, 1626–1636. 9. Harris, C. A., Andryuk, P. J., Cline, S. W., Mathew, S., Siekierka, J. J., and Goldstein, G. (1995) Genomics 28, 198–205. 10. Soullam, B., and Worman, H. J. (1993) J. Cell Biol. 120, 1093– 1100. 11. Nigg, E. A. (1992) Curr. Opin. Cell Biol. 4, 105–109. 12. Courvalin, J-C., Segil, N., Blobel, G., and Worman, H. J. (1992) J. Biol. Chem. 135, 19035–19038. 13. Foisner, R., and Gerace, L. (1993) Cell 73, 1267–1279. 14. Maison, C., Pyrpasopoulou, A., Theodoropoulos, P. A., and Georgatos, S. D. (1997) EMBO J. 16, 4839–4850. 15. Ellenberg, J., Siggia, E. D., Moreira, J. E., Smith, C. L., Presley, J. F., Worman, H. J., and Lippincott-Schwartz, J. (1997) J. Cell Biol. 138, 1193–1206. 16. Nguyen thi Man, Ellis, J. M., Love, D. R., Davies, K. E., Gatter, K. C., Dickson, G., and Morris, G. E. (1991) J. Cell Biol. 115, 1695–1700. 17. Georgatos, S. D., Pyrpasopoulou, A., and Theodoropoulos, P. A. (1997) J. Cell Sci. 110, 2129–2140. 18. Bridger, J. M., Kill, I. R., O’Farrell, M., and Hutchinson, C. J. (1993) J. Cell Sci. 104, 297–306. 19. Fricker, M., Hollinshead, M., White, N., and Vaux, D. (1997) J. Cell Biol. 136, 531–544. 20. Yang, L., Guan, T., and Gerace, L. (1997) J. Cell Biol. 139, 1077– 1087.
647
AID
BBRC 9209
/
695c$$$222
08-05-98 10:48:21
bbrcg
AP: BBRC