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BAF is required for emerin assembly into the reforming nuclear envelope

Tokuko Haraguchi1,*, Takako Koujin1, Miriam Segura-Totten2, Kenneth K. Lee2, Yosuke Matsuoka3, Yoshihiro Yoneda3, Katherine L. Wilson2 and Yasushi Hiraoka1

1 CREST Research Project of the Japan Science and Technology Corporation, Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan
2 Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA
3 Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Osaka 565-0871, Japan



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  		Fig. 1. Intracellular localization of emerin in HeLa cells. (a-c) Emerin stained with the antibody in fixed cells:(a) interphase; (b) metaphase; and (c) telophase stages of mitosis. (d-e) GFP-emerin in living cells: (d) interphase; (e) metaphase through telophase; time-lapse images in (e) were obtained at one-minute intervals in the same cell. Chromosomes were stained with DAPI in fixed cells, and with Hoechst 33342 in living cells. Bar, 10 µm.

 


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  		Fig. 2. Emerin ‘core’ localization does not depend on microtubules. Cells treated with nocodazole or vinblastine were fixed and stained with antibodies against endogenous emerin. Bar, 10 µm.

 


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  		Fig. 3. Molecular domains of emerin necessary for its localization. (a) Molecular structure of emerin and its truncations. GFP was fused to their C-terminus. Localization of each truncation at the NE and the ‘core’ is summarized: N-terminal 65 residues are necessary and sufficient for the ‘core’ localization of emerin; C-terminal domain is required for nuclear membrane localization. Examples are shown for {Delta}66-254 (b-e), for {Delta}1-64 (f,g), for {Delta}226-254 (h-k), and for {Delta}175-254 (l-o). In (g) time-lapse images at 1 minute intervals are shown. Bar, 10 µm.

 


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  		Fig. 4. Intracellular localization of BAF during mitosis. (a) HeLa cells stained with antibodies #3273 against human BAF (see Methods). b HeLa cells stained with anti-BAF peptide antibodies (Furukawa, 1999). c HeLa cells that express GFP-BAF and stained with antibodies against emerin. d Time-lapse images of a living HeLa cell that expresses GFP-BAF. Chromosomes were stained with DAPI in fixed cells, and with Hoechst 33342 in living cells. Bar, 10 µm.

 


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  		Fig. 5. Localization of GFP-fused mutant emerin-m24. (a) Molecular structure of GFP-fusion to mutant emerin-m24. (b) Localization of GFP-emerin-m24 in an interphase HeLa cell. (c-e) Localization of GFP-emerin-m24 and endogenous BAF in HeLa cells expressing the mutant emerin. GFP-tagged emerin-m24, and anti-BAF staining are shown in c, telophase in d and the next interphase in e. Chromosomes were stained with DAPI.

 


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  		Fig. 6. Emerin does not accumulate at nuclear envelopes reassembled in the presence of mutant BAF-G25E. HeLa cells were stained with antibodies against endogenous emerin. (a) A telophase cell expressing GFP-BAF-G25E. (b) Cells expressing GFP-BAF immediately after cytokinesis. (c) An interphase cell that went through mitosis in the presence of GFP-BAF-G25E is indicated by the arrow; cells with no expression of GFP-BAF-G25E are indicated by the arrowhead. (d) A late telophase cell with no expression of GFP-BAF-G25E observed in the same culture dish as for b. (e) HeLa cells expressing GFP-BAF (wildtype) shortly after cytokinesis. Chromosomes were stained with DAPI. Bars, 10 µm.

 


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  		Fig. 7. Reassembly of lamins and LAP2ß after cells go through mitosis in the presence of mutant BAF-G25E. HeLa cells were stained with antibodies against endogenous lamin A (a,b), lamin B (c), and LAP2ß (d,e). (a) Telophase cells expressing GFP-BAF-G25E and stained with antibodies against lamin A. (b) Untransfected telophase cell stained with antibodies against lamin A. (c) Telophase cell expressing GFP-BAF-G25E and stained with antibodies against lamin B. (d) Cells expressing (middle two cells), and not expressing (outer two cells), GFP-BAF-G25E and stained with antibodies against LAP2ß. (e) Untransfected cells stained with antibodies against LAP2ß. Chromosomes were stained with DAPI. Bars, 10 µm.

 

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