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First published online 15 July 2008
doi: 10.1242/jcs.033597

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Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed nuclear envelope assembly

¹ CREST Research Project, Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan
² Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka 560-0043, Japan
³ Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama 526-0829, Japan
⁴ Graduate School of Frontier Biosciences, Osaka University 1-3 Suita 565-0871, Japan

View larger version (29K): [in this window] [in a new window]   Fig. 1. Localization of NE proteins on the telophase chromosome. (A) Schematic diagram of the `core' region of NE proteins on telophase chromosomes. The region marked in red represents the core region where BAF is initially accumulated. The region marked in green represents the non-core region outside the core region. (B) Living HeLa cells fluorescently stained with Hoechst 33342 for chromosomes and with GFP fusions of NE proteins (as indicated on the left) were observed every minute. The time-lapse images obtained are shown from left to right, with time 0 indicating the metaphase-anaphase transition. BAF, lamin A and all LEM domain proteins are localized at the `core' region. Arrows indicate the time of core localization obtained from quantification of core localization shown in C. Scale bar: 10 µm. (C) Fluorescence intensity at the core region is plotted as a function of time in minutes after the metaphase-anaphase transition. Fluorescence intensity is presented as % maximum intensity after subtracting the intensity at 4 minutes. (D) The time of core localization was estimated from the plots shown in C. The time of core localization is defined by the time required, in seconds after the metaphase-anaphase transition, for intensity at the core to reach 80% of the maximum. The s.d. and the number of cells tested (in parentheses) is also shown. (E) Schematic diagram of NE formation. BAF (red) localizes first to the core region, prior to other LEM domain proteins.

View larger version (32K): [in this window] [in a new window]   Fig. 2. FRAP analysis of core-localizing NE proteins. (A) HeLa cells stably expressing core-localizing NE proteins were examined by FRAP analysis. Images of GFP-BAF are shown as an example. The bleached region is the 2 µm region indicated by the white square. (B-D) FRAP profiles of GFP-BAF (B), GFP-lamin A (C) and GFP-emerin (D). Closed circles, fluorescence intensity in the bleached region; open circles, fluorescence intensity in the unbleached region. Bars represent s.d. Number of cells examined was 7 for GFP-BAF, 5 for GFP-lamin A and 5 for GFP-emerin.

View larger version (47K): [in this window] [in a new window]   Fig. 3. FRET analyses of core-localizing NE proteins. (A-H) HeLa cells transiently expressing two fusion proteins, as indicated, were analyzed by the acceptor photobleaching method: FRET signals were detected by an increase of donor fluorescence after photobleaching the acceptor chomophore. (A,C,E,G) Typical examples of images at 480 nm for mCFP and 520 nm for mVenus. The bleached area is indicated by the white square; the inset in the upper right corner is an enlarged image of the bleached area. (B,D,F,H) Fluorescence spectra measured in the bleached area before and after acceptor photobleaching. (I) HeLa cells transiently expressing CFP-BAF and YFP-BAF were analyzed by the ratio-imaging method. Images were obtained every minute. Time 0 represents timing of the metaphase-anaphase transition. Ratio images are represented by the color code shown on the right. Scale bars:10 µm (A,C,E,G).

View larger version (102K): [in this window] [in a new window]   Fig. 4. Live-correlated EM analyses. (A) Time-lapse images of living HeLa cells expressing GFP-BAF were obtained every minute. Magenta represents chromosomes (Hoechst 33342) and green represents GFP-BAF. Cells were fixed after live-cell fluorescence imaging, and the same cell was subjected to EM analysis. (B) A series of deconvolved 3D images of the fixed cell. (C) A single section image from the indicated panel in B. (D) An EM image corresponding to the image in C. (E) Magnified view of the region indicated in D. (F) High-magnification EM images of the regions a-c indicated in E. In the lower panels, drawings are superimposed on the EM images indicating GFP-BAF (green), the NE (red), MTs (orange) and vesicles (purple arrowheads). Scale bars: 10 µm (A,C), 2 µm (D) and 200 nm (F).

View larger version (86K): [in this window] [in a new window]   Fig. 5. Live-correlated EM analyses. (A) Time-lapse images of living HeLa cells expressing GFP-BAF were obtained every minute. Magenta represents chromosomes (Hoechst 33342) and green represents GFP-BAF. Cells were fixed after live-cell fluorescence imaging, and the same cell was subjected to EM analysis. (B) A series of deconvolved 3D images of the fixed cell. (C) A single section image from the indicated panel in B. (D) An EM image corresponding to the image in C. (E,F) Magnified views of the regions indicated in D. (G) High-magnification EM images of the regions a-d indicated in E and F. In the lower panels, drawings are superimposed on the EM images indicating GFP-BAF (green), the NE (red), MTs (orange) and vesicles (purple arrowheads). Scale bars: 10 µm (A,C), 2 µm (D) and 200 nm (G). (H) Time-lapse images of living HeLa cells obtained every minute. Upper panels represent chromosomes stained with Hoechst 33342 and lower panels fluorescently conjugated NLS-BSA microinjected as described by Haraguchi et al. (Haraguchi et al., 2000). Time 0 represents the metaphase-anaphase transition. Arrows in the image taken at 7 minutes indicate the first detectable accumulation of fluorescent NLS signals.

View larger version (70K): [in this window] [in a new window]   Fig. 6. Live-correlated EM analyses. (A) Time-lapse images of living HeLa cells expressing GFP-BAF were obtained every minute. Magenta represents chromosomes (Hoechst 33342) and green represents GFP-BAF. Cells were fixed after live-cell fluorescence imaging, and the same cell was subjected to EM analysis. (B) A series of deconvolved 3D images of the fixed cell. (C) A single section image from the indicated panel in B. (D) An EM image corresponding to the image in C. (E,F) Magnified views of the regions indicated in D. (G) High-magnification EM images of the regions a-c indicated in E and F. In the lower panels, drawings are superimposed on the EM images indicating GFP-BAF (green), the NE (red), MTs (orange), and vesicles (purple arrowheads). (H) Schematic diagram of NE reformation during telophase. Upper panel: cartoon representation of NE formation at the core region. BAF (green) forms a stable complex on the surface of the telophase chromosome mass in the presence of MTs. Then, LEM-domain proteins containing NE precursor vesicles attach to the BAF complex, fuse with each other, and finally enclose the chromosomes at the core region as MTs disassemble. Lower panel: EM images of the stages depicted by the cartoon representations. Scale bars: 10 µm (A,C), 2 µm (D), 200 nm (G) and 100 nm (H).

View larger version (49K): [in this window] [in a new window]   Fig. 7. RNAi knockdown of BAF and lamin A. (A) Western blotting analysis of HeLa cells after siRNA treatment for BAF or lamin A. (B) Number of cells showing core localization after knockdown of BAF or lamin A. Loss of BAF abolished core localization of both emerin and lamin A. (C) Representative image data which was analyzed to generate the results shown in B. Scale bars: 10 µm. (D) Western blotting analysis of HeLa cells stably expressing GFP-BAF after siRNA treatment for BAF, lamin A or emerin. (E) Electron microscopy images of HeLa cells subjected to siRNA treatment for control luciferase (a) and BAF (c). The respective insets (b,d) are fluorescence images of the same specimen: magenta represents chromosomes and green represents BAF. The arrows show the BAF core (a,b), and the arrowheads indicate no accumulation of BAF (c,d). Scale bars: 2 µm (a,c), 10 µm (b,d). (F) Time-lapse images of living cells stably expressing GFP-BAF are shown for control RNAi (luciferase RNAi), lamin A RNAi, and emerin RNAi. Numbers on the top represent the time in minutes after the metaphase-anaphase transition. Numbers on the left or the bottom represent the lifetime of the core region. Scale bars: 10 µm. (G) HeLa cells were subjected to siRNA treatment for control luciferase, BAF, lamin A and emerin, as indicated on the left. After siRNA treatment, cells expressing GFP-BAF were fixed with formaldehyde and observed (GFP-BAF, labeled at the top); cells without GFP-BAF were fixed with methanol and endogenous proteins (emerin, lamin A and lamin B as indicated) were detected by indirect immunofluorescence with specific antibodies. Scale bar: 10 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 8. Live-correlated EM analyses of HeLa cells after siRNA treatment for BAF. (A) Time-lapse images for chromosomes and GFP-BAF were taken for control cells. Cells were fixed at 12 minutes after the metaphase-anaphase transition. Only images for GFP-BAF are shown. See supplementary material Fig. S4 for more details. (B) An EM image of the same cells shown in A. (C) A fluorescence micrograph of the same cells shown in A: selected focus from a series of 3D images. Magenta represents chromosomes (Hoechst 33342) and green represents GFP-BAF. (D) Magnified view of the region indicated in the larger square in B. Drawings are superimposed on the EM images indicating the NE (red) and MTs (orange). (E) High-magnification EM images of the small box indicated in B. (F) Time-lapse images for chromosomes and GFP-BAF were taken for BAF RNAi cells. Cells were fixed at 12 minutes after the metaphase-anaphase transition. Only images for GFP-BAF are shown. See supplementary material Fig. S4 for more details. (G) EM images of the same cells shown in F. (H) A fluorescence micrograph of the same cells in F: a selected focus from a series of 3D images. Magenta represents chromosomes (Hoechst 33342) and green represents GFP-BAF. (I) Magnified view of the region indicated in the largest square in G. (J) Drawings are superimposed on the EM images of I, indicating the NE (red) and MTs (orange). (K,L) High-magnification EM images of the small boxes indicated in G: K, left box; L, right box. (M) An EM image of another example of BAF siRNA-treated cells. (N) A fluorescence micrograph of the cells shown in M. Magenta represents chromosomes (Hoechst 33342) and green represents GFP-BAF. (O) Enlarged image of the boxed region in N and corresponding to the boxed region in M. (P) Magnified view of the boxed region in M and corresponding to the image shown in O. (Q) Drawings are superimposed on the EM images of P, indicating the NE (red) and chromosomes (magenta). Scale bars: 10 µm (A,F), 2 µm (B,C,G,H,M,N), 500 nm (D,J,P,Q) and 200 nm (L).

View larger version (60K): [in this window] [in a new window]   Fig. 9. Live-correlated EM analyses of HeLa cells expressing GFP-BAF after siRNA treatment (D-H) or luciferase siRNA treatment as a control (A-C). During time-lapse imaging of living HeLa cells expressing GFP-BAF, cells were fixed 7 minutes after the metaphase-anaphase transition and subjected to EM analysis. A magnified view of the region indicated in A is shown in B and C. Magnified views of the regions indicated in D are shown in E and F (left square region), and G and H (right square region). In C, F and H, drawings are superimposed on the EM images indicating GFP-BAF (green), the NE (red), and MTs (orange). Scale bars: 2 µm (A,D), 500 nm (B,C,E-H). (I) Model of NE formation in the control siRNA-treated (left) and BAF siRNA-treated cell (right). For details, see text.

View larger version (32K): [in this window] [in a new window]   Fig. 10. Microtubule-dependent accumulation of BAF at the core. (A) Localization of BAF on the spindle MTs in metaphase and at the core in telophase. (B) Localization of BAF in HeLa cells treated with nocodazole during anaphase (bottom) and in untreated control cells (top). (C) Model of spindle MT-mediated, BAF-dependent NE formation. See text for details. Scale bars: 10 µm (A,B).

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