2540 Research Article Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed assembly

Tokuko Haraguchi1,2,*, Tomoko Kojidani1, Takako Koujin1, Takeshi Shimi1, Hiroko Osakada1, Chie Mori1, Akitsugu Yamamoto3 and Yasushi Hiraoka1,2,4 1CREST 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 2Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka 560-0043, Japan 3Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama 526-0829, Japan 4Graduate School of Frontier Biosciences, Osaka University 1-3 Suita 565-0871, Japan *Author for correspondence (e-mail: [email protected])

Accepted19 May 2008 Journal of Cell Science 121, 2540-2554 Published by The Company of Biologists 2008 doi:10.1242/jcs.033597

Summary Assembly of the nuclear envelope (NE) in telophase is essential consistently abolished BAF accumulation at the core. In for higher eukaryotic cells to re-establish a functional nucleus. addition, RNAi of BAF eliminated the core assembly of lamin Time-lapse, FRAP and FRET analyses in human cells showed A and emerin, caused abnormal cytoplasmic accumulation of that barrier-to-autointegration factor (BAF), a DNA-binding precursor nuclear membranes and resulted in a significant delay , assembled first at the distinct ‘core’ region of the of NE assembly. These results suggest that the MT-mediated telophase and formed an immobile complex by BAF accumulation at the core facilitates NE assembly at the directly binding with other core-localizing NE , such as end of mitosis. lamin A and emerin. Correlative light and electron microscopy after live cell imaging, further showed that BAF formed an Supplementary material available online at electron-dense structure on the chromosome surface of the core, http://jcs.biologists.org/cgi/content/full/121/15/2540/DC1 close to spindle microtubules (MTs) prior to the attachment of precursor NE membranes, suggesting that MTs may mediate Key words: Barrier-to-autointegration factor, Chromatin, Emerin, core assembly of BAF. Disruption of the spindle MTs Lamin A, Nuclear envelope, Microtubule Journal of Cell Science

Introduction Molecular mechanisms of NE formation have been well described The nuclear envelope (NE) is a cellular structure that encloses in in vitro experimental systems (Hetzer et al., 2000; Harel et al., and provides a physicochemical framework for 2003; Hachet et al., 2004); however, compared with NE formation genetic activities such as expression and DNA replication. The in vitro, NE reformation in vivo is poorly understood. It is believed NE is a dynamic structure in eukaryotes, disassembling at prophase that it begins with attachment of precursor membranes to telophase and reassembling at telophase in each mitosis. The reassembly of chromosomes, followed by fusion of the membranes and reassembly the NE is crucial to re-establish a functional nucleus for the next of integral membrane proteins, the NPC, and into interphase and to ensure proper progression of the cell cycle. Several the NE. Chromatin-binding integral membrane proteins, such as lines of evidence have shown that defects in NE architecture can lamin B receptor (LBR) and LAP2β (official symbol, LAP2B) are cause diseases in animals. Recently, a variety of human disorders proposed to posses a role in early assembly of the NE (Gant and known as ‘nuclear envelopathy’ or ‘nuclear ’ have been Wilson, 1997; Ulbert et al., 2006). Assembly of LEM-domain NE reported (reviewed by Nagano and Arahata, 2000; Wilson et al., proteins such as emerin and LAP2β depends on barrier-to- 2001; Worman and Courvalin, 2005). These disorders involve autointegration factor (BAF), a chromatin-binding protein, in mutations in NE components. Mutations in the gene encoding lamin human cells (Haraguchi et al., 2001). A (official protein symbol, LMNA) cause 12 human diseases BAF forms dimers that bind nonspecifically to double-stranded (reviewed by Mattout et al., 2006) including Emery-Dreifuss DNA in vitro (Lee and Cragie, 1998) and can be phosphorylated muscular dystrophy (Bonne et al., 1999; Clements et al., 2000) and in its N-terminal region by the vaccinia-related kinases (Nichols Hutchinson-Gilford progeria syndrome (Eriksson et al., 2003; et al., 2006; Gorjánácz et al., 2007). BAF was first discovered as Mounkes et al., 2003). Emery-Dreifuss muscular dystrophy can also a cellular protein that prevents retroviral DNA from suicidal auto- be caused by mutations of the gene encoding emerin (official protein integration and ensures its integration into host DNA (Lee and symbol, EMD) (Bione et al., 1994). The fact that defects in the NE Cragie, 1998: Jacque and Stevenson, 2006). It was later described can cause many different diseases indicates that the NE is involved as a binding partner of LEM-domain NE proteins (Furukawa, in a wide variety of cellular functions. Thus, understanding the 1999; Lee et al., 2001; Shumaker et al., 2001). BAF is proposed dynamic architecture of the NE will provide insight into the to have a function in NE assembly in human cells (Haraguchi et regulatory mechanisms for these cellular functions. al., 2000) and C. elegans (Margalit et al., 2005; Gorjánácz et al., BAF directs nuclear envelope assembly 2541

2007) as well as in Xenopus oocyte extracts (Segura-Totten et al., That BAF accumulated first was confirmed by observation of 2002). Recently, it has also been reported to be important for the HeLa cells expressing mRFP-BAF transiently co-transfected with normal progression of S phase in human cells (Haraguchi et al., GFP-emerin or GFP-lamin A. In these cells, mRFP-BAF 2007). accumulated in the core region earlier than GFP-emerin or GFP- In human cells, BAF accumulates with emerin, at the central lamin A (data not shown). These results indicate that BAF appears regions of the assembling nuclear rim close to the spindle at the core region prior to the accumulation of other core- microtubules (MTs) during telophase when the NE is reforming. localizing NE proteins. Since BAF binds both emerin and lamin This region is named the ‘core’ region (Haraguchi et al., 2001). In A in vitro (Lee et al., 2001; Holaska et al., 2003), the early our previous study, we found that emerin, LAP2β and lamin A accumulation of BAF at the core region suggests the possibility accumulated in the core region during telophase in a BAF-dependent that BAF provides a structural foothold to allow assembly of manner (Haraguchi et al., 2001). LAP2α (official symbol, LAP2A) nuclear lamins and membrane-bound NE proteins. The time course also accumulates at the core (Dechat et al., 2004). Although the of assembly to the reforming NE at telophase is schematically core structure seems important for NE assembly of the LEM-domain summarized in Fig. 1E. proteins and lamin A, its structural basis and dynamics in living cells are largely unknown. To understand the structural basis and BAF forms an immobile structure at the core region biological significance of the core region, we have examined From our time-lapse analysis, we hypothesized that BAF might form physical and chemical properties of the core region by time-lapse a foothold on the chromosome surface to allow assembly of other microscopy of living cells, FRAP and FRET analyses, and EM NE proteins. To test this hypothesis, we examined the mobility of analysis combined with live-cell imaging. In addition, we used BAF at the ‘core’ region during telophase by FRAP analysis of RNAi to selectively remove individual components of the core HeLa cells stably expressing GFP-BAF. We previously reported structure, BAF, lamin A and emerin, to examine their functions in that GFP-BAF during interphase was highly mobile with a half time living cells. Here we report that BAF forms an electron-dense stable recovery of 270 mseconds and an immobile fraction (IM) of structure at the core region of the telophase chromosome in a approximately 3% (Shimi et al., 2004). GFP-BAF was also highly spindle-MT-dependent manner, and propose that this MT-dependent mobile during metaphase (data not shown). By contrast, GFP-BAF BAF accumulation at the core coordinates efficient rendezvous of became highly immobile during telophase with an IM of 72±16% nuclear membranes and chromosomes. (n=7) (Fig. 2A,B), and remained immobile until the core region disappeared at the end of M phase; GFP-BAF regained its high Results mobility in early G1 phase (data not shown). This result indicates BAF assembles first to the ʻcoreʼ region of the reforming that physical attributes of BAF are regulated during the cell cycle, telophase nuclear envelope with BAF forming an immobile structure at the core region Assembly of NE proteins at the end of mitosis is an essential step specifically in telophase, a characteristic also exhibited by LAP2α in the reformation of a functional nucleus in higher eukaryotic (Dechat et al., 2004). cells. In order to elucidate underlying molecular mechanisms of To further examine physical properties of the core structure, we NE reformation in human cells, we examined spatial and temporal determined the mobility of other core-localizing NE proteins, such

Journal of Cell Science sequences of NE assembly by monitoring the behavior of NE- as lamin A and emerin, at the core region. These NE components GFP fusion proteins in living cells. The NE proteins tested were showed a similar mobility to that of BAF with an IM of 74±14% BAF (which binds both chromatin and LEM-domain proteins); (n=5) for lamin A and 46±7% for emerin (n=5) (Fig. 2C,D). These LAP2β, emerin and MAN1 (membrane-bound LEM-domain NE results suggest that BAF may form a stable higher-order complex proteins); LAP2α (LEM-domain protein localized in the nucleus); together with these NE proteins at the core. lamin A and lamin B1 (major components of the nuclear lamina); LBR (classical integral nuclear membrane protein); and BAF directly interacts with BAF, lamin A and emerin at the core nucleoporin Nup35 (official protein symbol, NUP53). Time-lapse region observation of HeLa cells expressing each one of these NE fusion Because BAF, lamin A and LEM-domain NE proteins all become proteins showed that BAF, LAP2α, LAP2β, emerin, MAN1 and immobile at the core region, the core structure may consist of a lamin A accumulated at a specialized region of the telophase higher-order complex of BAF with lamin A and the LEM domain chromosome mass close to the spindle (the ‘core’ region; see Fig. NE proteins. To test whether BAF directly interacts with these 1A and images indicated by arrows in Fig. 1B). This accumulation NE proteins at the core region, we used fluorescence Förster occurred during a limited period of telophase, as described resonance energy transfer (FRET) analysis. In HeLa cells previously (Haraguchi et al., 2000; Haraguchi et al., 2001; Dechat transiently coexpressing mVenus-BAF and mCFP fusions of the et al., 2004). In contrast with these core-localizing proteins, lamin second protein, FRET signals were detected by acceptor B1, LBR and Nup35 did not show clear accumulation at the core photobleaching (Fig. 3). The results showed that mVenus-BAF region (Fig. 1B). produced FRET signals with mCFP-BAF (Fig. 3A,B), mCFP- The fluorescence intensity of NE proteins at the core region is lamin A (Fig. 3C,D) and mCFP-emerin (Fig. 3E,F). In addition, plotted in Fig. 1C; the timing of core localization was determined time-lapse ratio imaging between mCFP-BAF and YFP-BAF as the time, in seconds after the metaphase-anaphase transition, showed that FRET signals between BAF molecules dramatically at which 80% of maximum accumulation had occurred (Fig. 1D). increased in the core region during telophase (Fig. 3I). As BAF Among the core-localizing proteins, BAF accumulated earliest at forms a dimer or dodecamer in vitro (Zheng et al., 2000), this the core region, approximately 434 seconds after the metaphase- high FRET signal may represent a higher-order complex structure. anaphase transition. LAP2α, lamin A and emerin accumulated LAP2α fused with mCFP also produced FRET signals with approximately 30 seconds later, and LAP2β and MAN1 mVenus-BAF in the core (data not shown), consistent with the accumulated after approximately another 30 seconds (Fig. 1C,D). previous finding that LAP2α is colocalized with BAF at the core 2542 Journal of Cell Science 121 (15) Journal of Cell Science

Fig. 1. See next page for legend. BAF directs nuclear envelope assembly 2543

Fig. 1. Localization of NE proteins on the telophase chromosome. Table 1. FRET efficiency (A) Schematic diagram of the ‘core’ region of NE proteins on telophase chromosomes. The region marked in red represents the core region where BAF FRET efficiency (%)* is initially accumulated. The region marked in green represents the non-core Interphase NE Telophase core region region outside the core region. (B) Living HeLa cells fluorescently stained mCFP-BAF/mVenus-BAF 12.3±8.7 (n=7) 26.7±14.2 (n=15) 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 mCFP-BAF/mVenus-emerin 15.2±10.6 (n 11) 14.6±13.2 (n 29) mCFP-BAF/mVenus-emerin m24 ND 2.7±1.35 (n=10) 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 mCFP-BAF/mVenus-Lamin A 3.1±8.2 (n 10) 23.0±12.0 (n 31) at the ‘core’ region. Arrows indicate the time of core localization obtained μ *FRET efficiency (%) = [1–(donor intensity before acceptor from quantification of core localization shown in C. Scale bar: 10 m. (C) ϫ Fluorescence intensity at the core region is plotted as a function of time in photobleaching/donor intensity after acceptor photobleaching)] 100. minutes after the metaphase-anaphase transition. Fluorescence intensity is presented as % maximum intensity after subtracting the intensity at 4 minutes. probably forms a polymer or a higher-order complex structure (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 with BAF itself in the core region and also directly binds emerin, the metaphase-anaphase transition, for intensity at the core to reach 80% of the lamin A and LAP2α, assembling them into the core complex. 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 BAF forms an electron-dense complex on the surface of the the core region, prior to other LEM domain proteins. telophase chromosome mass To examine the core structure at high resolution, we used electron microscopy (EM). Because the core structure forms transiently, for structure (Dechat et al., 2004). A mutant emerin that does not bind only a few minutes, during telophase, we selected specimens by to BAF (emerin-m24) either in vivo (Haraguchi et al., 2001) or observing living HeLa cells expressing GFP-BAF. During live-cell in vitro (Lee et al., 2001) was used as a negative control. No FRET observation, the cell was fixed when the core structure formed and signals were detected between mCFP-emerin-m24 and mVenus- embedded in epoxy resin to prepare EM specimens (Fig. 4A; see BAF (Fig. 3G,H), suggesting that the detected FRET signals are Materials and Methods). Three-dimensional fluorescence the result of specific interactions. Estimated FRET efficiencies microscopy images of the fixed cell were also obtained (Fig. 4B). are summarized in Table 1. These results suggest that BAF Thin EM sections were compared with fluorescence microscopy images to identify the cell observed during live-cell observation (Fig. 4C,D) and details of the core structure were investigated at high resolution (Fig. 4E,F). Images shown in a, b, and c in Fig. 4F correspond to the regions indicated in Fig. 4E. This method provides an opportunity to combine the

Journal of Cell Science temporal information and molecular selectivity of fluorescence live-cell imaging with the high-resolution imaging of EM: this method is designated live correlative light electron microscopy (live CLEM). Examples fixed at later stages are shown in Figs 5 and 6. The reliability of this method was confirmed in an immunoelectron micrograph for GFP- BAF localization using anti-GFP antibody (supplementary material Fig. S1): GFP-BAF was localized in the core region of telophase cells as seen in the live CLEM imaging technique. We applied live CLEM to cells fixed at approximately 4 minutes Fig. 2. FRAP analysis of core-localizing NE proteins. (n=4), 7 minutes (n=17) and 10 (A) HeLa cells stably expressing core-localizing NE minutes (n=3) after the metaphase- proteins were examined by FRAP analysis. Images of GFP-BAF are shown as an example. The bleached anaphase transition, (Figs 4, 5 and 6, region is the 2 μm region indicated by the white respectively). The cell shown in Fig. square. (B-D) FRAP profiles of GFP-BAF (B), GFP- 4 was fixed at 4 minutes 15 seconds lamin A (C) and GFP-emerin (D). Closed circles, after the metaphase-anaphase fluorescence intensity in the bleached region; open transition, before BAF became circles, fluorescence intensity in the unbleached region. Bars represent s.d. Number of cells examined concentrated at the core. Comparison was 7 for GFP-BAF, 5 for GFP-lamin A and 5 for of EM images with those from GFP-emerin. fluorescence microscopy shows that 2544 Journal of Cell Science 121 (15) Journal of Cell Science

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). BAF directs nuclear envelope assembly 2545

BAF was localized to only a limited region of the chromosome the ER tubules, the precursors of nuclear membranes (Anderson mass (superimposed with the green region in panel b of Fig. 4F); et al., 2007). It should be emphasized that almost no NE was formed this region represents a trailing edge of the separating in the central region (b and c in Fig. 4F) where spindle MTs are chromosomes, which might correspond to the telomeres, as present, whereas the NE was well defined in the peripheral regions suggested by Foisner and colleagues (Dechat et al., 2004). In the where spindle MTs are few or not present at all. central region of the chromosomes where BAF localizes, spindle The cell shown in Fig. 5 was fixed at 7 minutes 10 seconds MTs approached and penetrated into the chromosome mass after the metaphase-anaphase transition, when distinct core (superimposed with the orange lines in regions b and c of Fig. 4F). localization of BAF was observed. Strikingly, an electron-dense At this time, the NE (superimposed with the red hatches) was not area, with a width of 24-64 nm, overlapping with the localization being formed in the central region of the chromosome mass where of BAF (superimposed with green) was clearly seen on the surface the mitotic spindle was present; rather, the most peripheral regions of the chromosome mass (Fig. 5G). At this time, well-defined (‘non-core’ region in Fig. 1A) of the chromosome mass were being NE could be observed in this region of the chromosome mass, enveloped by the NE (see panel a of Fig. 4F; also see supplementary but the NE was not yet fully assembled at the sites of the electron- material Fig. S2A,B). The purple arrowheads indicate vesicular dense areas (panel a in Fig. 5G). Vesicular membranes could be membranes. These vesicular membranes are presumably part of seen near these electron-dense areas (indicated by purple Journal of Cell Science

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). 2546 Journal of Cell Science 121 (15)

arrowheads in Fig. 5G); some vesicles with a diameter of arrowheads in panel c in Fig. 5G and panel b in Fig. 6G). It could approximately 50 nm appeared to attach to the BAF-containing also be seen that some MTs had penetrated into the chromosome electron dense areas (see panel c in Fig. 5G and panel b in Fig. mass in the gap between two BAF-containing electron-dense areas 6G). In addition, vesicular membranes at the site of the reforming (panel b in Fig. 5G), and others attached to the surface of the NE at the core region appeared to be attached to MTs (see electron-dense area (panel d in Fig. 5G). At the same time, most Journal of Cell Science

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. BAF directs nuclear envelope assembly 2547 Journal of Cell Science

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). 2548 Journal of Cell Science 121 (15) Journal of Cell Science

Fig. 7. See next page for legend. BAF directs nuclear envelope assembly 2549

Fig. 7. RNAi knockdown of BAF and lamin A. (A) Western blotting analysis BAF has a direct role on NE formation at the core region, and of HeLa cells after siRNA treatment for BAF or lamin A. (B) Number of cells lamin A and emerin affect the lifetime of the core structure showing core localization after knockdown of BAF or lamin A. Loss of BAF To determine the contribution of BAF to core formation and 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: subsequent NE formation, we examined the effects of BAF 10 μm. (D) Western blotting analysis of HeLa cells stably expressing GFP- knockdown by RNAi in HeLa cells. BAF was selectively removed BAF after siRNA treatment for BAF, lamin A or emerin. (E) Electron in cells treated with BAF siRNA (Fig. 7A,D). In those cells, core microscopy images of HeLa cells subjected to siRNA treatment for control localization of emerin and lamin A was effectively inhibited (Fig. luciferase (a) and BAF (c). The respective insets (b,d) are fluorescence images 7B); examples of microscopic images are shown in Fig. 7C. In of the same specimen: magenta represents chromosomes and green represents BAF. The arrows show the BAF core (a,b), and the arrowheads indicate no addition, we applied live CLEM analysis to BAF siRNA-treated accumulation of BAF (c,d). Scale bars: 2 μm (a,c), 10 μm (b,d). (F) Time- HeLa cells expressing GFP-BAF, and observed cell structures lapse images of living cells stably expressing GFP-BAF are shown for control especially focusing on the electron-dense structure, the NE, RNAi (luciferase RNAi), lamin A RNAi, and emerin RNAi. Numbers on the chromosomes, the MTs and membranes (Fig. 7E, Figs 8 and 9; also 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. see supplementary material Fig. S4). In BAF siRNA-treated cells Scale bars: 10 μm. (G) HeLa cells were subjected to siRNA treatment for (see Fig. 7D), the electron-dense structure at the periphery of the control luciferase, BAF, lamin A and emerin, as indicated on the left. After central region of the chromosome mass was lost (n=4; Fig. 7E, siRNA treatment, cells expressing GFP-BAF were fixed with formaldehyde compare arrowheads in cells at 7 minutes after the metaphase- and observed (GFP-BAF, labeled at the top); cells without GFP-BAF were anaphase transition with arrows in the control cells), and fixed with methanol and endogenous proteins (emerin, lamin A and lamin B as indicated) were detected by indirect immunofluorescence with specific chromosomes remained condensed (Fig. 8, compare G or M with antibodies. Scale bar: 10 μm. B), indicating that BAF is required for formation of the core structure and for chromosome decondensation. In addition, the NE at the core region had not formed in BAF siRNA-treated cells even at 12 minutes after the metaphase-anaphase transition (n=10; see Fig. 8I,J) of the non-core region of telophase chromosomes was overlaid whereas it was almost fully reassembled in luciferase siRNA-treated with NE (supplementary material Fig. S2C,D), suggesting that control cells (n=4; Fig. 8D). In the BAF-depleted cells it was also NE reformation in the core and non-core regions are regulated frequently observed that relatively long MTs remained in the by distinct mechanisms. chromosomal region at this late period (compare Fig. 8J with 8D; It should be pointed out that the core region is not fully also compare Fig. 9F with 9C). Finally, abnormal inclusion of covered by NE at 7 minutes; complete reassembly of the NE cytoplasm inside the nucleus was also often seen these cells (Fig. does not occur until approximately 10 minutes (Fig. 6). This is 8P,Q; pale regions in nucleus). surprising because nuclear transport started at 7-8 minutes (Fig. Interestingly, in addition to these phenotypes, BAF depletion 5H, arrows; also see Haraguchi et al., 2000). At this time, the caused a significant reduction of tubular membranes near the core NPCs that have been targeted to the NE were localized region where the spindle MTs persisted, in contrast to an abundance exclusively outside the core region (Fig. 1B; also see of tubular membranes in the presence of BAF (compare Fig. 9E,F supplementary material Fig. S2C,D). Collectively, these results with 9B,C). Instead, in BAF RNAi cells, abnormal piles of three-

Journal of Cell Science suggest that the NPCs on the reforming NE at 7-8 minutes after dimensionally extended ER membranes in the cytoplasm were the metaphase-anaphase transition are functional and that nuclear generated (Fig. 9G,H; also see Fig. 8K,L, compare these with Fig. transport through the NPCs may begin in the absence of a fully 8E): in many cases these abnormal membranes seemed to be NE- reassembled NE. like membranes assembling away from the chromosomes (Fig. The cell shown in Fig. 6 was fixed at 10 minutes 11 seconds 9G,H). These findings are schematically summarized in Fig. 9I. after the metaphase-anaphase transition. At this time, most of the Taken together, these results indicate that BAF has a direct effect chromosome mass was surrounded by the NE; the BAF-containing on NE formation at the core region, as suggested previously electron-dense surface (green) was also enveloped within the NE (Haraguchi et al., 2000), and suggest that the role of BAF in NE (red). This is consistent with previous findings that emerin- formation is to coordinate the recruitment of NE precursor containing and LBR-containing membranes fuse at this time membranes to the chromosome surface. (Haraguchi et al., 2000). Most spindle MTs have disappeared (panels RNAi knockdown of lamin A did not eliminate core localization a and b Fig. 6G), but some MTs were still present close to the of BAF or emerin, but did reduce the frequency of core localization chromosomes. In some cases, MT cables proximate to the electron- (Fig. 7B). To test whether this reduced frequency reflected a dense chromosome areas were disrupted by the presence of the NE shortened life of the core structure, we determined the duration of (panel c Fig. 6G). Importantly, electron-dense structures formed on the core structure (Fig. 7F) in cells with specific RNAi-mediated the surface of the telophase chromosome mass in HeLa cells not knockdown of BAF, lamin A or emerin, as shown in Fig. 7D. Its expressing exogenous GFP-BAF (supplementary material Fig. S3), duration was significantly shortened from 8 minutes 9 seconds indicating that this structure is a native one, and not an artefact of (n=10) in control luciferase siRNA cells to 1 minute 42 seconds GFP-BAF. (n=7) in lamin A siRNA cells, suggesting that lamin A stabilizes A proposed order of NE formation at the core region is the core structure (Fig. 7F). By contrast, in emerin siRNA cells, the summarized in Fig. 6H. The NE forms directly on the chromosomes core structure remained intact significantly longer (more than 20 outside the core region, but forms on the BAF-containing dense minutes) (n=7) compared with control RNAi cells (n=7) (Fig. 7F), structures at the core region. It appears that accumulation of the often persisting up to early G1 phase (data not shown), suggesting electron-dense material eliminates MT attachment to the that emerin destabilizes the core structure. chromosome mass as the electron-dense core grows. The BAF- Furthermore, knockdown of these proteins also affected containing electron-dense structure then disappears after the NE localization to the NE in the subsequent interphase. Knockdown covers the entire chromosome mass. of BAF disrupted NE localization of emerin and lamin A, 2550 Journal of Cell Science 121 (15)

knockdown of lamin A disrupted NE localization of BAF and The spindle MTs mediate BAF assembly to the core region emerin, and knockdown of emerin disrupted NE localization of BAF forms an electron-dense core structure close to the MT- BAF and lamin A (Fig. 7G). These effects are specific because attaching regions (Fig. 5). Therefore, the MTs could be involved the same treatment did not affect lamin B localization (Fig. 7G). in BAF assembly to the core region. To test this idea, we first Taken together, these results suggest that BAF has a direct role examined whether BAF colocalizes with the spindle MTs during on core formation and that lamin A and emerin affect the stability mitosis. Indirect immunofluorescence staining of cells fixed with of the core structure. trichloroacetic acid (TCA) showed that in mitotic cells BAF

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. Journal of Cell Science (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). BAF directs nuclear envelope assembly 2551

localizes to the spindle MTs from metaphase to telophase (Fig. 10A). was lost (Fig. 10B), indicating that the spindle MTs are required Since this result shows colocalization of BAF with the spindle MTs for BAF assembly to the core. Taken together, these results suggest for the first time, we used BAF knockdown by BAF RNAi to that BAF is associated with the spindle MTs during metaphase confirm our initial observation; BAF RNAi treatment effectively through anaphase and assembles to the core region in early removed the signal, indicating that BAF, at least in part, colocalizes telophase, in a spindle-MT-dependent manner, where it forms a with the spindle MTs (supplementary material Fig. S5). To further stable complex with other core-localizing proteins such as lamin A test a direct role of the spindle MTs in core formation, we examined and emerin. Based on these findings, we propose a model, the effect of nocodazole, a MT-depolymerizing reagent, on BAF schematically illustrated in Fig. 10C, in which spindle MT function assembly to the core region. In cells treated with nocodazole during organizes BAF spatially, thereby engendering BAF-directed NE anaphase, the spindle MTs disappeared and BAF core assembly assembly.

Discussion In this report, we have demonstrated that the BAF-dependent core structure is an immobile complex that appears as an electron-dense structure directly on chromosomes with a width of 24- 60 nm. This study shows for the first time that the spindle MTs are involved in NE reformation at the end of mitosis: spindle MTs mediate BAF assembly to the core region where BAF recruits selective NE components such as lamin A and emerin. The core structure is important in establishing a normal interphase NE and, consequently, normal nuclear function. Live CLEM observation revealed that the timing of NE reformation is different in the core region from that in the non-core region: NE reformation is significantly later in the core region (compare Figs 4-6 with supplementary material Fig. S2). This suggests that

Journal of Cell Science there are at least two different mechanisms for NE assembly; one that acts at the core region, and a second that acts at the non-core region. RanGTP would appear to be involved in NE reformation at the non-core region because it is known to be involved in the assembly of the NPCs in in vitro Xenopus oocyte extracts

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. 2552 Journal of Cell Science 121 (15)

reformation by recruiting selective NE protein-bearing membranes in the presence of the ‘inhibitory’ spindle MTs. This hypothesis is further supported by the fact that loss of BAF by RNAi resulted in removal of most of the membranes from the area near the core region during telophase (Fig. 9E,F), and abnormal assembly of NE-like membranes in the cytoplasm far from the chromosomal region (Fig. 9G,H), and also caused lamin A and emerin to disperse into the cytoplasm (Fig. 7C,G). In addition, abnormal inclusion of cytoplasm inside the nucleus was frequently observed in cells lacking BAF (Fig. 8P,Q). As BAF is a key molecule for core formation, BAF targeting to the core region must be an important step for core formation. Recently, Gorjánácz and colleagues have reported that BAF-1 (C. elegans BAF homolog) is localized in a chromosomal region similar to the ‘core’ during NE formation in dividing cells of early C. elegans embryos, and its ‘core’-like localization is abolished by siRNA depletion of VRK-1 (C. elegans vaccinia-related kinase homolog) (Gorjánácz et al., 2007). Their results suggest that phosphorylation of BAF is essential for its ‘core’-like localization in C. elegans embryos. Human BAF can be phosphorylated in vitro on its N- terminal conserved residues, Ser4 (Bengtsson and Wilson, 2006) or all three amino acid residues Thr2-Thr3-Ser4, by vaccinia-related kinases (Nichols et al., 2006). Paradoxically, however, initial studies have indicated that phosphorylation of human BAF abolishes its interaction with DNA in vitro (Nichols et al., 2006), reduces its binding to the LEM proteins in vitro (Nichols et al., 2006), and disrupts emerin localization to the NE in vivo (Bengtsson and Wilson, 2006), suggesting that the N-terminal phosphorylated form of human BAF cannot attach to the chromosomal region at the ‘core’. One possibility to reconcile these results is that BAF phosphorylated by VRK1 binds spindle MTs, and then is able to localize to the core region once it is dephosphorylated at the end of mitosis. This hypothesis is supported by the following findings: (1) BAF associates with the spindle MTs (Fig. 10A); (2) BAF core accumulation is

Journal of Cell Science abolished by destruction of the spindle MTs (Fig. 10B); and (3) phosphorylated BAF is localized on the spindle MTs but not in the Fig. 10. Microtubule-dependent accumulation of BAF at the core. (A) core region (T.H., unpublished result). However, it remains to be Localization of BAF on the spindle MTs in metaphase and at the core in elucidated whether phosphorylation by Vrk1 is required for telophase. (B) Localization of BAF in HeLa cells treated with nocodazole association with the spindle MTs. during anaphase (bottom) and in untreated control cells (top). (C) Model of Cell-cycle dependent phosphorylation of emerin also regulates spindle MT-mediated, BAF-dependent NE formation. See text for details. Scale bars: 10 μm (A,B). the interaction of emerin with BAF (Hirano et al., 2005; Ellis et al., 1998), suggesting that modification of emerin or other core- localizing proteins may also contribute to the core formation and structural features of the ‘core’. Since mitotic progression is (Zhang et al., 2002; Walther et al., 2003) and our observations regulated by the spatial and temporal coordination of several cell- indicate that NPCs assemble exclusively in the non-core region. cycle-regulating kinases and phosphatases, the combination of We propose that BAF is a factor involved in NE assembly at the several protein modifications may cooperatively enforce core core region. formation in telophase. Live cell imaging of various ER- or NE-associated integral Cooperative actions of BAF and core-localizing proteins in NE membrane proteins have shown that most membranes are excluded organization are supported by several observations. BAF, lamin A from the area of the spindle MTs until early telophase (Ellenberg and emerin form a three-way complex in vitro (Holaska et al., 2003) et al., 1997; Yang et al., 1997; Haraguchi et al., 2000; Anderson and in vivo in C. elegans (Liu et al., 2003; Margalit et al., 2005; and Hetzer, 2007; Anderson and Hetzer, 2008). From these Gorjánácz et al., 2007). Retention of lamin A and emerin in the observations, we speculate that the mitotic spindle, or their reforming NE requires their initial localization at the core region, associated proteins, may have an inhibitory effect on NE and this localization is BAF dependent (see Fig. 7G). Core reformation, presumably by somehow repelling precursor localization is also required for BAF to establish its own proper membranes. As a consequence of this, fewer precursor membranes nuclear localization (Zheng et al., 2000; Margalit et al., 2005; would be available for NE reformation in the core region. Here, Gorjánácz et al., 2007). Furthermore, the lifetime of the core we report that BAF associates with the spindle MTs, and that its structure seems to depend on specific core-localizing proteins such targeting to the core region requires spindle MTs (Fig. 10B). These as emerin, lamin A and LAP2α. Loss of emerin by RNAi prolonged findings strongly support the idea that BAF acts to direct core NE core lifetime (see Fig. 7F). A persistent core structure is also BAF directs nuclear envelope assembly 2553

observed in HeLaS3 cells expressing high levels of lamin A, and confirmed using an ABI 310 or ABI 3100 Genetic Analyser (Applied Biosystems, this persistent core structure is enriched in emerin and lamin A Norwalk, CT). Primer sequences are listed in supplementary material Table S1. (Maeshima et al., 2006). We also observed a prolonged core lifetime Fluorescence microscopy in cells overexpressing lamin A or LAP2α (data not shown). By Cells were grown in a glass-bottom culture dish (MatTech, Ashland, MA). GFP fusion contrast, loss of lamin A by RNAi significantly reduced the lifetime plasmids (0.1-0.2 μg) were transfected into cells with LipofectaminePlus (Gibco BRL) according to the manufacturer’s methods except that the incubation time with DNA of the core structure (see Fig. 7F). These results suggest that emerin was reduced to 1.5 hours. destabilizes the core, whereas lamin A and LAP2α stabilize the Time-lapse, multicolor images in living cells, as well as indirect core structure. The balance between their competing activities may immunofluorescence images in fixed cells, were obtained using the DeltaVision microscope system based on an Olympus wide-field fluorescence microscope IX70 be important for regulating the formation of the transient core (Applied Precision, Seattle, WA). For temperature control during live observation, structure, the formation of the mature NE and the eventual formation the microscope was kept at 37°C in a temperature-controlled room as described of a functional nucleus. previously (Haraguchi et al., 1999). A series of three-dimensional image data were In summary, we conclude that BAF assembles on the surface of obtained, and deconvolved with the SoftWorx software equipped with the DeltaVision as described previously (Haraguchi et al., 2001; Haraguchi et al., 2004). the telophase chromosome mass in regions where the chromosomes are interacting with spindle MTs, and that this assembly is MT FRAP and FRET analyses dependent and requires coordinated BAF phosphorylation followed FRAP and FRET studies were done using a laser-scanning confocal microscope, Zeiss LSM510 META (Carl Zeiss, Yena) equipped with a Kr laser (Coherent, Santa Clara, by BAF dephosphorylation. Chromosome-associated BAF makes CA) (Haraguchi et al., 2002; Hiraoka et al., 2002). FRAP experiments were carried a higher-order complex with itself, lamin A and emerin, resulting out as described previously (Shimi et al., 2004). Briefly, HeLa cells stably expressing in formation of an electron-dense structure at the core which is GFP fusion proteins were imaged using a water-immersion objective lens (C- ϫ required for proper reformation of the NE in this region and for the Apochromat 40 /NA1.2) on a Zeiss LSM510 META. Photobleaching was performed with full power of 488 nm light from the argon laser 10 iterations for a defined region re-establishment of a functional nucleus. Furthermore, BAF- of each cell. Image data were collected through a pinhole of 2.37 Airy units dependent NE reformation at the core, which involves LEM-domain (corresponding to an optical section 2 μm thick) with 488 nm argon laser at 0.5% proteins containing NE precursor vesicles attaching to the BAF full power. For FRET experiments, living HeLa cells transiently expressing pairs of NE proteins complex and then fusing with each other, is mechanistically distinct fused with mCFP and mVenus were imaged using a water-immersion objective lens from NE reformation at the non-core region of the chromosome (C-Apochromat 40ϫ/NA1.2) on a Zeiss LSM510 META. FRET was detected by mass. Understanding the dynamics and the structural details of NE acceptor photobleaching (Wouters et al., 2001). mVenus was photobleached by 514 nm light from the argon laser, with full power, 50 iterations for a defined area (2.1 reformation at the end of mitosis will provide new insights into the μm square) in each cell. After photobleaching, time-lapse images were collected on development of lamin-A-dependent disease and other NE-associated the META detector from 470 nm to 545 nm through a pinhole of 2.18 Airy units human diseases. (corresponding to an optical section 1.8 μm thick) by exciting with 413 nm light from the Kr laser at 0.3-0.9% full power. FRET efficiency was determined for fluorescence intensity of mCFP and mVenus after linear unmixing as described Materials and Methods previously (Shimi et al., 2004). For time-lapse FRET ratio imaging, a fluorescence Cells and reagents microscope IX70 (Olympus) equipped with a Nipkow disc confocal unit, CSU21 HeLa cells were obtained from the Riken Cell Bank (Tsukuba, Japan) and maintained (Yokogawa, Japan) was used. Cells were illuminated with a 430 nm diode laser to in DME medium containing 10% calf serum. Double-stranded siRNA for BAF, lamin excite CFP, and image data for CFP and YFP were simultaneously obtained on a A and emerin were purchased from Qiagen (Hilden, Germany). Mouse monoclonal CoolSnapHQ CCD camera (Photometrics, Roper, Tucson, Texas) through a dual anti-lamin A/C antibodies TiM92 (IgM) were generated as described previously viewer (Optical Insights), which separates emission light for wavelengths of 480 nm

Journal of Cell Science (Haraguchi et al., 2001). Mouse monoclonal anti-lamin A/C antibodies MMS-107P (corresponding to CFP) and 535 nm (corresponding to YFP). Ratio images between (IgM) were purchased from Covance (Berkeley, CA). Mouse monoclonal anti-lamin CFP and YFP were obtained by MetaMorph software (Universal Imaging Corporation, B (101-B7) antibodies were obtained from MatriTech (Cambridge, MA). Rabbit Downingtown, PA). polyclonal anti-emerin antibodies were a gift of the Arahata lab (Tokyo, Japan). Anti- BAF antibodies (PU38143) were generated against recombinant BAF in a rabbit and Correlative light electron microscope analysis after live affinity purified. Plasmids encoding mCFP and mVenus (monomeric versions of ECFP observation (live CLEM) and Venus), which were made by substitution of Ala for Lys (Zacharias et al., 2002), Cells stably expressing GFP-BAF were cultured in a special glass-bottomed dish were gifts of Nagai, Sawano and Miyawaki (RIKEN, Wako, Japan). LAP2α-YFP with an addressing grid (grid size, 175 μm) on the coverslip (Iwaki, Japan). After and GFP-lamin B1 were gifts of R. Foisner (Medical University Vienna) and R. staining with Hoechst 33342, live cells were monitored on DeltaVision; at the desired Goldman (Northwestern University), respectively. time point after the metaphase-anaphase transition, cells were fixed with glutaraldehyde at a final concentration of 2.5% for 1 hour. Three-dimensional images Plasmid construction (50-60 focal planes at 0.2 μm intervals) were obtained using an Olympus oil- Methods for constructing DNA plasmids encoding LBR-GFP, GFP-emerin, GFP- immersion objective lens (PlanApo ϫ60/NA1.4) and computationally processed by BAF, GFP-LAP2β and GFP-MAN1 were described previously (Haraguchi et al., 2000; three-dimensional deconvolution (Agard et al., 1989). Electron microscope (EM) Haraguchi et al., 2001, Shimi et al., 2004). To construct GFP-lamin A, the coding observation of the same cell was carried out as follows. Cells were post-fixed with region was PCR-amplified from the RT-PCR product of lamin A using primers lam- 1% OsO4 (Nisshin EM, Tokyo, Japan) in phosphate buffer, pH 7.4, for 1 hour, washed 1 and lam-2 and inserted into the pEGFP-C1 vector (Clontech Laboratories, Palo briefly with distilled water, and sequentially dehydrated with 50 and 70% ethanol. Alto, CA). To construct mCFP and mVenus fusion proteins, the coding region of After staining with 2% uranyl acetate in 70% ethanol for 1 hour, cells were again mCFP or mVenus was PCR-amplified from ECFP-A206K/pRSETB or Venus- sequentially dehydrated with 90% and 100% ethanol, and then embedded in epoxy A206K/pRSETB, respectively, with primers cfp-1 and cfp-2 and inserted into the resin by incubating with 50% (v/v) Epon812 (TAAB, Berkshire, UK) in ethanol for pEGFP-C1 vector digested with NheI and BglII restriction enzymes to replace EGFP. 30 minutes and 100% Epon812 for 1 hour. The epoxy block containing the same cell The resulting mCFP and mVenus vectors were used for construction of mCFP-BAF, observed by a fluorescence microscope was trimmed according to the grid reference mCFP-lamin A, mCFP-emerin, mCFP-emerin-m24 and mVenus-BAF. To construct on the coverslip, and sliced to ultra-thin sections with a thickness of 80 nm using a mCFP-BAF or mVenus-BAF, the coding region of BAF was PCR-amplified from microtome (Leica microsystems, Germany). Thin sections were stained with 2% uranyl pET15b-BAF (Lee and Craigie, 1998) using primers baf-1 and baf-2 and inserted acetate (Merck) for 1 hour and a commercial ready-to-use solution of lead citrate into the above described mCFP or mVenus vectors, respectively. To construct mVenus- (Sigma) for 1 minute. Image data were collected using an electron microscope Hitachi emerin or mVenus-emerin-24m, the coding region of emerin was PCR-amplified from H7600 (80 kV, Hitachi, Japan). a GFP-emerin construct (Haraguchi et al., 2000) using primers eme-1 and eme-2 and inserted into the mVenus vector. To construct mVenus-lamin A, the coding region of Knockdown by RNAi lamin A was PCR-amplified from the GFP-lamin A construct using primers lam-3 Two to five micrograms of oligomeric double-stranded RNA for BAF, lamin A or emerin and lam-4 and inserted into the mVenus vectors. The coding sequence of human were transfected into HeLa cells cultured in a 35 mm culture dish using Oligofectamine Nup35 was obtained from mRNA in HeLa cells by RT-PCR using primers nup-1 and (Invitrogen, Carlsbad, CA), Lipofectamine PLUS (Invitrogen), or RNAiFect (Qiagen) nup-2, cloned into the TA cloning vector pCR2.1 (Invitrogen), and re-cloned to the transfection reagents according to the manufacturer’s instructions. The cells were pEGFP-C3 vector at the EcoRI sites. The DNA sequences of all fusion plasmids were incubated for 3 days in DMEM culture medium containing 10% calf serum. 2554 Journal of Cell Science 121 (15)

We are grateful to the Riken Cell Bank for HeLa cells, A. Miyawaki Harel, A., Chan, R. C., Lachish-Zalait, A., Zimmerman, E., Elbaum, M. and Forbes, and H. Nagai for mCFP and mVenus, R. Foisner for LAP2α-YFP, A. D. J. (2003). Importin beta negatively regulates nuclear membrane fusion and nuclear Gajewski for initial work on FRET between LAP2α and BAF, R. pore complex assembly. Mol. Biol. Cell 14, 4387-4396. Hetzer, M., Bilbao-Cortés, D., Walther, T. C., Gruss, O. J. and Mattaj, I. W. (2000). Goldman for GFP-lamin B1, P. Traktman for anti-phosphorylated BAF GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol. Cell 5, 1013- antibody, and N. Nakamura for anti-GFP antibody. Especially we thank 1024. K. L. Wilson for valuable discussion throughout this research. We also Hirano, Y., Segawa, M., Ouchi, F. S., Yamakawa, Y., Furukawa, K., Takeyasu, K. and thank D. B. Alexander, M. Gorjánácz and I. W. Mattaj for critical Horigome, T. (2005). 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