Cell Nuclei Spin in the Absence of Lamin B1
2007; Elsevier BV; Volume: 282; Issue: 27 Linguagem: Inglês
10.1074/jbc.m611094200
ISSN1083-351X
AutoresJulie Y. Ji, Richard Lee, Laurent Vergnes, Loren G. Fong, Colin L. Stewart, Karen Reue, Stephen G. Young, Qiuping Zhang, Catherine M. Shanahan, Jan Lammerding,
Tópico(s)Genomics and Chromatin Dynamics
ResumoMutations of the nuclear lamins cause a wide range of human diseases, including Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome. Defects in A-type lamins reduce nuclear structural integrity and affect transcriptional regulation, but few data exist on the biological role of B-type lamins. To assess the functional importance of lamin B1, we examined nuclear dynamics in fibroblasts from Lmnb1Δ/Δ and wild-type littermate embryos by time-lapse videomicroscopy. Here, we report that Lmnb1Δ/Δ cells displayed striking nuclear rotation, with ∼90% of Lmnb1Δ/Δ nuclei rotating at least 90° during an 8-h period. The rotation involved the nuclear interior as well as the nuclear envelope. The rotation of nuclei required an intact cytoskeletal network and was eliminated by expressing lamin B1 in cells. Nuclear rotation could also be abolished by expressing larger nesprin isoforms with long spectrin repeats. These findings demonstrate that lamin B1 serves a fundamental role within the nuclear envelope: anchoring the nucleus to the cytoskeleton. Mutations of the nuclear lamins cause a wide range of human diseases, including Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome. Defects in A-type lamins reduce nuclear structural integrity and affect transcriptional regulation, but few data exist on the biological role of B-type lamins. To assess the functional importance of lamin B1, we examined nuclear dynamics in fibroblasts from Lmnb1Δ/Δ and wild-type littermate embryos by time-lapse videomicroscopy. Here, we report that Lmnb1Δ/Δ cells displayed striking nuclear rotation, with ∼90% of Lmnb1Δ/Δ nuclei rotating at least 90° during an 8-h period. The rotation involved the nuclear interior as well as the nuclear envelope. The rotation of nuclei required an intact cytoskeletal network and was eliminated by expressing lamin B1 in cells. Nuclear rotation could also be abolished by expressing larger nesprin isoforms with long spectrin repeats. These findings demonstrate that lamin B1 serves a fundamental role within the nuclear envelope: anchoring the nucleus to the cytoskeleton. Mutations in nuclear lamins and lamin-associated proteins cause a panoply of human diseases (laminopathies) including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction system disease, Dunnigan-type familial partial lipodystrophy, limb-girdle muscular dystrophy, Charcot-Marie tooth disorder type 2, and Hutchinson-Gilford progeria syndrome (1Bonne G. Di Barletta M.R. Varnous S. Becane H.M. Hammouda E.H. Merlini L. Muntoni F. Greenberg C.R. Gary F. Urtizberea J.A. Duboc D. Fardeau M. Toniolo D. Schwartz K. Nat. Genet. 1999; 21: 285-288Crossref PubMed Scopus (1105) Google Scholar, 2Bione S. Maestrini E. Rivella S. Mancini M. Regis S. Romeo G. Toniolo D. Nat. 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Pak E. Durkin S. Csoka A.B. Boehnke M. Glover T.W. Collins F.S. Nature. 2003; 423: 293-298Crossref PubMed Scopus (1614) Google Scholar). The mechanism by which these ubiquitously expressed proteins cause such diverse phenotypes is unclear. This is not particularly surprising, though, because the functions of the nuclear lamins themselves are currently incompletely understood. Lamins are the principal components of the nuclear lamina, an intermediate filament meshwork that lines the inner nuclear membrane (8Aebi U. Cohn J. Buhle L. Gerace L. Nature. 1986; 323: 560-564Crossref PubMed Scopus (685) Google Scholar, 9Burke B. Stewart C.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 575-585Crossref PubMed Scopus (355) Google Scholar). Lamins are associated with chromatin, other integral proteins of the inner nuclear membrane, inner portions of the nuclear pore complexes (NPCs), 2The abbreviations used are: NPC, nuclear pore complex; MEF, mouse embryo fibroblast; LBR, lamin B receptor; KASH, Klarsicht/ANC-1/Syne-1 homology; GFP, green fluorescent protein; EGFP, enhanced GFP; RFP, red fluorescent protein; ER, endoplasmic reticulum; Az, sodium azide; DOG, 2-deoxyglucose; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. and several transcription factors such as SREBP1, retinoblastoma protein, and MOK (10Goldman R.D. Gruenbaum Y. Moir R.D. Shumaker D.K. Spann T.P. Genes Dev. 2002; 16: 533-547Crossref PubMed Scopus (493) Google Scholar). Thus, lamins are critical for the structural integrity of the nucleus and also play a role in DNA replication, chromatin organization, and transcriptional regulation (11Hutchison C.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 848-858Crossref PubMed Scopus (249) Google Scholar). In mammalian cells, the major A-type lamins, lamins A and C, are alternatively spliced products of LMNA, whereas the major B-type lamins, lamin B1 and lamin B2, are encoded by two distinct genes, LMNB1 and LMNB2, respectively (12Stuurman N. Heins S. Aebi U. J. Struct. Biol. 1998; 122: 42-66Crossref PubMed Scopus (596) Google Scholar). Although the A- and B-type lamins share a similar structure, they differ in their behavior during cell division and their patterns of expression. B-type lamins are found in all cell types and are expressed throughout development, whereas A-type lamins are not present in early embryos (13Broers J.L. Machiels B.M. Kuijpers H.J. Smedts F. van den Kieboom R. Raymond Y. Ramaekers F.C. Histochem. Cell Biol. 1997; 107: 505-517Crossref PubMed Scopus (172) Google Scholar). Within the nucleus, lamin B1 binds directly to chromatin and histones (14Taniura H. Glass C. Gerace L. J. Cell Biol. 1995; 131: 33-44Crossref PubMed Scopus (234) Google Scholar) and interacts with several chromatin-binding inner nuclear membrane proteins (e.g. lamina-associated proteins, lamin B receptor (LBR), and the nuclear pore protein nucleoporin 153) (15Smythe C. Jenkins H.E. Hutchison C.J. EMBO J. 2000; 19: 3918-3931Crossref PubMed Scopus (119) Google Scholar). Following mitosis, B-type lamins assemble first into the nuclear lamina, followed by lamin A, and subsequently by lamin C (16Hutchison C.J. Alvarez-Reyes M. Vaughan O.A. J. Cell Sci. 2001; 114: 9-19Crossref PubMed Google Scholar). Few data exist on the biological role of the B-type lamins. B-type lamins may have a direct role in DNA synthesis (16Hutchison C.J. Alvarez-Reyes M. Vaughan O.A. J. Cell Sci. 2001; 114: 9-19Crossref PubMed Google Scholar), and silencing of lamin B by siRNA causes cell death in human cells and in Caenorhabditis elegans (17Liu J. Ben-Shahar T.R. Riemer D. Treinin M. Spann P. Weber K. Fire A. Gruenbaum Y. Mol. Biol. Cell. 2000; 11: 3937-3947Crossref PubMed Scopus (336) Google Scholar, 18Harborth J. Elbashir S.M. Bechert K. Tuschl T. Weber K. J. Cell Sci. 2001; 114: 4557-4565Crossref PubMed Google Scholar). For these reasons, B-type lamins are generally assumed to be essential. Mice deficient in lamin B1 (Lmnb1Δ/Δ), which were created with a gene-trap embryonic stem cell line, die in the perinatal period with defects in lung and bone (19Vergnes L. Peterfy M. Bergo M.O. Young S.G. Reue K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10428-10433Crossref PubMed Scopus (303) Google Scholar). Lmnb1Δ/Δ embryonic fibroblasts display nuclear shape abnormalities, chromosomal abnormalities, and impaired differentiation into adipocytes (19Vergnes L. Peterfy M. Bergo M.O. Young S.G. Reue K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10428-10433Crossref PubMed Scopus (303) Google Scholar). In humans, duplication of LMNB1 causes autosomal dominant leukodystrophy (20Padiath Q.S. Saigoh K. Schiffmann R. Asahara H. Yamada T. Koeppen A. Hogan K. Ptacek L.J. Fu Y.H. Nat. Genet. 2006; 38: 1114-1123Crossref PubMed Scopus (308) Google Scholar). To further elucidate the function of lamin B1, we decided to examine nuclear shape and dynamics in fibroblasts from Lmnb1Δ/Δ and wild-type embryos by quantitative time-lapse videomicroscopy. We made a stunning observation: nuclei spin in the absence of lamin B1, and we show that this nuclear rotation could be rescued by transfection with a GFP-lamin B1 fusion protein. Using fluorescence labeling of discrete nuclear envelope components, we found that the nuclear rotation includes rotational movement of chromatin, the nuclear lamina, the inner nuclear membrane, NPC proteins, as well as the endoplasmic reticulum (ER) immediately adjacent to the nucleus, but not the extended ER or the surrounding cytoskeleton. Furthermore, the nuclear rotation was energy-dependent, required an intact cytoskeleton, and the rotation could be reduced by transfection of Lmnb1Δ/Δ cells with larger nesprin isoforms. These data suggest a critical role for lamin B1 as a molecular anchor at the nuclear-cytoplasm interface, specifically in facilitating physical coupling between the outer nuclear membrane and the surrounding cytoskeleton. Cell Culture and Reagents—Wild-type and Lmnb1-deficient (Lmnb1+/+ and Lmnb1Δ/Δ, respectively) mouse embryo fibroblasts (MEFs) were derived from Lmnb1Δ/Δ mouse embryos and wild-type littermates (19Vergnes L. Peterfy M. Bergo M.O. Young S.G. Reue K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10428-10433Crossref PubMed Scopus (303) Google Scholar). Lmna+/+ and Lmna-/- MEFs were obtained from Dr. Colin Stewart (21Sullivan T. Escalante-Alcalde D. Bhatt H. Anver M. Bhat N. Nagashima K. Stewart C.L. Burke B. J. Cell Biol. 1999; 147: 913-920Crossref PubMed Scopus (968) Google Scholar). All cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (HyClone, Logan, UT) and penicillin/streptomycin (Invitrogen) at 37 °C. To study the role of the cytoskeleton in nuclear dynamics, cells were treated with media containing either 200 nm taxol, 300 nm nocodazole, or 200 nm cytochalasin D (all from Sigma) before starting time-lapse imaging experiments. For recovery studies, drugs were removed and replaced with fresh media before the second time-lapse experiment. In all cases, fluorescence immunohistochemistry confirmed that each drug was disrupting its intended target cytoskeletal component, while leaving other structures intact. For ATP inhibitor studies, subconfluent Lmnb1Δ/Δ MEFs were treated with media containing 5 mm sodium azide (Az) and 1 mm 2-deoxyglucose (DOG), both from Sigma, before starting the time-lapse imaging experiment. Cell nuclei were labeled with Hoechst 33342; the endoplasmic reticulum was stained with ER-Tracker Blue-White DPX; and mitochondria were stained with MitoTracker Green FM (all from Molecular Probes). Plasmid Construct and Transfection—The pGFP-Lmnb1 construct was generated by inserting the human lamin B1 sequence into the multiple cloning site of the pEGFPC1 vector (Clontech), generating lamin B1 fused to the carboxyl terminus of EGFP. The nesprin constructs were generated by cloning human cDNAs for nespirn-1α,-2α, and -2β into pEGFPC1 vectors. Nesprin-1αKASH (amino acids: nespirn-1α-(918-982)) or 2αKASH (amino acids: nespirn-2α-(483-542)) contain only the KASH domain sequences for each isoform. All nesprins are fused to the carboxyl terminus of EGFP. The pGFP-emerin plasmid was a gift from Dr. Chris Hutchison of University of Durham; the pRFP-Lmna and pLBR-GFP plasmids were provided by Dr. Howard Worman of Columbia University; and the pPOM121-GFP plasmid was provided by Dr. Brian Burke of the University of Florida. GFP-actin and GFP-tubulin were from Dr. Frank Gertler of the Massachusetts Institute of Technology. All constructs contain full-length human proteins, which, except for LBR and POM121, are placed at the carboxyl terminus of fluorescence tags. Transfection was conducted with GeneJammer Transfection Reagent (Stratagene) at a ratio of 3 μl to 1 μg of DNA. Time-lapse Imaging and Fluorescent Confocal Microscopy—Lmnb1+/+, Lmnb1Δ/Δ, and Lmna-/- MEFs were grown to subconfluent density in 35-mm polystyrene cell culture dishes (Corning), sealed with parafilm, and equilibrated to room temperature. Images of cells were acquired automatically at either 20× or 60× magnification on an Olympus IX-70 microscope with a digital charge-coupled device camera (CoolSNAP HQ, Roper Scientific) for 100 frames at 5-min intervals (corresponding to 8 h and 20 min). Phase contrast images alone were acquired with ImagePro image acquisition software (Media Cybernetics). Consecutive phase contrast and fluorescent images were acquired with an automated, motorized shutter system (Prior ProScan II) controlled by IPLab version 3.7 (Scanalytics) software. Fluorescence images were processed with Deconvolution 7.0 (Vaytech Image). For confocal microscopy, cells were plated on 35-mm glass-bottom culture dishes (MatTek) and imaged with a Plan-Apochromat 63×/1.4 oil differential interference contrast oil-immersion objective on a Zeiss Axiovert 100M microscope. Laser scanning microscopy was done with an argon laser module. Photobleaching and image acquisition were controlled with the Zeiss LSM510 software. Image Acquisition and Manipulation—Phase contrast and fluorescence time-lapse images and confocal microscopy were carried out as described above. Time-lapse images were taken with an Olympus LCPlanF 20× phase contrast objective (numerical aperture, 0.40). Confocal images were acquired with a Plan-Apochromat 63× oil immersion objective (numerical aperture, 1.4). All imaging experiments were carried out at room temperature, and cells were maintained in complete culture media. Videos of time-lapse images were generated with MATLAB, and fluorescence grayscale images from IPLab were colorized in MATLAB for the appropriate fluorochromes (green for GFP, and red for RFP). Photographic films from Western blot studies were digitized on an Epson Perfection 2450 scanner with linear intensity settings. Digital images were processed in Adobe Photoshop (version 6.0) by adjusting the linear image intensity display range. Nuclear Movement Analysis—Customized MATLAB algorithms were used to track the position of three to six distinct nucleoli within each nucleus as described previously (22Lammerding J. Hsiao J. Schulze P.C. Kozlov S. Stewart C.L. Lee R.T. J. Cell Biol. 2005; 170: 781-791Crossref PubMed Scopus (284) Google Scholar). For each frame, the centroid of selected nucleoli was calculated, and the linear conformal image transformation was computed that best mapped the current centroid positions to the original positions while minimizing the least-square error. The linear conformal transformations can account for a combination of translation, rotation, and scaling, and also preserve the relative position of objects to each other. The deviation from the best fit (i.e. the error between the least-square fit transformation and the actual nucleoli positions) was used as a measure of nuclear deformation, as it describes the extent of nuclear deformation from its initial shape independent of absolute nuclear movement or uniform changes in size. Nuclei of each cell type were analyzed for the time-averaged rotational movement (in degree), translational movement (in microns), and nuclear shape deformation (in microns) as well as normalized nuclear size, or the scaling factor. Time-lapse videos were also qualitatively scored by an observer blinded to genotype, as either rotating or not rotating. A rotating nucleus was defined as rotating at least 90° in either direction within the 8-h, 20-min time frame. Western Analysis—Cells were lysed in radioimmune precipitation assay buffer with 1 mm dithiothreitol, 0.5 mm phenyl-methylsulfonyl fluoride, and protease inhibitor mixture (Sigma) at 1:1000 dilution. Equal amounts of protein in 12 μl of sample buffer were electrophoresed on 10% Bis-Tris polyacrylamide gels (Invitrogen), and then transferred onto polyvinylidene difluoride membrane (PerkinElmer Life Sciences). Blots were probed with either primary rabbit polyclonal antibody against human lamin B1 (sc20682, Santa Cruz Biotechnology, 1:500 dilution), goat polyclonal antibody against human lamin A/C (sc6215, Santa Cruz Biotechnology, 1:500 dilution), rabbit polyclonal antibody against human emerin (Abcam, 1:3000 dilution), goat polyclonal horseradish peroxidase-conjugate antibody against GFP (Abcam, 1:500 dilution), or rabbit anti-actin antibody (Sigma, 1:5000 dilution), followed by chemiluminescence detection (PerkinElmer Life Sciences). Immunofluorescence Microscopy—Cells were grown on glass slides, transfected with GFP constructs of nesprin isoforms, and fixed in 4% paraformaldehyde for 10 min, 48 h after the transfection. Cells were then permeabilized for 10 min with either 0.2% Triton X-100 in phosphate-buffered saline, or 0.003% digitonin (Sigma) in water (in experiments involving selective permeabilization of the plasma membrane only). The cells were then blocked with 1% bovine serum albumin and labeled with appropriate primary and secondary antibodies. Fluorescence images were acquired at 20× magnification using an Olympus IX-70 microscope with a digital charge-coupled device camera (CoolSNAP HQ, Roper Scientific) driven by IPLab version 3.7 (Scanalytics) software. Cells were probed with either goat polyclonal antibody against human lamin A/C (sc6215, Santa Cruz Biotechnology) followed by Alexa Fluor 568 (red) rabbit anti-goat IgG (A11079, Invitrogen) or rabbit polyclonal antibody against GFP (ab6556, Abcam) followed by Alexa Fluor 350 (blue) goat anti-rabbit IgG (A11046, Invitrogen). All antibodies were used at 1:200 dilution in 1% bovine serum albumin. Statistical Analysis—All experiments were performed at least three independent times. Statistical analyses were performed with the PRISM 3.0 and INSTAT (GraphPad, San Diego, CA). The unpaired Student's t test (allowing for different variance) was used to analyze if the means of two data groups were statistically different. For all experiments, a two-tailed p value of <0.05 was considered significant. All data are expressed as mean ± S.E. Lmnb1Δ/Δ Nuclei Spin—To assess the functional importance of lamin B1, we examined nuclear dynamics in fibroblasts derived from littermate Lmnb1Δ/Δ and wild-type mouse embryos with time-lapse videomicroscopy. Representative videos of wild-type, Lmnb1Δ/Δ, and lamin A/C-null (Lmna-/-) cells are included in Video 1 (supplemental material). Remarkably, a high percentage of Lmnb1Δ/Δ nuclei displayed rotational movement around an axis perpendicular to the image plane (i.e. the nuclei rotated parallel to the plane of the cell substrate). Rotation in an orthogonal axis was never observed. The rotational motion was intermittent, and the degree and speed of rotation varied; the direction of spinning was random. Occasionally, nuclei underwent rapid rotation and turned several times before stopping. Fig. 1A shows a series of 14 images (corresponding to 65 min) capturing one nucleus with rapid counterclockwise rotation of up to 145°. As demonstrated in Video 1, the positions of nucleoli relative to each other did not change in the rotating nuclei, despite the striking rotation. Also, despite the spinning, Lmnb1Δ/Δ nuclei maintained a circular shape and appeared normally positioned within the cell, suggesting that the entire nucleus rotates as a solid body. In contrast, Lmna-/- nuclei displayed dynamic nuclear deformation but little rotational movement; wild-type cells displayed stable nuclear shape and only minimal nuclear rotation. To quantify the frequency of nuclear rotation, we counted the fraction of nuclei that rotated at least 90° during an 8-h observation period. The majority (90 ± 2.7%) of Lmnb1Δ/Δ cells met this criterion, whereas wild-type and Lmna-/- cells rarely displayed any nuclear rotation (p < 0.005) (Fig. 1B). Subsequently, we analyzed nuclear dynamics in more detail (e.g. nuclear rotation, translation, and deformation) based on the trajectories of selected nucleoli within each nucleus. We calculated the incremental rotation at each time point and determined the absolute angles of nuclear rotation at the end of each time-lapse period for all of the nuclei observed. On average, Lmnb1Δ/Δ nuclei rotated 90.0 ± 20.8° in either direction, with one nucleus turning as much as 593° (i.e. more than 1.5 full turns) (Fig. 1C). Despite the intermittent nature of nuclear rotation, the time-averaged angle of rotation for Lmnb1Δ/Δ nuclei was larger than in wild-type and Lmna-/- nuclei. Both Lmnb1Δ/Δ and Lmna-/- cells had slightly increased nuclear translational motion (Fig. 1D), predominantly caused by increased cell movement. However, because Lmna-/- and Lmnb1Δ/Δ cells did not differ statistically in their translational movement, this effect appears to be unrelated to nuclear rotational movement, which was unique to Lmnb1Δ/Δ cells. Unlike Lmna-/- cells, Lmnb1Δ/Δ cells did not show increased time-averaged nuclear deformation compared with wild-type cells (Fig. 1E), and nuclear fragility was normal (Fig. S1, supplemental material). Rotation of Lmnb1Δ/Δ Nuclei Is Attenuated by the Expression of a GFP-lamin B1 Fusion Protein—To confirm that nuclear spinning was attributable to the loss of wild-type lamin B1, we analyzed nuclear movement in Lmnb1Δ/Δ cells transfected with a GFP-lamin B1 fusion construct. Experiments were done within 48 h post-transfection, enough time for mitosis and nuclear envelope reformation to occur in most cells. Nuclei expressing GFP-lamin B1 exhibited reduced nuclear rotation compared with nontransfected Lmnb1Δ/Δ controls (45% reduction, p = 0.02), but expression of a GFP-emerin fusion construct did not alter nuclear rotation (Fig. 1F) and failed to restore wild-type behavior. Similarly, transfection with a red fluorescent protein-lamin A fusion protein (RFP-lamin A) did not prevent nuclear rotation (not shown). Average translational movement was not affected by any of the constructs. Western analysis confirmed the presence of both GFP-lamin B1 and GFP-emerin 1 day after transfection (Fig. 1G). Expression of GFP-lamin B1, however, did not completely restore the wild-type phenotype (45% reduction in nuclear rotation), possibly due to variations in transfection efficiency or expression of GFP-lamin B1. These data indicate that rotation in Lmnb1Δ/Δ nuclei is due to the loss of functional lamin B1. Nuclear Rotation in Lmnb1Δ/Δ Cells Includes Chromatin and the Nuclear Envelope—Although time-lapse analysis of phase-contrast images is well suited to quantify the extent of nuclear rotation based on nucleoli movement, it is insufficient to determine if the rotational movement encompasses the entire nuclear interior and the nuclear envelope. Therefore, we selectively labeled chromatin, the nuclear lamina, the inner nuclear membrane, and NPCs with fluorescent probes and then analyzed, by time-lapse videomicroscopy, which nuclear structures participated in the nuclear rotation in Lmnb1Δ/Δ fibroblasts. Staining cells with Hoechst 33342, a DNA minor groove-binding fluorescent dye, demonstrated that the entire chromatin contents were involved in the nuclear rotation (Fig. 2A and Video 2). This finding was in keeping with our observation that nucleoli retained their relative position to each other. Because lamins can directly bind to DNA and thus attach the nuclear lamina to chromatin (14Taniura H. Glass C. Gerace L. J. Cell Biol. 1995; 131: 33-44Crossref PubMed Scopus (234) Google Scholar), we investigated whether the nuclear lamina also rotated in the Lmnb1Δ/Δ fibroblasts. Time-lapse microscopy of cells expressing RFP-lamin A revealed distinct rotation of the nuclear lamina (Video 3). These results were further confirmed by time lapse-analysis of partially photobleached nuclei in RFP-lamin A-labeled Lmnb1Δ/Δ cells. In one example, images taken immediately after photobleaching showed a darkened band through the fluorescent nuclear lamina (Fig. 2F), which turned ∼50° clockwise during the subsequent 78-min period of time-lapse confocal laser scanning microscopy (see also Video 7, supplemental material). Subsequently, we tracked rotation of the inner nuclear membrane by fluorescent time-lapse videomicroscopy in cells transfected with GFP-tagged lamin B receptor (LBR-GFP) or emerin (GFP-emerin). LBR and emerin reside at the inner nuclear membrane (23Georgatos S.D. EMBO J. 2001; 20: 2989-2994Crossref PubMed Scopus (24) Google Scholar) and therefore can serve as an indicator for inner nuclear membrane movement. We found that both LBR, which interacts with lamin B1, and emerin, which binds to lamin A, participated in the nuclear rotation in Lmnb1Δ/Δ cells (Fig. 2C and 3A). Rotation of the inner nuclear membrane was clearly demonstrated by the movement of the fluorescent nuclear periphery in the supplemental Videos 4 and 8. Importantly, the inner nuclear membrane (marked by GFP-emerin) rotation occurred in unison with the rotation of the nuclear interior, as indicated by the numbered nucleoli in the phase-contrast images (Fig. 3A). We also attempted photo-bleaching studies on GFP-emerin- and LBR-GFP-labeled nuclei, but the high diffusional mobility of these inner nuclear membrane proteins and the resulting rapid fluorescence recovery (<10 min) did not allow long term observations of the photobleached sections. Lamin B1 can bind to NPC proteins such as nucleoporin 153 (15Smythe C. Jenkins H.E. Hutchison C.J. EMBO J. 2000; 19: 3918-3931Crossref PubMed Scopus (119) Google Scholar), so that loss of lamin B1 could potentially allow the inner nucleus to rotate relative to the nuclear pores and the outer nuclear envelope. To address this possibility, we followed the movement of GFP-fused nuclear pore complex protein POM121 in Lmnb1Δ/Δ cells with inverse fluorescence recovery after photobleaching. POM121 is a pore complex protein that serves to anchor NPCs to the nuclear membrane (24Soderqvist H. Imreh G. Kihlmark M. Linnman C. Ringertz N. Hallberg E. Eur. J. Biochem. 1997; 250: 808-813Crossref PubMed Scopus (30) Google Scholar); in these experiments, the entire nuclear fluorescence is extensively photobleached except for a small region of the nuclear envelope (25Rabut G. Doye V. Ellenberg J. Nat. Cell Biol. 2004; 6: 1114-1121Crossref PubMed Scopus (359) Google Scholar), so that only POM121-GFP stably incorporated into NPC within that region remain fluorescent while eliminating fluorescence from soluble and excess GFP-tagged proteins that can diffuse freely within the nuclear membrane. We tracked nuclear movement up to 1 h after photobleaching during which time fluorescence recovery was not observed, and incorporation of newly produced POM121-GFP occurred only very slowly. Video 5 (Fig. 2D) shows distinct rotational movement of a small section of fluorescently labeled NPCs. The lack of quick fluorescence recovery confirms stable incorporation into NPCs and correlates well with previous reports of slow turnover (26Daigle N. Beaudouin J. Hartnell L. Imreh G. Hallberg E. Lippincott-Schwartz J. Ellenberg J. J. Cell Biol. 2001; 154: 71-84Crossref PubMed Scopus (324) Google Scholar) and low dissociation rates of GFP-tagged POM121 following inverse fluorescence recovery after photobleaching (25Rabut G. Doye V. Ellenberg J. Nat. Cell Biol. 2004; 6: 1114-1121Crossref PubMed Scopus (359) Google Scholar). The clockwise directional turning of fluorescent NPCs in Video 5, with fluorescence extension at one end while retracting at the other end, suggests that nuclear membrane movement is due to rotation and not membrane diffusion. An additional video of a nucleus expressing POM121-GFP, in which a distinct fluorescent bleb on the nuclear periphery revealed rotational movement of NPCs, is included in the supplemental materials (Video 13). To further confirm that the outer nuclear membrane, like the NPCs and the inner nuclear membrane, also turn during nuclear rotation, we followed the movement of a GFP-fused nesprin isoform, nesprin-2α, as an outer nuclear membrane marker (Fig. 2E and Video 6). Nesprin-2 isoforms are present at the outer nuclear membrane (27Zhang Q. Ragnauth C.D. Skepper J.N. Worth N.F. Warren D.T. Roberts R.G. Weissberg P.L. Ellis J.A. Shanahan C.M. J. Cell Sci. 2005; 118: 673-687Crossref PubMed Scopus (217) Google Scholar), and we showed independently that expression of GFP-nesprin-2α does not interfere with nuclear rotation in Lmnb1Δ/Δ cells (see Fig. 5A). Following time-lapse image analysis, rotation of the outer nuclear membrane was clearly demonstrated by the movement of GFP-nesprin-2α (Video 6). Because the outer nuclear membrane is continuous with the ER, we monitored ER movement in cells labeled with ER-Tracker (Molecular Probes). These experiments revealed that only the ER immediately surrounding the nucleus displayed some rotational movement along with the nucleus, particularly at the start of rotation, but that the majority of the ER remained stationary throughout the rotation, suggesting that the lipid membranes can flow sufficiently on the slow experimental time scales (Fig. 3B and Video 9). The Nucleus Rotates Relative to the Stationary Cytoskeleton—To address the possibility that nuclear rotation occurred as part of overall cellular and cytoskeletal movement, we performed time-
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