Full-length Sec61β sequence was amplified from HeLa cDNA library (Invitrogen, Carlsbad, CA) by PCR using primers 5′ACGAATTCTATGCCTGGTCCGACCCC3′ and 5′CCGGTACCCTACGAACGAGTGTACTTGC3′. Both Sec61β and human LBR (IMAGE clone 3925138; Open Biosystems, Thermo Biosystems, Huntsville, AL) were fused with GFP at the amino terminus in pAcGFP1-C1 vector (Clontech, Mountain View, CA). Human H2B (IMAGE clone 40002352; Open Biosystems) was fused with mCherry at the carboxyl terminus in the vector pGW1myc2a-mCherry. Alanine mutations at Ser-71, Ser-86, Thr-118, and Thr-200 of LBR were generated with the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA). Deletion of the globular I domain in LBR was generated by PCR using primers 5′GGCGTTAGATCTAGGCAAAGGAAAGGTGGC3′ and 5′GGGAATTCGAGCATTAGTAGATGTATGG3′. Deletion of the globular II domain in LBR was generated by two steps of PCR. The first step amplified the amino-terminal region (amino acids 1–104) using primers 5′GGCAGATCTATGCCAAGTAGGAAATTTGC3′ and 5′CACCAGGTACTCCTCCAAAGGCCTGGTGGGAAGCAGAAGC3′, as well as the carboxyl-terminal region (amino acids 209–615) using primers 5′GCTTCTGCTTCCCACCAGGCCTTTGGAGGAGTACCTGGTG3′ and 5′GGGAATTCGAGCATTAGTAGATGTATGG3′. Both PCR products were then mixed and used as the template in the second PCR using primers 5′GGCAGATCTATG CCAAGTAGGAAATTTGC3′ and 5′GGGAATTCGAGCATTAGTAGATGTATGG3′. For the generation of GST-fused recombinant proteins, amino-terminal fragment of LBR (amino acids 1–208) and its variants were amplified from the full-length constructs in pAcGFP1-C1 vector by PCR using primers 5′GGCAACGGATCCATGCCAAGTAGGAAATTTGC3′ and 5′GGTTGAATTCCTCCAAGTCCTTTGCCCG3′ and were cloned into pGEX-2T at BamHI and EcoRI sites. For the generation of recombinant proteins with N-terminal 6His and C-terminal mCherry fusions, LBR fragments were subcloned into pET28a vector at BamHI and EcoRI sites and mCherry cloned at EcoRI and XhoI sites.
HeLa cells were grown in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37°C in a humidified incubator supplemented with 5% CO2. For live imaging, cells were seeded on the glass coverslip coated with 1 μg/ml poly-l-lysine (Sigma-Aldrich, St. Louis, CA). On the following day the cells were transfected with the expression plasmids using Effectene transfection reagent (Qiagen, Valencia, CA). Two days after transfection, the glass coverslip with cells was set up in a POC-R chamber (LaCon, Staig, Germany) for microscopy. For treatment of inhibitors during live-cell imaging, the prewarmed medium containing 200 μM roscovitine (CalBiochem, La Jolla, CA), 80 nM calyculin A (CalBiochem), 20 μM ZM447439 (Tocris Bioscience, Ellisville, MO), or an equivalent volume of the solvent DMSO was injected into the chamber through a perfusion tubing, and the existing medium in the chamber was simultaneously withdrawn through a second tubing.
Image acquisition and processing
Images were acquired with Observer Z1 microscope (Carl Zeiss, Jena, Germany) equipped with 63 × LCI Plan-Neofluor numerical aperture–1.3 water objective and AxioCam MR charge-coupled device camera controlled by AxioVision, release 4.8, software (Carl Zeiss). The cells in the POC-R chamber were maintained with 5% CO2
at 37°C during time-lapse imaging. For NE reassembly, Z-stack images (14 sections, 1–1.5 μm apart) were collected every minute. For membrane dissociation after NEBD, Z-stack images (12 sections, 1–1.2 μm apart) were collected every 2 min. The images were processed by Huygens deconvolution software. The initial attachment of ER was defined as the first time point that ER contacted with the chromatin, which extended on the chromatin surface on the following time points and eventually became a part of the NE. The distance between chromatin surface and NE/ER membrane was measured as described previously (Muhlhausser and Kutay, 2007
) for the central four sections of the Z-stack. Briefly, a radial grid of 20 lines with an angle of 18° between lines was placed in the center of the chromatin mass, and the distance between chromatin and membrane was manually measured along the grid lines by AxioVision, release 4.8, software.
Purification of recombinant proteins
The expression constructs of pGEX-2T-LBR were transformed into Escherichia coli strain BL21-DE3 RIL. The bacteria were cultured in 1 l of Luria-Bertani (LB) medium to 0.6 OD600 before inducing the protein expression with 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 2.5 h. The cells were then lysed by sonication in lysis buffer A (phosphate-buffered saline [PBS] containing 50 mM EDTA, 10 μg/ml each of leupeptin, pepstatin, and chymostatin, 1 mM phenylmethylsulfonyl fluoride [PMSF]), followed by the addition of 1% Triton X-100. The GST-fusion proteins were purified through 1 ml of glutathione agarose and eluted with 10 mM glutathione in 50 mM Tris (pH 8.0). The purified proteins were concentrated by dialysis in PBS containing 50% glycerol.
For 6His-LBR-mCherry, the plasmids were transformed into BL21 strain. The protein expression was induced in 1 l of culture with 0.2 mM IPTG at 25°C for 4 h. The cells were lysed by sonication in lysis buffer B (50 mM sodium phosphate, pH 8, 300 mM NaCl, 10 μg/ml each of leupeptin, pepstatin, and chymostatin, and 1 mM PMSF), followed by the addition of 0.5% Triton X-100. The proteins were purified through 1.5 ml of Ni-NTA (Qiagen, Valencia, CA). After washing with wash buffer (50 mM sodium phosphate, pH 6, 300 mM NaCl, and 0.5% Triton X-100) containing 25 mM imidazole, the proteins were eluted with 0.3 M imidazole in wash buffer. The purified proteins were concentrated by dialysis in PBS containing 50% glycerol.
In vitro phosphorylation of LBR
The purified recombinant GST-LBR1-208 proteins and GST were used as the substrates for Cdk1. The assay was performed in the kinase buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1 mM Na3VO4, 5 mM β-glycerophosphate, 2 mM dithiothreitol) supplemented with 50 μM ATP, 1 ng/μl Cdk1 (Cell Signaling Technology, Beverly, MA), 0.2 μg/μl substrate, and 1 μCi [γ-32P]ATP at 30°C for 20 min. The reaction was resolved by 10% SDS–PAGE. The gel was stained by Coomassie blue (Sigma-Aldrich) and dried for autoradiography. The 32P incorporation was quantified by Typhoon FLA 9000 (GE Healthcare, Piscataway, NJ) with ImageQuant TL 7.0 software (GE Healthcare), and protein levels were determined with the ImageJ software (National Institutes of Health, Bethesda, MD).
The extracts from Xenopus
eggs were prepared as described (Murray, 1991
) with the following modifications. For interphase cytosol, dejellied eggs were activated with 10 μM calcium ionophore A23187 (Sigma-Aldrich) in 0.2 × MMR (0.2 M NaCl, 4 mM KCl, 2 mM MgSO4
, 4 mM CaCl2
, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 0.2 mM EDTA, pH 7.8). Eggs were then washed several times with 0.2 × MMR and then with extraction buffer (50 mM HEPES, pH 7.8, 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl2
, and 10 μg/ml each of leupeptin, pepstatin, and chymostatin,). Packed eggs were centrifuged at 12,000 × g
for 15 min in an SW55Ti rotor (Beckman Coulter, Brea, CA) at 2°C. The crude cytoplasmic fraction from the centrifugation was then centrifuged at 200,000 × g
for 90 min in a TLS55 rotor (Beckman Coulter) to separate the cytosol from the membrane fractions. The cytosol was then centrifuged again at 200,000 × g
for 30 min to remove residual membrane. The resulting high-speed supernatant (HSS) was then used for the chromatin-binding assay or for phosphorylating LBR proteins. The in vitro chromatin-binding assay was performed as described (Takano et al., 2002
). Briefly, HSS was first heated at 95°C for 10 min, cooled on ice for 5 min, and then centrifuged at 20,000 × g
for 15 min to remove denatured proteins. The heated cytosol containing nucleoplasmin was then used to decondense demembranated frog sperm nuclei that were prepared as described (Murray, 1991
). For each chromatin-binding reaction, 40 μl of heated cytosol were incubated with sperm nuclei at a density of ~15,000 per μl of cytosol for 30 min at 25°C, followed by incubation with 0.5 μg of 6His-LBR-mCherry proteins for 30 min on ice. The samples were then diluted with nine volumes (360 μl) of ice-cold extraction buffer containing 0.1% Triton X-100 and layered over 1 ml of 30% sucrose made in extraction buffer containing 0.1% Triton X-100. The samples were centrifuged at 15,000 × g
for 15 min in a JS13.1 rotor (Beckman Coulter). Chromosomal pellets were washed in 500 μl of the sucrose solution and spun again for 5 min. The pellets were solubilized by heating at 95°C for 5 min in SDS–PAGE sample buffer for immunoblot analysis with anti-mCherry antibody (provided by Chao-Wen Wang, Academia Sinica, Taipei, Taiwan).
For prior treatment with cytosol, 6His-LBR-mCherry proteins were first bound to Ni-NTA beads and then incubated with mitotic or interphase HSS for 75 min at 25°C. The beads were then washed four times with wash buffer (50 mM sodium phosphate, pH 6, 300 mM NaCl, and 0.5% Triton X-100), and the proteins were eluted with 0.3 M imidazole in wash buffer.
HeLa cells were synchronized at mitosis with 100 ng/ml of nocodazole for 16 h. Mitotic cells were harvested by physical shake-off, replated, and treated with DMSO, roscovitine (200 μM), or ZM447439 (20 μM) for 20 min in a 37°C humidified incubator. The cells were lysed with lysis buffer (10 mM potassium phosphate, pH, 7.2, 1 mM EDTA, 5 mM ethylene glycol tetraacetic acid, 50 mM β-glycerophosphate, 1 mM sodium vanadate, 10 mM MgCl2, 0.5% Triton X-100, 0.1% sodium deoxycholate, 10% glycerol, 1 mM PMSF, and 10 μg/ml each of leupeptin, pepstatin, and chymostatin). A total of 300 μg of lysate was used for immunoprecipitation with anti-Cdc2 antibody (sc-54; Santa Cruz Biotechnology, Santa Cruz, CA), anti–AIM-1 antibody (BD Transduction Laboratories, BD Biosciences, San Diego, CA) and anti-Plk antibody (N-19) (Santa Cruz Biotechnology), with 20 μl of protein G–Sepharose (GE Healthcare), for 1 h. The immunoprecipitates were washed four times with lysis buffer and once with kinase buffer. The kinase assay was performed in 10-μl reaction in kinase buffer containing 50 μM ATP, 1 μCi [γ-32P]ATP, and substrates (2 μg of histone H1, 2 μg of histone H3, and 0.6 μg of casein for the kinase assay of Cdk1, Aurora B, and Plk1, respectively) at 30°C for 20 min.
HeLa cells were transfected with plasmids expressing GFP or GFP-LBR variants along with H2B-mCherry as described for live imaging. The cells were harvested 2 d after transfection and lysed in lysis buffer. The Western blots were probed with anti-LBR rabbit monoclonal antibody (E398L) (Abcam, Cambridge, MA), anti–α-tubulin antibody (Upstate, Millipore, Billerica, MA), and anti-GFP antibody (provided by Chao-Wen Wang, Academia Sinica).