Protocols

Protocols (in progress)

Preparation of Xenopus embryonic extracts
Xenopus laevis eggs are fertilized in vitro, the jelly coats are removed with 2% cysteine, and the embryos are developed in MMR/5 (Newmeyer and Wilson, 1991). In early gastrulation (~10 h after fertilization at 23°C), viable embryos are rinsed four times in extract buffer (250 mM sucrose, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, and 50 µg/ml cycloheximide [ICN Biomedicals]), transferred to a 2-ml straight-sided Eppendorf tube, allowed to settle, and excess buffer is removed. Leupeptin, chymostatin, pepstatin (50 µg/ml; Sigma-Aldrich), and cytochalasin B (5 µg/ml; Sigma-Aldrich) are added. The embryos (1–2 ml) are packed at 200 g for 25 s at 4°C in a refrigerated centrifuge (Allegra GR; Beckman Coulter) and excess buffer is removed. Embryos are lysed by centrifugation at 8,300 g for 9 min at 4°C in a fixed horizontal rotor (H6002; Beckman Coulter). A clear straw-colored phase (the extract) between the lipid layer and the dense pigment granules is collected by puncturing the tube with a 21-gauge needle. Extracts containing ~2,000 nuclei/µl are frozen in N2l and stored at -70°C in 40-µl aliquots.

Xenopus Interphase Extracts and In Vitro Nuclear Assembly Reactions
Xenopus laevis egg interphase extracts are prepared as described above. The extract is frozen in liquid nitrogen in 70-µl aliquots. For all nuclear assembly reactions, aliquots of interphase extract are thawed rapidly and brought to 15 mM Hepes, pH 7.4. For ATP generation, the extract is made 1 mM ATP (Sigma Chemical Co., St. Louis, MO), 10 mM phosphocreatine (Sigma Chemical Co.), and 50 µg/ml creatine phosphokinase (Sigma Chemical Co.) (see 43). Bacterially expressed protein of interest in protein buffer is added to the assembly reaction to a final concentration of 200 µg/ml. Controls consisted of the addition of an equal volume of protein buffer to parallel assembly reactions. The volume of protein solution or PB alone added to an assembly reaction is maintained at 10% of the final volume of the reaction mixture. 15 min after the addition of protein or PB to an assembly reaction, demembranated sperm chromatin is added to initiate nuclear assembly. Sufficient sperm chromatin is added to achieve a final concentration of 1,000 nuclei per µl. Unless otherwise specified, nuclei are either fixed for immunofluorescence or prepared for electrophoretic analysis at 90-120 min after initiating the assembly reaction as described below.
In some experiments, assembled nuclei are removed from one assembly reaction and transferred to another. To accomplish this, the assembly reaction mixture is diluted 50-fold with NWB (200 mM sucrose, 15 mM Hepes, pH 7.4, 50 mM NaCl, 2.5 mM MgCl2, and 1 mM DTT), and the nuclei are recovered by centrifugation for 3 min at 1,600 g (5). The resulting pellets are then resuspended in fresh nuclear assembly reaction mixture.

Immunostaining of Xenopus Nuclei Assembled In Vitro
Nuclei assembled in vitro are fixed for 10 min at 20°C by diluting the assembly reaction mixture 10-fold in NWB and adding 0.1 vol of 100 mM ethylene glycol bis [succinimidylsuccinate] in DMSO. Fixation is stopped by adding 1 M Tris HCl, pH 7.4, to achieve a final concentration of 25 mM. Alternatively, nuclei are fixed with 4% paraformaldehyde in NWB for staining with XMCM3. The nuclei are transferred to poly-L-lysine–coated coverslips by centrifugation through a sucrose cushion (30% sucrose, 15 mM Tris-HCl, pH 7.4, 50 mM NaCl, 2.5 mM MgCl, 1 mM DTT). After fixation, coverslips are placed in 0.1% NP-40 or 0.1% Triton X-100 in PBS for 2 min, and then rinsed twice for 2 min in PBS at room temperature. 30-µl aliquots of primary antibodies, diluted 1:20 in PBS, are overlaid onto coverslips. After a 30-min incubation at 37°C, coverslips are washed four times with PBS and incubated for an additional 30 min at 37°C with a 1:50 dilution of the appropriate fluorochrome-labeled secondary antibody. The coverslips are then washed five times in PBS and mounted in 50 mM Tris Base, pH 9.0, 50% glycerol, and 2 mg/ml of p-phenylenediamine (Sigma Chemical Co.).

Transcription assays of Xenopus nuclei in extracts
Frozen aliquots are thawed (24°C), buffered with 15 mM Hepes (pH 7.4), and supplemented with an ATP-generating system (Spann et al., 1997), nucleotides (0.5 µM ATP, 0.5 µM CTP, 0.5 µM GTP, and 0.25 µM UTP), 5 mM MgCl2, RNasin (0.25 U/µl; Amersham Pharmacia Biotech), and protein of interest (1 µM), or an equal volume of dialysis buffer (Moir et al., 2000). Transcription is assayed after 1 h by adding 0.6 µM BrUTP (Sigma-Aldrich) and 0.5 U/µl of RNasin. After 15 min, nuclei are fixed and processed for immunofluorescence as described above. Alternatively, transcription products are sized by substituting 0.3 µM [ ³²P]UTP (3,000 mCi/mM; Amersham Pharmacia Biotech) for BrUTP in the embryonic extract. 15 min later, total RNA is prepared from 40 µl of extract using a SNAP kit (Invitrogen) and resolved by electrophoresis on a 0.8% agarose denaturing gel. The dried gel is used for autoradiography. Effects of -amanitin (10 µg/ml; Roche) are tested by adding the drug 20 min before [ ³²P]UTP.

DNA Replication Assay
Replication assays typically consisted of 10–15 µl of extract containing lamin-disrupted or control nuclei (1,000 nuclei/µl). The AraC arrest of replication is relieved by adding dCTP (0.8 mM final concentration) (New England Biolabs, Inc.), and replication products are labeled by the incorporation of [ ³²P]dATP (75 uCi/ml, 3,000 Ci/mM) (Amersham Pharmacia Biotech). The reactions are stopped by adding 0.5 ml of 4°C NWB, and the nuclei are isolated by centrifugation at 14,000 g for 30 s. Pellets of nuclei are resuspended in a buffer containing SDS and proteinase K (1 mg/ml) (Stratagene) (Dasso and Newport 1990 ; Walter and Newport 1997 ) and incubated at 37°C for 3 h. DNA is resolved on 0.8% agarose TBE gels (Tris-borate, EDTA). Alternatively, the sizes of replication products are determined by alkaline gel electrophoresis (50 mM NaOH, 1 mM EDTA, 1% agarose, 20 V/cm) (Strausfeld et al. 1994 ).

Immunostaining for intermediate filaments
Cells on coverslips are rinsed in PBS and fixed in either methanol (–20°C) for 4 minutes or 3.5% formaldehyde (Tousimis) at room temperature for 5 minutes. Following formaldehyde fixation, cells are permeabilized with 0.05% NP-40 for 5 minutes. Cells are then washed with PBS and processed for indirect immunofluoresence. Subsequently, cells are incubated with the appropriate primary antibody for 30 min at 37°C in a moist chamber, washed with PBS, and then incubated with the appropriate secondary antibody for 15-30 min. Following staining, coverslips are washed in PBS and mounted on glass slides in gelvatol containing 100 mg/ml Dabco [1,4-diazabicyclo [2.2.2] octane; (Aldrich Chemical).

The Preparation of IF-enriched Cytoskeletons, SDS-PAGE, and Immunoblotting
IF-enriched cytoskeletal fractions are prepared from spreading, and fully spread cells maintained at either 37°C or 4°C according to a modified version of the protocol of Starger and Goldman (1977) . For immunofluorescence studies, cells grown to 50% confluency on coverslips are lysed for 15 s in IF lysis buffer (PBS containing 0.6 M KCl, 5 mM EDTA, 5 mM EGTA, and protease inhibitors [1 mM PMSF, 1 mM TAME, 1 mg/ml leupeptin, pepstatin, and aprotinin; Sigma Chemical Co.]) containing 0.1% TX-100, and fixed and processed for immunofluorescence as described above.
For biochemical studies, cells grown to 80% confluency in 100-mm plastic dishes are washed with PBS, and then lysed in IF lysis buffer containing 1% TX-100. The insoluble fraction containing IF is pelleted at 15,000 g at 4°C in a Sorvall RC-2B centrifuge. Chromatin and actin in the pellet are removed by treatment with 5 mg/ml DNase for 30 min. After centrifugation at 18,000 g in a Sorvall centrifuge, the pellet, enriched in IF and associated proteins, is used to assay for the presence of kinesin by immunoblotting. Whole cell extracts are prepared by solubilizing live cells in boiling Laemmli buffer containing protease inhibitors (1 mg/ml leupeptin, pepstatin, and aprotinin). The samples are separated on 7.5% polyacrylamide gels according to the method of Laemmli (1970) .

Platinum replica EM for IF
For preservation of MTs and IFs, cells on coverslips are extracted with PEM buffer (100 mM Pipes, pH 6.9, 1 mM MgCl2, 1 mM EGTA) containing 1% Triton X-100, 4% PEG, 1 mg/ml DNase 1 (Sigma-Aldrich), and 10 µg/ml Taxol (Sigma-Aldrich) for 5 min (RT). For MT-free IF-enriched cytoskeletal preparations, cells are extracted in PEM buffer with 1% Triton X-100, 0.4 M NaCl, and 1 mg/ml DNase 1 for 10 min. To remove any residual actin, all preparations are treated with recombinant gelsolin NH2-terminal do main (a gift of Dr. Gary Borisy, Northwestern University School of Medicine) (Verkhovsky and Borisy, 1993). Cells are then fixed with 2% glutaraldehyde and stained with 0.1% tannic acid and 0.2% uranyl acetate. These preparations are then processed by critical point drying followed by rotary shadowing with platinum and carbon (Svitkina et al., 1995). Replicas are removed from the coverslips and transferred to copper grids as described (Svitkina et al., 1995). Immunostaining with primary and secondary gold-coupled antibodies is performed after glutaraldehyde fixation as described (Svitkina et al., 1995). Controls for these preparations involved all of the various steps and incubations described above using either no antibodies or secondary gold-coupled antibodies alone.

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Protocols		Last modified May 2004 Protocols (in progress) *Preparation of Xenopus* embryonic extracts** Xenopus laevis eggs are fertilized in vitro, the jelly coats are removed with 2% cysteine, and the embryos are developed in MMR/5 (Newmeyer and Wilson, 1991). In early gastrulation (~10 h after fertilization at 23°C), viable embryos are rinsed four times in extract buffer (250 mM sucrose, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, and 50 µg/ml cycloheximide [ICN Biomedicals]), transferred to a 2-ml straight-sided Eppendorf tube, allowed to settle, and excess buffer is removed. Leupeptin, chymostatin, pepstatin (50 µg/ml; Sigma-Aldrich), and cytochalasin B (5 µg/ml; Sigma-Aldrich) are added. The embryos (1–2 ml) are packed at 200 g for 25 s at 4°C in a refrigerated centrifuge (Allegra GR; Beckman Coulter) and excess buffer is removed. Embryos are lysed by centrifugation at 8,300 g for 9 min at 4°C in a fixed horizontal rotor (H6002; Beckman Coulter). A clear straw-colored phase (the extract) between the lipid layer and the dense pigment granules is collected by puncturing the tube with a 21-gauge needle. Extracts containing ~2,000 nuclei/µl are frozen in N2l and stored at -70°C in 40-µl aliquots. Xenopus Interphase Extracts and In Vitro Nuclear Assembly Reactions Xenopus laevis egg interphase extracts are prepared as described above. The extract is frozen in liquid nitrogen in 70-µl aliquots. For all nuclear assembly reactions, aliquots of interphase extract are thawed rapidly and brought to 15 mM Hepes, pH 7.4. For ATP generation, the extract is made 1 mM ATP (Sigma Chemical Co., St. Louis, MO), 10 mM phosphocreatine (Sigma Chemical Co.), and 50 µg/ml creatine phosphokinase (Sigma Chemical Co.) (see 43). Bacterially expressed protein of interest in protein buffer is added to the assembly reaction to a final concentration of 200 µg/ml. Controls consisted of the addition of an equal volume of protein buffer to parallel assembly reactions. The volume of protein solution or PB alone added to an assembly reaction is maintained at 10% of the final volume of the reaction mixture. 15 min after the addition of protein or PB to an assembly reaction, demembranated sperm chromatin is added to initiate nuclear assembly. Sufficient sperm chromatin is added to achieve a final concentration of 1,000 nuclei per µl. Unless otherwise specified, nuclei are either fixed for immunofluorescence or prepared for electrophoretic analysis at 90-120 min after initiating the assembly reaction as described below. In some experiments, assembled nuclei are removed from one assembly reaction and transferred to another. To accomplish this, the assembly reaction mixture is diluted 50-fold with NWB (200 mM sucrose, 15 mM Hepes, pH 7.4, 50 mM NaCl, 2.5 mM MgCl2, and 1 mM DTT), and the nuclei are recovered by centrifugation for 3 min at 1,600 g (5). The resulting pellets are then resuspended in fresh nuclear assembly reaction mixture. Immunostaining of Xenopus Nuclei Assembled In Vitro Nuclei assembled in vitro are fixed for 10 min at 20°C by diluting the assembly reaction mixture 10-fold in NWB and adding 0.1 vol of 100 mM ethylene glycol bis [succinimidylsuccinate] in DMSO. Fixation is stopped by adding 1 M Tris HCl, pH 7.4, to achieve a final concentration of 25 mM. Alternatively, nuclei are fixed with 4% paraformaldehyde in NWB for staining with XMCM3. The nuclei are transferred to poly-L-lysine–coated coverslips by centrifugation through a sucrose cushion (30% sucrose, 15 mM Tris-HCl, pH 7.4, 50 mM NaCl, 2.5 mM MgCl, 1 mM DTT). After fixation, coverslips are placed in 0.1% NP-40 or 0.1% Triton X-100 in PBS for 2 min, and then rinsed twice for 2 min in PBS at room temperature. 30-µl aliquots of primary antibodies, diluted 1:20 in PBS, are overlaid onto coverslips. After a 30-min incubation at 37°C, coverslips are washed four times with PBS and incubated for an additional 30 min at 37°C with a 1:50 dilution of the appropriate fluorochrome-labeled secondary antibody. The coverslips are then washed five times in PBS and mounted in 50 mM Tris Base, pH 9.0, 50% glycerol, and 2 mg/ml of p-phenylenediamine (Sigma Chemical Co.). *Transcription assays of Xenopus* nuclei in extracts Frozen aliquots are thawed (24°C), buffered with 15 mM Hepes (pH 7.4), and supplemented with an ATP-generating system (Spann et al., 1997), nucleotides (0.5 µM ATP, 0.5 µM CTP, 0.5 µM GTP, and 0.25 µM UTP), 5 mM MgCl2, RNasin (0.25 U/µl; Amersham Pharmacia Biotech), and protein of interest (1 µM), or an equal volume of dialysis buffer (Moir et al., 2000). Transcription is assayed after 1 h by adding 0.6 µM BrUTP (Sigma-Aldrich) and 0.5 U/µl of RNasin. After 15 min, nuclei are fixed and processed for immunofluorescence as described above. Alternatively, transcription products are sized by substituting 0.3 µM [ ³²P]UTP (3,000 mCi/mM; Amersham Pharmacia Biotech) for BrUTP in the embryonic extract. 15 min later, total RNA is prepared from 40 µl of extract using a SNAP kit (Invitrogen) and resolved by electrophoresis on a 0.8% agarose denaturing gel. The dried gel is used for autoradiography. Effects of -amanitin (10 µg/ml; Roche) are tested by adding the drug 20 min before [ ³²P]UTP. DNA Replication Assay Replication assays typically consisted of 10–15 µl of extract containing lamin-disrupted or control nuclei (1,000 nuclei/µl). The AraC arrest of replication is relieved by adding dCTP (0.8 mM final concentration) (New England Biolabs, Inc.), and replication products are labeled by the incorporation of [ ³²P]dATP (75 uCi/ml, 3,000 Ci/mM) (Amersham Pharmacia Biotech). The reactions are stopped by adding 0.5 ml of 4°C NWB, and the nuclei are isolated by centrifugation at 14,000 g for 30 s. Pellets of nuclei are resuspended in a buffer containing SDS and proteinase K (1 mg/ml) (Stratagene) (Dasso and Newport 1990 ; Walter and Newport 1997 ) and incubated at 37°C for 3 h. DNA is resolved on 0.8% agarose TBE gels (Tris-borate, EDTA). Alternatively, the sizes of replication products are determined by alkaline gel electrophoresis (50 mM NaOH, 1 mM EDTA, 1% agarose, 20 V/cm) (Strausfeld et al. 1994 ). Immunostaining for intermediate filaments Cells on coverslips are rinsed in PBS and fixed in either methanol (–20°C) for 4 minutes or 3.5% formaldehyde (Tousimis) at room temperature for 5 minutes. Following formaldehyde fixation, cells are permeabilized with 0.05% NP-40 for 5 minutes. Cells are then washed with PBS and processed for indirect immunofluoresence. Subsequently, cells are incubated with the appropriate primary antibody for 30 min at 37°C in a moist chamber, washed with PBS, and then incubated with the appropriate secondary antibody for 15-30 min. Following staining, coverslips are washed in PBS and mounted on glass slides in gelvatol containing 100 mg/ml Dabco [1,4-diazabicyclo [2.2.2] octane; (Aldrich Chemical). The Preparation of IF-enriched Cytoskeletons, SDS-PAGE, and Immunoblotting IF-enriched cytoskeletal fractions are prepared from spreading, and fully spread cells maintained at either 37°C or 4°C according to a modified version of the protocol of Starger and Goldman (1977) . For immunofluorescence studies, cells grown to 50% confluency on coverslips are lysed for 15 s in IF lysis buffer (PBS containing 0.6 M KCl, 5 mM EDTA, 5 mM EGTA, and protease inhibitors [1 mM PMSF, 1 mM TAME, 1 mg/ml leupeptin, pepstatin, and aprotinin; Sigma Chemical Co.]) containing 0.1% TX-100, and fixed and processed for immunofluorescence as described above. For biochemical studies, cells grown to 80% confluency in 100-mm plastic dishes are washed with PBS, and then lysed in IF lysis buffer containing 1% TX-100. The insoluble fraction containing IF is pelleted at 15,000 g at 4°C in a Sorvall RC-2B centrifuge. Chromatin and actin in the pellet are removed by treatment with 5 mg/ml DNase for 30 min. After centrifugation at 18,000 g in a Sorvall centrifuge, the pellet, enriched in IF and associated proteins, is used to assay for the presence of kinesin by immunoblotting. Whole cell extracts are prepared by solubilizing live cells in boiling Laemmli buffer containing protease inhibitors (1 mg/ml leupeptin, pepstatin, and aprotinin). The samples are separated on 7.5% polyacrylamide gels according to the method of Laemmli (1970) . Platinum replica EM for IF** For preservation of MTs and IFs, cells on coverslips are extracted with PEM buffer (100 mM Pipes, pH 6.9, 1 mM MgCl2, 1 mM EGTA) containing 1% Triton X-100, 4% PEG, 1 mg/ml DNase 1 (Sigma-Aldrich), and 10 µg/ml Taxol (Sigma-Aldrich) for 5 min (RT). For MT-free IF-enriched cytoskeletal preparations, cells are extracted in PEM buffer with 1% Triton X-100, 0.4 M NaCl, and 1 mg/ml DNase 1 for 10 min. To remove any residual actin, all preparations are treated with recombinant gelsolin NH2-terminal do main (a gift of Dr. Gary Borisy, Northwestern University School of Medicine) (Verkhovsky and Borisy, 1993). Cells are then fixed with 2% glutaraldehyde and stained with 0.1% tannic acid and 0.2% uranyl acetate. These preparations are then processed by critical point drying followed by rotary shadowing with platinum and carbon (Svitkina et al., 1995). Replicas are removed from the coverslips and transferred to copper grids as described (Svitkina et al., 1995). Immunostaining with primary and secondary gold-coupled antibodies is performed after glutaraldehyde fixation as described (Svitkina et al., 1995). Controls for these preparations involved all of the various steps and incubations described above using either no antibodies or secondary gold-coupled antibodies alone.