Chapter 17

Systems for the Study of Nuclear Assembly, DNA Replication, and Nuclear Breakdown in Xenopus laevis Em Extracts CARL SMYTHE AND JOHN W. NEWPORT Department of BioIoey Universily of California, San Diego La JoIla, California 92093

I. Introduction 11. Obtaining Xenopus Eggs and Isolation of Sperm Chromatin A. Induction of Oocyte Maturation and Ovulation 9. Egg Collection C. Preparation of Demembranated Sperm Chromatin 111. Preparation of Xenopus Extracts and Assays Associated with Nuclear Assembly/Disassembly A. The S Phase Extract B. The Fractionated S Phase Extract C. Nuclear Assembly Assays D. The M Phase Extract E. Preparation of a Crude MPF Fraction F. Nuclear Disassembly Assays G. The Oscillating Extract H. Monitoring Progress of Nuclear Assembly and Disassembly I. Histone H1 Kinase Assays J. DNA Replication Assays IV. Conclusions and Perspectives References

449 METHODS IN CELL BIOLOGY. VOL. 35

Copyright 0 1991 by Academic Press. lnc. All rtghts of reproduction in any form reserved.

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I. Introduction A definitive characteristic of the eukaryotic cell is the existence of a compartment containing the genomic DNA of that cell. The eukaryotic nucleus is bounded by a nuclear envelope, consisting of an inner and outer membrane. The nuclear envelope contains many nuclear pores, which control the transport of macromolecules into and out of the nucleus, thus regulating nuclear content and communication with the cytoplasmic environment. The nuclear lamina located between the inner nuclear membrane and chromatin serves to support nuclear shape and creates sites for attachment and organization of the chromatin. In eukaryotic cells which undergo “open” mitoses, all of these nuclear structures must be disassembled and reassembled once during the cell cycle. Although much is known about the physical composition and organization of the intact nucleus (Newport and Forbes, 1987; Gerace and Burke, 1988), much less is understood about the temporal regulation of nuclear assembly, DNA replication, and subsequent nuclear disassembly during mitosis. In the amphibian Xenopus laeuis, egg fertilization is followed by a period of rapid and synchronous cell division, which occurs in the complete absence of transcription or of any increase in total mass of the egg. Thus, during oogenesis, the egg must synthesize and store a large reservoir of nuclear components for use during the rapid proliferation which occurs during early development. The existence of such a reservoir of nuclear components has permitted the development of systems for the study of nuclear dynamics in uitro. Described below are conditions for preparing cell-free extracts from Xenopus eggs which allow the study of these nuclear dynamics and which have proved fruitful in elucidating structural and functional requirements for nuclear reassembly, DNA replication, and nuclear diassembly.

11. Obtaining Xenopus Eggs and Isolation of Sperm Chromatin All reagents may be obtained from Sigma Chemical Co.

A. Induction of Oocyte Maturation and Ovulation Female Xenopus frogs are maintained in water tanks (45 cm x 28 cm x 28 cm) containing 40 liters of 0.1 M NaCl at an approximate density of one animal per liter. In order to induce the maturation of oocytes, each frog

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45 1

is primed by injection with 100 units (0.5 ml) of pregnant mare serum gonadotropin 3- 10 days before eggs are required. Gonadotropin is introduced into the dorsal lymph sac by subcutaneous injection using a 25-gauge needle. If necessary, frogs may be restrained by immersion in ice-cold water for 5 minutes prior to injection. Mature eggs may be obtained by injecting each frog with 500 units (0.5 ml) of human chorionic gonadotropin 12-16 hours before use. The route of injection is the same as that used for priming.

B. Egg Collection After injection to induce ovulation, each frog is transferred to an individual tank containing 2.5 liters of either 100 mM NaCl (for S and M extracts) or modified amphibian Ringer’s solution (MMR; 100 mM NaCl, 2 mM KCl, 1 m M MgS04, 2 mM CaCI,, 0.1 mM EDTA, 5 m M HEPES, pH 7.8, for oscillating extracts). Frogs generally begin to lay eggs 8-10 hours after injection and may continue to do so for a period of up to 15 hours. Maintaining frogs in individual tanks ensures that poor-quality eggs laid by a particular frog may be discarded. Eggs are recovered from each tank by pouring off the excess solution and transferring the eggs to a glass beaker.

C. Preparation of Demembranated Sperm Chromatin Xenopus sperm provide an abundant source of chromatin, which after removal of plasma and nuclear membranes may be easily purified. The following is a modification of the method of Lohka and Masui (1983). Solutions Buffer T: 15 m M PIPES, 15 mM NaCl, 5 mM EDTA, 7 mM MgCl,, 80 mM KCl, 0.2 M sucrose, pH 7.4 (4 ml) Buffer R: Buffer T + 3% bovine serum albumin (3 ml) Buffer S: Buffer T + 20 mM maltose + 0.05% lysolecithin (0.3 ml) Apparatus Clinical centrifuge Procedures 1. Anesthetize a male frog by immersion in ice-containing water for 5 minutes. Sacrifice the animal by cranial dislocation. Open peritoneal cavity via a mid-line incision through the abdominal wall using dissection scissors and move the organs of the intestinal tract to one side. Remove the pair of testes, off-white in colour, and each 5-8 mm in length, which are located in the mid body, on either side of the mid line, and ventral to the kidneys. 2. Rinse testes in buffer T and transfer testes to a microcentrifuge tube containing 1 ml of buffer T. Mince testes with forceps to release sperm into buffer T.

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3. Centrifuge the suspension in a clinical centrifuge at 170 g (1000 rpm) for 10 seconds. Retain the supernatant containing the sperm and reextract the pellet with 1 ml of buffer T. Recentrifuge as before and combine supernatants. 4. Centrifuge suspension at 1350 g (2800 rpm) for 2 minutes. The resultant pellet consists of a whitish upper region consisting predominantly of the sperm, and a lower pink layer containing red blood cells and somatic cells. Resuspend only the upper portion of the pellet in 0.5 ml of buffer T by trituration with a Pipetman p200, and repeat centrifugation and resuspension steps twice. 5. Resuspend pellet in 100 p1 of buffer T and add 300 p1 of buffer S and incubate at 23°C for 5 minutes. 6. Add 1.2 ml of buffer R and centrifuge at 300 g (1300 rpm) for 10 minutes. 7. Resuspend pellet in 200 p1 of buffer R and dilute with 1000 p1 of buffer R and recentrifuge at 300 g (1300 rpm) for 10 minutes. 8. Finally, resuspend sperm chromatin in 50 p1 of buffer T. Dilute an aliquot of the sperm chromatin preparation 100-fold in buffer T and estimate the sperm chromatin concentration using a hemocytometer. Typically, concentrations of 40,000 per microliter are obtained. Sperm may be stored in 5-p1 aliquots at - 70°C indefinitely.

111. Preparation of Xenopus Extracts and Assays Associated with Nuclear Assembly/Disassembly All of the radiochemicals described below may be obtained from ICN. Histone H1, pepstatin A, chymostatin, aprotinin, and leupeptin are from Boehringer. Bacteriophage 1 DNA is obtained from New England Biolabs. Versilube F-50 oil is obtained from Andpak-EMA (San Jose, CA). All other reagents may be obtained from Sigma Chemical Co.

A. The S Phase Extract Mature Xenopus eggs are physiologically arrested in metaphase of the second meiotic division and therefore contain high levels of maturation (or mitosis) promoting factor (MPF). M P F consists of at least two components, et al., 1988) and cyclin (Draetta et al., the protein kinase ~ 3 4 ' ~(Dunphy '~ 1989), and a complex of these polypeptides is essential for the mitotic activity of ~34'~''. Egg lysis in the absence of phosphatase inhibitors or CaZ+ chelators results in a transient release of Ca2+from intracellular stores. This brings about the specific proteolysis of cyclin, which causes the distruction of MPF, allowing the extract to exit from M phase and enter S phase.

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Eggs require no homogenization but rather are lysed by centrifugation; the yolk cortex and much of the pigment are sedimented in the pellet, while cytoplasmic constituents are released into the supernatant. Lysis is performed in the presence of cycloheximide to prevent any further protein synthesis which might allow the renewed accumulation of cyclin. Addition of either demembranated sperm chromatin (Lohka and Masui, 1984) or protein-free bacteriophage A DNA (Newport, 1987)to the cytoplasmic S extract allows the assembly of functional nuclei around these DNA templates. Such nuclei formed in uitro are morphologically indistinguishable from normal eukaryotic nuclei, and undergo semiconservative DNA replication (Blow et ul., 1989). Stock Solutions. Storage temperatures are indicated in parentheses. Cytochalasin B, 5 mg/ml in DMSO (-20°C). Protease inhibitor cocktail: Aprotinin, leupeptin, each 5 mg/ml in water ( - 70°C). Solutions. Volumes required are indicated in parentheses. 2% cysteine, pH 7.7 (100 ml) MMR: 100 mM NaCl, 2 mM KCl, 1 mM MgSO,, 2 mM CaCl,, 0.1 mM EDTA, 5 mM HEPES, pH 7.8 (200 ml) Lysis buffer: 250 mM sucrose, 2.5 mM MgCl,, 50 mM KCl, 50 pg/ml cycloheximide, I mM dithiothreitol, 10 mM HEPES, pH 7.7 (50 ml) Appurutus Clinical centrifuge Sorvall RC refrigerated centrifuge (or equivalent), HB-4 swinging bucket rotor Light microscope Procedures 1. Dejelly about 25 ml of eggs in 100 ml of 2% cysteine, pH 7.7 (22°C). Allow the eggs to stand for up to 5 minutes, gently swirling them at intervals to facilitate removal of the jelly coat. Dejellying takes 3-5 minutes and is complete when eggs are tightly packed. The total volume of packed eggs will be about 5 ml. 2. Decant cysteine and wash eggs three times with 50 ml of MMR (22°C) to remove residual cysteine and jelly coats. Remove all but 20 ml of final MMR wash and transfer eggs to a petri dish. It is essential that eggs be suspended gently by swirling prior to transfer between containers as packed eggs tend to stick to glass and subsequently lyse. 3. Examine eggs under a light microscope and discard abnormal eggs using a Pasteur pipette. Normal eggs are 1.2-1.5 mm in diameter and consist of discrete animal (dark pigmented) and vegetal (pale yellow) regions of roughly equal size. Abnormal eggs may be grossly enlarged and/or display nonuniform pigmentation.

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4. Rinse eggs three times with lysis buffer (22°C) and transfer eggs in a minimal volume to a 15-ml polypropylene centrifuge tube. Pack the eggs by centrifugation at 170 g (lo00 rpm) for 30 seconds in a clinical centrifuge and remove excess buffer initially with a Pasteur pipette and finally with absorbent tissue paper. Add leupeptin and aprotinin to a final concentration of 10pg/ml each and cytochalasin B (which prevents unwanted actin gelation in the extract) to 5 pg/ml. 5. Lyse the eggs by centrifugation at 12,000 g (Sorvall HB-4 rotor, 10,000 rpm or equivalent, 4"C, 10 minutes). Withdraw the cytoplasmic extract (2.5 ml) by puncturing the side of the tube (Fig. 1A) using a 21-gauge needle and a 5-ml syringe. The cytoplamic extract varies in color depending on precise egg pigmentation, and may either be gray or cream. The extract may be recentrifuged (10,OOO rpm, 4"C, 10 minutes) to remove remaining contaminating yolk and particulate matter, and is then stored on ice until required. The nuclear reconstitution extract prepared in this manner consists of 75% cytoplasm and 25% lysis buffer and thus, 1 egg equivalent is approximately 0.625 p1 of extract. The extract is stable for nuclear reconstitution experiments for up to 3 hours and may be diluted by up to 50% with lysis buffer with no loss in nuclear reconstitution efficiency.

A

00 0

packandnmove

0

ee

+

excess buffer

centrifuge

+

16,oOOg, 10 min

pellet (yolk platelets)

Xenopus eggs

B

lipid crude cytoplasmic fraction

lipid nuclear assembly membranes mitochondria glycogen and ribosomes

FIG. I . Flow diagram for the preparation of (A) the S phase extract and (B) membrane and cytosol fractions for nuclear assembly.

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B. The Fractionated S Phase Extract The crude extract described above contains all of the molecular components necessary to assemble nuclei around metaphase chromosomes, sperm chromatin, or protein-free DNA (Newport, 1987). The membranes and soluble components required for this process may be separated from each other and from other cellular components by further fractionation and this allows long-term storage of the nuclear reassembly components (Wilson and Newport, 1988). Apparatus Beckman TL- 100 table-top ultracentrifuge (or equivalent), TLS-55 swinging bucket rotor Procedures 1. Centrifuge the crude cytoplasmic fraction (step 5 above) at 260,000 g (TLS-55 rotor or equivalent, 55,000 rpm, 1 hour, 4°C) This procedure generates a series of layers containing different subcellular components as indicated in Fig. 1B. 2. Remove the soluble fraction using a Pipetman plO00, taking care to minimize the removal of any of the underlying membrane components. The soluble fraction may be recentrifuged at 260,000 g (25 minutes, 4°C) to remove any residual membrane. The resulting membrane-free supernatant (1 ml) may either be used for nuclear reassembly assays directly or frozen in aliquots at - 70°C. Frozen aliquots should be no larger than 25 pl, as the slower rate of thawing obtained with larger volumes reduces the efficiency of subsequent nuclear assembly. 3. Remove the uppermost golden, translucent membrane fraction (Fig. 1B, 100 pl) using a Pipetman p200 with a sawn-off tip, taking care not to remove any of the tan-colored layer directly below. Resuspend the membrane fraction in lysis buffer (1 ml), containing protease inhibitor cocktail, and layer over lysis buffer +0.5 M sucrose (200 pl) and recentrifuge at 34,000 g (TLS-55 rotor, 20,000 rpm, 20 minutes, 4°C). The membrane pellet is resuspended in lysis buffer +0.5 M sucrose to 0.1 volume of the original cytoplasmic extract and frozen in aliquots not larger than 5 pl.

-

C. Nuclear Assembly Assays Stock Solutions.

Storage temperatures are indicated in parentheses.

ATP regenerating system components (-20°C) 0.2 M phosphocreatine, in 10 mM potassium phosphate buffer, pH 7.0 0.2 M ATP, pH 7.0 0.5 mg/ml creatine phosphokinase in 10 mM HEPES, pH 7.5, 50% glycerol

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Procedures. Rapidly thaw both soluble and membrane fractions. The soluble fraction is supplemented with an ATP regenerating system (20 m M phosphocreatine, 2 mM ATP, pH 7.0, 5 pg/ml creatine kinase). A typical reaction consists of 20 pl of soluble fraction, 2-3 p1 of membrane fraction, and 0- 1500 sperm chromatin/$ of extract. For reassembly around protein-free DNA, bacteriophage i. DNA (5-15 ng/p1 of extract) may be used. Reactions may be incubated for up to 5 hours at room temperature (22°C) and nuclear assembly monitored as described in Section III,H. Timing of Events in the Assembly of Nuclei. The formation of nuclei is an ordered process involving discernible intermediate steps. The rate of complete nuclear reconstitution may vary slightly from extract to extract. If protein-free DNA is used for nuclear reassembly, then the DNA is first assembled into nucleosomes (0-40 minutes), and further organized to form a distinctive condensed sphere (40-80 minutes), involving the formation of a nuclear scaffold. Only after the chromatin is fully condensed do the nuclear lamina and membrane vesicles begin to assemble around the DNA and subsequently form an intact nuclear envelope (80- 150 minutes). In contrast, membrane vesicles and the lamina begin binding to sperm chromatin as soon as the latter is added to the extract and thus, using this template, mature nuclei are formed more quickly (-60 minutes). DNA replication (Newport, 1987; and Fig. 2) occurs over a 30- to 60-minute period after the formation of an intact nuclear envelope.

D. The M Phase Extract M P F is known to be responsible for maintaining mature unfertilized Xenopus eggs in meiotic metaphase (Masui and Markert, 1971).The activity of M P F (consisting of cyclin and p34'"3 is regulated by mechanisms of phosphorylation/dephosphorylation (Dunphy and Newport, 1989; Morla et al.. 1989; Gautier et al., 1989), and its extraction in stable form requires conditions which prevent the proteolytic destruction of the cyclin component, and which maintain its active phosphorylation state. This is achieved by lysing eggs in the presence of the Cat+ chelator EGTA, together with the phosphatase inhibitor P-glycerophosphate and ATPyS. After egg lysis, M P F is recovered in a high-speed supernatant fraction. Addition of this M phase extract to reconstituted nuclei formed previously in an S phase extract brings about the mitotic disassembly of these nuclei, as judged by the loss of the nuclear envelope, lamin depolymerization, and chromosome condensation (Newport and Spann, 1987; Lohka and Maller, 1985; Miake-Lye and Kirschner, 1985). Stock Solutions. Storage temperatures are indicated in parentheses. Cytochalasin B, 5 mg/ml in DMSO ( - 20°C) Protease inhibitor cocktail: aprotinin, leupeptin, each 5 mg/ml in water ( - 70°C) ATPyS, 100 mM in 50 mM Hepes, pH 7.8,O.l mM DTT (-20°C)

17.

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60

90

120

457

150

Time (min)

FIG.2. Nuclear envelope formation is a prerequisite for efficient DNA replication. Sperm chromatin was added to each extract and the extracts were incubated at 22°C to initiate nuclear assembly (where appropriate). DNA replication was determined at the times indicated by measuring the incorporation of C3*P]dCTP into DNA during a 30-minute pulse. The incorporation of label into DNA was measured by scintillation counting of the DNA after agarose gel electrophoresis. DNA replication was measured in a complete S phase extract, a high-speed membrane-free supernatant obtained by centrifugation of the S phase extract at 200,000 g for 1 hr, and the high-speed supernatant supplemented with the membrane fraction. Membranes were added to a level equivalent to that present in the complete S phase extract. No DNA replication was observed with the membrane fraction alone.

Solutions 2% cysteine, pH 7.7 (100 ml) MMR:100 mM NaCI, 2 mM KCI, 1 mM MgS04, 2 mM CaCI,, 0.1 mM EDTA, 5 m M HEPES, pH 7.8 (200 ml) Buffer M: 240 mM sodium P-glycerophosphate, 60 mM EGTA, 45 mM MgCI,, 1 mM dithiothreitol, pH 7.3 (50 ml) Apparatus Clinical centrifuge Sorvall RC centrifuge (or equivalent), HB-4 rotor Beckman TL-100 table-top ultracentrifuge (or equivalent), TLS-55 swinging bucket rotor Procedure 1. Dejelly, wash, and sort eggs as described in steps 1-3 of Section III,A.

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2. Rinse eggs three times with buffer M (22°C) and transfer eggs in a minimal volume to a 15-ml centrifuge tube. Pack the eggs by centrifugation at 170 g (1000 rpm) for 30 seconds in a clinical centrifuge and remove excess buffer initially with a Pasteur pipette and finally with absorbent tissue paper. Add leupeptin and aprotinin to a final concentration of 10 pg/ml each, cytochalasin B (which prevents unwanted actin gelation in the extract) to 5 pg/ml, and ATPyS tofinal concentration of 0.5 mM. 3. Lyse the eggs by centrifugation at 16,000 g (Sorvall HB-4 rotor or equivalent, 10,000 rpm, 10 minutes 4°C). Withdraw the cytoplasmic extract (2.5 ml) by puncturing the side of the tube (Fig. 1A) using a 21-gauge needle and a 5-ml syringe. 4. Dilute the cytoplasmic extract with an equal volume of ice-cold 0.33 x buffer M containing 0.5 mM ATPyS and 10 pg/ml aprotinin/leupeptin. Centrifuge the diluted extract at 260,000 g (TLS-55 rotor or equivalent, 55,000 rpm, 1 hour, 4°C). 5. Remove the soluble fraction using a Pipetmen p1000. The soluble fraction may be recentrifuged at 260,000 g (25 minutes, 4°C) to remove any residual membrane. The resulting membrane-free supernatant (1 ml) may either be used for nuclear disassembly assays directly or frozen in 100-pl aliquots at - 70°C.

E. Preparation of a Crude MPF Fraction The soluble supernatant described above contains active MPF, capable of bringing about nuclear disassembly in uitro. A partially purified and concentrated preparation of M P F can be obtained as follows.

1. Add 0.43 volume of 3.6 M ammonium sulfate dissolved in 0.33 x buffer M to the soluble fraction obtained in Section III,C, step 7. 2. Centrifuge the precipitated proteins at 20,000 g (TLS-55 rotor, 15,000 rpm, 10 minutes, 4°C). 3. Discard the supernatant and redissolve the pellet in 1 volume of 0.33 x buffer M (containing 0.1 mM ATPy S) equivalent to one fifth of the volume of the original soluble fraction (Section III,D, step 5). 4. Dialyze this fraction against 30 volumes of 0.33 x buffer M containing 0.1 mM ATPyS for 6 hours. The M P F preparation may be frozen in 100-pl aliquot at - 70°C and is stable for at least 6 months.

F. Nuclear Disassembly Assays M phase extracts are capable of bringing about the mitotic disassembly of purified rat liver or thymus nuclei (Newport and Spann, 1987),in addition

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to Xenopus nuclei reconstituted as described in Section III,A and C (Dunphy and Newport, 1988). The time required for complete nuclear membrane breakdown and chromosome condensation depends on the type of exogenous nuclei added. The most rapid disassembly occurs with reconstituted nuclei (20-25 minutes). In contrast, disassembly of rat liver nuclei, prepared by the method of Blobel and Potter (1966), may take up to 3 hours (Newport and Spann, 1987). For routine nuclear disassembly experiments using reconstituted nuclei, it is useful to prepare nuclei which may then be frozen at - 70°C and subsequently used when required. Procedures 1 . Prepare reconstituted nuclei as described in Section II1,A and C using sperm chromatin (final concentration of lOOO/pl of extract) as a template. Incubate nuclear assembly components (up to 500 pl as required) for 1 hour at 22"C, to allow DNA decondensation and nuclear envelope formation. 2. Divide the incubation mixture into covenient aliquots (not greater than 50 pl), freeze in liquid nitrogen, and store at - 70°C. 3. Rapidly thaw frozen reconstituted nuclei when required, and mix 5 pl with an equal volume of M phase extract or partially purified M P F fraction. Incubate the mixture for up to 1 hour at 22°C and monitor the release of the nuclear envelope from the nuclei as described in Section III,H.

G. The Oscillating Extract In Xenopus eggs, the first 12 cell divisions following fertilization are independent of new transcription and are biphasic, consisting of consecutive S and M phases with no measurable G, or G2 phases. The oscillation between S phase and mitosis is regulated by the periodic activation and inactivation of MPF, which consists of at least two components, p34'*' and cyclin. Unlike ~ 3 4 ' ~ 'which ~ , is present in constant amounts during the cell cycle, levels of cyclin vary during the cycle; the protein accumulates during S phase and is abruptly degraded at the metaphase/anaphase transition. Progression into M phase has long been known to have a protein synthesis requirement, and this extract preserves b o d t h e protein synthetic capability of the egg and the ability to destroy cyclin specifically at the metaphase/anaphase transition. Eggs are arrested in second meiotic metaphase and contain high levels of MPF, stabilized by another cytoplasmic effector (Masui and Markert, 1971), termed cytostatic factor (CSF). During normal fertilization, sperm not only contributes the paternal genome, but also provides the stimulus for embryonic development (know as egg activation) by bringing about the destruction of CSF and allowing normal cell-cycle oscillations to commence. The metaphase arrest may be overcome by parthenogenetic activation of an egg and, in the procedure described below, synchronous activation of eggs is achieved by

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electrical stimulation. The resulting extract faithfully mimics the cell cycle of the intact fertilized egg with respect to nuclear breakdown and reformation, DNA replication, and M P F activation and inactivation (Hutchinson et al., 1987; Murray and Kirschner, 1989). Stock Solutions. Storage temperatures are indicated in parentheses. Cytochalasin B, 5 mg/ml in DMSO (- 20°C) Protease inhibitor cocktail: pepstatin A, chymostatin, leupeptin, each 5 mg/ml in water ( - 70°C) ATP regenerating system (-20°C): 150 mM creatine phosphate, 20 mM ATP, 2 mM EGTA, 20 mM MgCI,, pH 7.7 Solutions 2% cysteine (100 ml), pH 7.7 0.2 x MMR: 20 mM NaCI, 0.4 mM KCl, 0.2 mM MgSO,, 0.4 mM CaCl,, 0.02 mM EDTA, 1 mM HEPES, pH 7.8 (lo00 ml) Extract buffer (XB): 50 mM sucrose, 100 mM KCI, 0.1 mM CaCl,, 1 mM MgCI,, 10 mM K-HEPES, pH 7.7 (200 ml) Apparatus Activation chamber: The chamber consists of a plexiglass box (11 cm x 11 cm x 5 cm). The bottom of the interior and the underside of the lid contain plate electrodes made of stainless steel. The bottom electrode is covered to a depth of 1 cm with 2% agarose in 0.2 x MMR (Fig. 3). The eggs rest on the agarose and are covered with 600 ml of 0.2 x MMR to allow electrical contact with the upper electrode. Activation is achieved using a 12 V (AC) power supply Timer/clock Ice/water bath Wide-bore Pasteur pipette: Cut the tip of a Pasteur pippette with a glass saw and remove the lower part of the tip to make a pipette with a bore of 4-5 mm

To 12v AC

upper electrode 0.2 x MMR agarose cushion

lower electrode FIG.3. Electrical activation chamber for the parthenogeneticactivation of Xenopus eggs.

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Narrow-bore Pasteur pipette: Using a Bunsen flame, draw out a Pasteur pipette to generate a pipette with a bore of -0.5 mm Clinical centrifuge Beckman L-8 preparative ultracentrifuge (or equivalent), SW 50 swinging bucket rotor, or equivalent which accepts 5-ml tubes Procedures 1. Rinse about 25 ml of eggs, previously laid into MMR, with deionized water (22°C)and allow eggs to stand in water for 10 minutes. This ensures that the subsequent activation step is quantitative. 2. Add 1 ml of Versilube F-50 oil to each of two 5-ml ultracentrifuge tubes (Beckman SWSO). Layer 2 ml of XB (22OC) containing the protease inhibitor cocktail (final concentration of 5 pg/ml per inhibitor) and cytochalasin B (final concentration of 5 pg/ml) on top of the oil. 3. Dejelly and sort eggs as described in Section III,A,4 steps 1-3, 4. Activate eggs either by electrical stimulation or with the Ca ionophore A231 87. For electrical activation, transfer eggs to activation chamber filled with 0.2 x M M R (22°C). Activation is achieved by applying two 3-second pulses (1 2 V AC) separated by a 5-second pause. Successful activation of eggs is judged by the contraction of the pigment in the animal hemisphere. At the time of activation, set the timer running and ensure that all subsequent procedures up to step 6 are completed within I5 minutes of egg activation. Following activation, remove eggs from the activation chamber, place in a petri dish, and rinse eggs with four changes of XB (22°C). 5. Using a wide-bore Pasteur pipette, transfer the eggs into the centrifuge tubes (step 2, above) with a minimal volume of suspension buffer, to ensure that cytochalasin B and protease inhibitors are kept as concentrated as possible. Eggs should fall through the XB and come to rest above the oil layer. 6. Centrifuge the eggs in a clinical centrifuge at 300 g (1300 rpm) for 60 seconds followed by 1350 g (280 rpm) for 20 seconds. This packs the eggs at the bottom of the tube and allows the Versilube oil to rise above the eggs, thus separating them from excess buffer which must be removed using a Pasteur pipette. Small pockets of buffer which remain trapped between the eggs and the wall of the tube should be removed using a narrow-bore Pasteur pipette. Incubate the packed eggs at 22°C until 15 minutes has elapsed since activation. 7. Transfer the centrifuge tubes containing the eggs to an ice/water bath and incubate at 0°C for a further 15 minutes. 8. Lyse the eggs by centrifugation at 12,000 g (Beckman SW50 rotor or equivalent, 10,000 rpm, 10 minutes, 2°C). Withdraw the cytoplasmic extract (volume 1.5-2.0 ml), which may be found either above or below the oil layer, by puncturing the side of the tube (Fig. 4) using a 21-gauge needle and a 5-ml syringe. The cytoplasmic extract varies in color depending on precise egg pigmentation, and may be either gray or cream.

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CARL SMYTHE AND JOHN W. NEWPORT Excess Extract Buffer

-

?+

Versilube

F-50Oil

centrifuge

centrifuge

300g. 6 0 s 1350g, 20s

12,OOOg, 10 min

+

lipid crude cytoplasmic fraction Versilube F-50 oil pellet (yolk platelets)

FIG.4. Flow diagram for the preparation of an oscillating extract from Xenopus eggs.

9. Add 0.05 volume of ATP regenerating system, and recentrifuge at 12,000 g (Beckman SW50 rotor or equivalent, 10,OOO rpm, 10 minutes, 2°C) to remove remaining contaminating yolk and particulate matter. The extract may then be stored on ice for up to 2 hours until required. 10. Following the addition of demembranated sperm chromatin to the required concentration (lOO-5oO/pl), the cell-cycle oscillations may be initiated by warming the extract to 22°C. Timing of Events in an Oscillating Extract. When demembranated sperm chromatin is added to such extracts, while the extract is in interphase, the chromatin acts as a template for the formation of intact nuclei and the DNA undergoes replication. After 50-90 minutes in S phase, the extract initiates mitosis. At this time, the previously intact nuclei undergo nuclear envelope breakdown and chromosome condensation (Fig. 5). After approximately 20 minutes in mitosis, the extract returns to S phase as indicated by the reassembly of nuclear structure around the condensed chromatin. The chromatin decondenses and assumes an interphase morphology and an intact nuclear envelope is again observed. Cell-free oscillating extracts carry out two to three such cycles with uniform periods of 60-100 minutes, depending on the extract and the concentration of DNA present (Dasso and Newport, 1990).

H. Monitoring Progress of Nuclear Assembly and Disassembly Nuclear assembly and disassembly is readily followed either by phasecontrast microscopy or fluorescence microscopy using a light microscope (e.g., Zeiss Photomicroscope 111) with exciter-barrier reflector combinations suitable for 3,3'-dihexyloxacarbocyanine (DHCC; fluorescein channel), and bisbenzamide (Hoechst 33258). Samples (1-2 pl) are removed at appropriate times and diluted on a microscope slide with an equal volume of fix/ stain buffer (200 mM sucrose, 10 mM HEPES, pH 7.5, 7.4% formaldehyde)

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FIG. 5 . Assembly and disassembly of nuclei in an oscillating extract. An oscillating extract was made from Xenopus eggs as described in Section 111,G. Sperm chromatin was added to the extract at a concentration of SOOjpl of extract. The sperm chromatin decondensed and nuclear assembly occurred, with the extract remaining in S phase for 70 minutes, as judged by the decondensed appearance of the DNA (A) and the presence of a nuclear envelope (C).The extract then entered mitosis for a 20-minute period as indicated by the condensation of the DNA (B) and the loss of the nuclear envelope (D). Following the mitotic period, the extract returned to S phase as judged by the reformation of nuclear envelope and decondensed chromatin (not shown). The nuclear envelope was observed by phase-contrast microscopy, while the DNA was visualized by fluorescencemicroscopy after staining with bisbenzamide. Bar, 5 pm.

containing either bisbenzamide (1 pg/ml) for staining DNA, and/or the lipophilic dye DHCC ( 1 pg/ml) for visualizing membranes.

I. Histone H1 Kinase Assays The p34'd'2component of M P F is a protein kinase, and it has been established that the growth-associated histone HI kinase (Arion et al., 1988) is identical to MPF. Thus, changes in the activity of M P F during the course of the cell cycle can be conveniently monitored by measuring histone H1 kinase activity (Fig. 6). As CAMP-dependent protein kinase is also a histone H1 kinase, specificity for ~ 3 4 ' ~is ' maintained ~ by the addition, to the assay mixture, of an inhibitor peptide of the former enzyme.

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CARL SMYTHE AND JOHN W. NEWPORT

- aphidicolin 200

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105 120 150 180

Time (min)

FIG.6. Cell-cycle-dependent variation of histone H1 kinase activity. Histone H1 kinase activity was measured at the indicated times in an oscillating extract containing sperm chromatin (4OO//.d) in the absence (upper panel) and presence (lower panel) of the DNA replication inhibitor aphidicolin (40pg/ml). Kinase activity was quantitated by laser densitometry of an autoradiogram following gel electrophoresis of histone H1-containing samples. In the upper panel, mitosis refers to the time intervals in which the extracts werejudged to be in mitosis by visual observation of the nuclei. The aphidicolin-treated extract failed to enter mitosis.

Stock Solutions. Storage temperatures are given in parentheses. The letters A-F refer to the assay components in Table I.

A. 200 m M HEPES, 50 m M EGTA, 100 m M MgCI,, pH 7.3 (4°C) B. Histone H 1,2 mg/ml in H 2 0 (- 20°C) C. Protein kinase inhibitor peptide (PKI), 100 pM in H,O, (-20°C) D. ATP, pH 7.0,2 m M H,O (-20°C) E. [y-32P]ATP, which, when added to solution D, is sufficient to make the final specific activity z 500,000-1,000,000 cpm/nmol(-2o"C) F. H,O EB buffer: 80 m M sodium glycerophosphate, 10 mM MgCl,, 5 m M EGTA, pH 7.5 (4°C)

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TABLEI ASSAYMIXTURE FOR MEASURING HISTONEHI KINASEACTIVITY OF p34cd'' Number of assays required Assay components Buffer Histone HI PKI ATP H2O a

(A) (B) (C)

(D+ E) (F)

1 assay" 2.0 2.0 2.0

2.0 2.0

10 assays"

20 20 20 20 20

All volumes given are in microliters.

Procedure 1. In order to assay histone H1 kinase activity in any of the above extracts, samples (2 pl) are removed from the extract as appropriate, diluted into an equal volume of EB buffer, and immediately frozen in liquid nitrogen for subsequent analysis. 2. Mix the reaction components according to Table I, such that there are 10 pl of reaction mixture per assay. 3. Maintain samples frozen in a dry ice/ethanol bath until immediately prior to assay. Initiate reactions at 1-minute intervals, by diluting each sample 60-fold with EB buffer and immediately adding 10 pl of the diluted enzyme to 10 pl of assay mixture. The incubation is continued for 10 minutes at 22°C. Stop reaction either by step 4 or 5. 4. Add 20 p1 of 2 x SDS sample load buffer and electrophorese on 10% SDS-polyacrylamide gel. After electrophoresis, discard that portion of the gel containing the dye front (and free [32P]ATP), fix the gel in 7% acetic acid, 10% methanol, and, after drying the gel, expose to preflashed X-ray film for direct autoradiograph y. Alternatively: 5. Remove 15 pl of reaction mixture and pipet onto 1.5 cm x 1.5 cm phosphocellulose paper (Whatman, P81). Terminate reaction by immersion in 1% phosphoric acid. Wash filters for two 15-minute periods in 1% phosphoric acid, then 95% ethanol, dry, and count in scintillation fluid.

J. D N A Replication Assays DNA replication may be assayed in any of the above extracts as follows. Stock Reagents

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[B-~~PI~A (-20°C) TP Replication sample buffer: 8 mM EDTA, 0.13% phosphoric acid, 10% Ficoll, 5% SDS, 0.2% bromophenol blue, 80 mM Tris-C1, pH 8.0 (22°C) Proteinase K, 10 mg/ml in H,O (-20°C) Procedures 1. Remove 10-p1 aliquots of extract incubation at 15-minute intervals and add each to 1 pCi of [ c ( - ~ ~ P ] ~ A T P . 2. Allow incubation to continue for a further 15 minutes. Terminate the reaction by the addition of 10 pl of replication sample buffer. 3. Digest each reaction mixture with proteinase K (final concentration 1 mg/ml) for 2 hours at room temperature. 4. Electrophorese on 0.8%agarose gel an after drying the gel, expose to preflashed X-ray film for direct autoradiography. The extent of DNA replication may be quantitated by laser densitometry of the autoradiogram, or by excising and counting relevant segments of the gel.

IV. Conclusions and Perspectives Using the extracts described above, synthetic nuclei may be reconstituted around protein-free DNA or sperm chromatin. The in uitro assembled nuclei are morphologically indistiguishable from normal eukaryotic nuclei; they are surrounded by a double membrane, containing functional nuclear pores, and are lined with a peripheral nuclear lamina (Newport, 1987). Fractionation studies have shown that nuclear envelope assembly is initiated by the binding to chromatin of a specific set of membrane vesicles, which are distinct from the majority of endoplasmic reticulum-derived vesicles. The association of these vesicles with chromatin is mediated by a trypsin-sensitive integral membrane receptor (Wilson and Newport, 1988), and this association is regulated by reversible phosphorylation in a cell-cycle-dependent manner (Pfaller et al., 1991). Inhibitors may be added to, and particular proteins may be depleted from, these extracts in order to test theories of nuclear assembly, disassembly, and the cell-cycle control of these processes. Thus, the addition of the DNA topoisomerase inhibitor VM26 prevents DNA condensation and subsequent nuclear envelope assembly (Newport, 1987). Following initial envelope formation and insertion of nuclear pores, inhibition of transport through the nuclear pores by addition of wheat germ agglutinin (Finlay et al., 1987) blocks the import of the embryonic lamin L,,,, which prevents subsequent enlargement of the nuclear envelope. Immunodepletion of lamin Llllfrom the extract results in the formation of nuclei which are unable to replicate their

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DNA (Newport el al., 1990). Inhibition of DNA replication by aphidicolin prevents the subsequent activation of MPF and entry into mitosis, indicating the presence of a control system that monitors the replication state of DNA and regulates the activation of MPF (Dasso and Newport, 1990). Such approaches yield the conclusion that the structural organization within the nucleus is built up sequentially, such that the completion of one layer of organization serves as the foundation for the next, and that checkpoints exist to ensure that key stages are completed before the next stage is initiated. Future applications of these systems will undoubtedly involve the development of assays to measure discrete steps in the pathways of nuclear assembly, genome replication, and nuclear disassembly. The progressive biochemical dissection of these processes, coupled with genetic and immunological approaches, will facilitate the identification of, and assignment of function to, proteins involved in processes as diverse as nuclear vesicle binding to chromatin, nuclear vesicle fusion and envelope growth, nuclear transport, as well as signal transduction pathways which regulate cell-cycle progression. ACKNOWLEDGMENTS The authors would like to thank their colleagues, Eva Meier, Colin Macaulay, Mary Dasso, Sally Kornbluth, Philippe Hartl, and Fang Fang for much helpful advice on the compilation of these methodologies. Work on this subject in J. W. N’s laboratory was supported by a National Institutes of Health grant (GM334234).

REFERENCES Arion, D., Meijer, L., Brizuela. L., and Beach, D. (1988). cdc2 is a component of the M-phase specific HI kinase: Evidence for identity with MPF. Cell (Cambridge. Mass.) 55, 371-378. Blobel, G., and Potter, V. (1966). Nuclei from rat liver: Isolation method that combines purity with high yield. Science 154, 1662-1665. Blow, J. J., Sheehan, M. A., Watson, J. V., and Laskey, R. A. (1989). Nuclear structure and the control of DNA replication in the Xenopus embryo. J. Cell Sci. Suppl. 12, 183-185. Dasso, M., and Newport, J. W. (19%). Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in uitro: Studies in Xenopus. Cell (Cambridge, Mass.) 61,811-823. Draetta, G., Luca, F., Westendorf, J., Ruderman, J., and Beach, D. (1989). cdc2 protein kinase is complexed with cyclin A and B: Evidence for proteolytic inactivation of MPF. Cell (Cambridge. Mass.) 56, 829-838. Dunphy, W. G., and Newport, J. W. (1988).Mitosis-inducing factors are present in a latent form during interphase in the Xenopus embryo. J . Cell Biol. 106,2047-2056. Dunphy, W. G., and Newport, J. W. (1989). Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell (Cambridge. Mass.) 58, 181-191. Dunphy, W. G., Brizuela, L., Beach, D., and Newport, J. W. (1988). The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis Cell (Cambridge. Mass.) 55, 423 -43 1.

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Finlay, D. R., Newmeyer, D. D., Price, T. M., and Forbes, D. J. (1987). Inhibition of in uitro nuclear transport by a lectin that binds to nuclear pores. J . Cell Biol. 104, 189-200. Gautier, J., Matsukawa, T., Nurse, P., and Maller, J. (1989). Dephosphorylation and activation of Xenopus p34 protein kinase during the cell cycle. Nature (London) 339,626-629. Gerace, L., and Burke, B. (1988). Functional organisation of the nuclear envelope. Annu. Rev. Cell Biol.4, 335-374. Hutchinson, C., Cox, R., Drepaul, R., Gomperts, M., and Ford, C. (1987). Periodic DNA synthesis in cell-free extracts in Xenopus eggs. EMBO J. 6,2003-2010. Lohka, M., and Maller, J. (1985). Induction of nuclear envelope breakdown, chromosome condensation and spindle formation in cell-free extracts. J . Cell Biol. 101, 518-523. Lohka, M., and Masui, Y. (1983). Formation in uitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220,719-721. Lohka, M., and Masui, Y. (1984). Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. J . Cell Biol. 98, 1222-1230. Masui, Y.,and Markert, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177, 129-146. Miake-Lye, R., and Kirschner, M. (1985). Induction of early mitotic events in a cell-free system. Cell (Cambridge, Mass.) 41, 165-175. Morla, A. O., Draetta, G., Beach, D., and Wang, J. Y. J. (1989). Reversible tyrosine phosphorylation of cdc2: Dephosphorylation accompanies activation during entry into mitosis. Cell (Cambridge, Mass.) 58, 193-203. Murray, A. W., and Kirschner, M. W. (1989). Cyclin synthesis drives the early embryonic cell cycle. Nature (London) 339,287-292. Newport, J. W. (1987). Nuclear reconstitution in uitro: Stages of assembly around protein-free DNA. Cell (Cambridge, Mass.) 48,205-217. Newport, J. W., and Forbes, D. J. (1987). The nucleus: Structure, function and dynamics. Annu. Rev. Biochem. 56, 535-565. Newport, J. W., and Spann, T. (1987). Disassembly of the nucleus in mitotic extracts: Membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell (Cambridge, Mass.) 48,219-230. Newport, J. W., Wilson, K. L., and Dunphy, W. G. (1990). A lamin-independent pathway for nuclear envelope assembly. J. Cell Biol. 111,2247-2259. Pfaller, R., Smythe, C., and Newport, J. W. (1991). Assembly/disassembly of the nuclear envelope membrane: Cell-cycle dependent binding of nuclear membrane vesicles to chromatin in oitro. Cell (Cambridge, Mass.) 65, 209-217. Wilson, K. L., and Newport, J. W. (1988). A trypsin sensitive receptor on membrane vesicles is required for nuclear envelope formation in uitro. J . Cell Biol. 107, 57-68.